method for producing wheat flour, wheat flour, food ingredient, food product, food production method

专利摘要:
WHEAT GRAIN, METHOD TO PRODUCE WHOLE FLOUR WHEAT OR FLOUR, INTEGRAL FLOUR OF T RIGO OR FLOUR PRODUCED FROM WHEAT GRAIN, FOOD INGREDIENT, FOOD PRODUCT, COMPOSITION OR MIXTURE, METHOD OF STARCH METHOD WHEAT OR WHEAT GRAIN PLAN, METHOD TO PRODUCE A FOOD OR DRINK. A wheat grain (Triticum aestivum) comprising a germ, starch and one, two or three SBEIIa proteins is provided, said germ comprising two identical alleles of a SBEIIa-A gene, two identical alleles of the SBEIIa-B gene and two identical alleles of the SBEIIa- D gene, in which the starch has an amylose content of at least 50% in (w / w) as a proportion of the extractable starch from the grain and in which at least one of the SBEIIa proteins is produced in the endosperm of the developing wheat and has enzymatic activity that gave starification.
公开号:BR112013011025B1
申请号:R112013011025-2
申请日:2011-11-04
公开日:2021-03-02
发明作者:Ahmed Regina；Matthew Kennedy Morell；Pierre Georges Louis Berbezy；Elisabeth Marie-Anne Ida Chanliaud；Bernard Duperrier
申请人:Arista Cereal Technologies Pty Ltd；
IPC主号:

专利说明:

This order claims the priority of the Order for
North American Provisional Patent No. 61 / 410,288, filed on November 4, 2010, the contents of which are incorporated herein by reference. FIELD
The specification describes methods of obtaining hexaploid wheat plants that have high amylose starch and the use of such plants, and particularly grain or starch from them in a variety of food and non-food products. HISTORIC
The bibliographic details of the publications mentioned by the author of this specification are collected at the end of the description.
Reference to any prior technique in this specification is not, and should not be regarded as, knowledge or any form of suggestion that the prior technique is part of the general common knowledge in any country.
In the last decade, much has been learned about the molecular, genetic and cellular events that underlie plant life cycles and plant production. One vegetable product in particular is the grain of the wheat. Wheat grain is a staple food in many countries and it supplies at least 20% of the kilojoule food for the total world population. Starch is the main component of wheat grain and is used in a wide variety of food and non-food products. The characteristics of starch vary and they play an important role in determining the suitability of wheat starch for end use
particular. Despite this huge global consumption and despite an increase in awareness of the importance of starch functionality in the quality of the final product, research into genetic variation in wheat and its precise impact on the characteristics of starch lag behind other commercially important plant crops.
Common wheat (Triticum aestivum) is a hexaploid that has three pairs of homologous chromosomes defining genomes A, B and D. The endosperm of the grain comprises 2 haploid complements of a maternal cell and 1 of a paternal cell. The wheat grain germ comprises a haploid complement of each of the maternal and paternal cells. Hexaploidy was considered a significant obstacle in the research and development of useful wheat variants. In fact, very little is known about how the homologous genes of wheat interact, how their expression is regulated and how the different proteins produced by the homologous genes work separately or together.
Cereal starch is made up of two polymers of glucose, amylose and amylopectin. The ratio of amylose to amylopectin appears to be a major determinant (i) in the health benefit of wheat grain and wheat starch and (ii) in the final quality of products that comprise wheat starch.
Amylose is an essentially linear polymer of α-1,4-linked glucose unit, while amylopectin is highly branched with α-1,6 glycosidic unit bonds that link linear chains.
Starches with a high amylose content are of particular interest for their health benefits. It has been found that foods that are high in amylose, by the way, are naturally higher in resistant starch, a form of dietary fiber. AR is starch or starch digestive products that are not digested or absorbed in the small intestine. Resistant starch is increasingly seen as having an important role in promoting intestinal health and protecting against diseases such as colorectal cancer, type II diabetes, obesity, heart disease and osteoporosis. Starches with a high amylose content have been developed in certain grains such as corn and barley for use in food as a means of promoting intestinal health. The beneficial effects of resistant starch result from the provision of a nutrient to the large intestine in which the intestinal microflora is given to an energy source that is fermented to form short-chain fatty acids, by the way. These short-chain fatty acids provide nutrients for colonocytes, increase the absorption of certain nutrients through the large intestine and promote physiological activity of the colon. Generally, if resistant starches or other dietary fibers are not supplied to the colon, it becomes metabolic and relatively inactive. Thus, products with a high amylose content have the potential to facilitate increasing fiber consumption. Some of the potential health benefits of consuming high amylose wheat grain, or products such as starch, include its role in regulating sugar and insulin and lipid levels, promoting intestinal health, producing less valuable food caloric that promotes satiety, improving laxation, volume of water in the stool, promoting the growth of probiotic bacteria and increasing the excretion of fecal bile acid.
Starchy foods contain very little AR. Breads made using wild-type wheat flour and a conventional formulation and baking process contained <1% RA. In comparison, breads baked using the same process and storage conditions, but containing wheat with a high content of modified amylose had levels of AR as much as 10 times higher (see International Publication No. WO 2006/069422). Vegetables, which are one of the few rich sources of AR in the human diet, contain levels of AR that are normally <5%. Therefore, consumption of wheat bread with a high amylose content in quantities normally consumed by adults (for example, 200 g / d) would easily supply at least 5 to 12 g of RA. Thus, the incorporation of high amylose wheat into food products has the potential to make a considerable contribution to the dietary RA intakes of developed nations, where average daily RA intakes are estimated to be only about 5g.
Starch is widely used in the food, paper and chemical industries. The physical structure of starch can have an important impact on the nutritional and handling properties for industrial or food or non-food products. Certain characteristics can be considered as an indication of the structure of the starch including the distribution of the amylopectin chain length, the degree and type of crystallinity, and properties such as gelatinization temperature, viscosity and swelling volume. Changes in the amylopectin chain length can be an indicator of crystallinity, gelatinization or altered amylopectin retrogradation.
While chemically or otherwise modified starches can be used in foods that provide functionality not normally provided by unmodified sources, such processing has a tendency to alter other components of value or lead to the perception of being undesirable due to the processes involved in the modification. Therefore, it is preferable to provide sources of constituents that can be used in unmodified form in food.
Starch is initially synthesized in plants in photosynthetic chloroplasts of tissues such as leaves, in the form of transient starch. This is mobilized during the dark periods to supply carbon to export to the dissipating organs and energy metabolism, or to store in organs such as seeds or tubers. The long-term synthesis and storage of starch occurs in the amyloplasts of the storage organs, such as the endosperm, where the starch is deposited as semicrystalline granules up to 100 μm in diameter. The granules contain amylose and amylopectin, the former typically as an amorphous material in the natural starch granule, while the latter is semicrystalline through the stacking of linear glycoside chains. The granules also contain some of the proteins involved in the starch biosynthesis.
Starch synthases in the endosperm are performed in four essential stages. ADP-glucose pyrophosphorylase (ADGP) catalyzes the synthesis of ADP-glucose from glucose-1-phosphate and ATP. The starch synthases then promote the transfer of ADP-glucose to the end of a glucose unit bound to α-1,4. Third, starch branching enzymes (SBE) form new α-1,6 bonds in α-polyglycans. The starch debranching enzymes (SDBE) then remove some of the branching bonds through a mechanism that has not been fully resolved.
While it is clear that at least these four activities are required for normal starch granule synthesis in larger plants, multiple isoforms of enzymes participating in one of the four activities are found in the endosperm of larger plants. The specific roles for some isoenzymes have been proposed based on mutational analysis or by modifying levels of gene expression using transgenic approaches (Abel et al, 1996; Jobling et al, 1999; Schwall et al, 2000). However, the precise contributions of each isoform of each activity to starch biosynthesis are not yet known, and these contributions appear to differ markedly between species.
In the endosperm of the cereal, two isoforms of ADP-glucose pyrophosphorylase (ADGP) are present, one form within the amyloplast, and one form in the cytoplasm. Each shape is made up of two types of subunits. The shrunken (sh2) and fragile (bt2) mutants in corn represent lesions in the major and minor subunits, respectively.
Some efforts have focused on starch synthase enzymes to investigate strategies to modulate the amylose / amylopectin ratio in wheat (see Sestili et al. 2010).
Four classes of starch synthase (SS) are found in the cereal endosperm, an isoform exclusively located within the starch granule (granule-bound synthase (GBSS)) two forms that are divided between the granule and the soluble fraction (SSI and SSII ) and a fourth form that is totally located in the soluble fraction (SSIII). GBSS has been shown to be essential for amylose synthesis, and mutations in SSII and SSIII have been shown to alter the structure of amylopectin.
A mutant wheat plant entirely lacking the SGP-1 protein (SSIIa) was produced by crossbred strains that were missing in the specific forms of the SGP-1 protein (SSII) genome A (BIIII) (Yamamori et al, 2000 ). Examination of SSII null seeds showed that the mutation resulted in changes in the structure of amylopectin, deformed starch granules, and a high relative amylose content for about 30 to 37% of the starch, which was an increase of about 8 % in relation to the wild type level (Yamamori et al., 2000). Amylose was measured by colorimetric measurement, amperometric titration (both for iodine binding) and a concanavalin A method. The starch of the SSII null mutant exhibited a decreased gelatinization temperature compared to the starch of an equivalent, non-mutant plant. The starch content was reduced from 60% in the wild type to below 50% in the SSII null grain.
In maize, dull mutation 1 causes reduced starch content and increased amylose levels in the endosperm, with the extent of the change depending on genetic inheritance, and increased degree of branching in the remaining amylopectin. The gene corresponding to the mutation was identified and isolated by a transposition element labeling strategy using the transposition element modifier (Mu) and presented to encode the synthesis of the projected starch synthase II (SSII) of the enzyme. The enzyme is now recognized as a member of the SSIII family in cereals. The mutant endosperm had reduced levels of SBEIIa activity associated with the dull mutation 1. It is not known whether these findings are relevant to other cereals.
Barley strains having a high proportion of amylose in grain starch have been identified. These include Glacial with High Amylose Content (AC38) which has a relative amylose content of about 45%, and chemically induced mutations in the barley SSIIa gene that raised amylose levels in the kernel starch to about 65 to 70% (WO 02/37955 Al; Morell et al, 2003). The starch showed reduced gelatinization temperatures.
Two main classes of SBEs are known in plants, SBEI and SBEII. SBEII can be further categorized into two types in cereals, SBEIIa and SBEIIb. Additional forms of SBEs are also reported in some cereals, 149 kDa of putative SBEI for wheat and 50/51 kDa of SBE for barley.
Sequence alignment reveals a high degree of sequence similarity at both nucleotide and amino acid levels and allows for grouping into the classes of SBEI, SBEIIa and SBEIIb. SBEIIa and SBEIIb generally exhibit about 80% of the identity of the nucleotide sequence to each other, particularly in the central regions of the genes.
In maize and rice, phenotypes with a high amylose content have been shown to result from lesions in the SBEIIb gene, also known as the amylose extender gene (ae) (Boyer and Preiss, 1981, Mizuno et al, 1993; Nishi et al, 2001). In these SBEIIb mutants, the starch grains of the endosperm showed an abnormal morphology, the amylose content was significantly high, the frequency of the residual amylopectin branch was reduced and the proportion of short chains (<DP17, especially DP8-12) was lower . In addition, the gelatinization temperature of the starch has been increased. In addition, there was a significant association of the material that was defined as an “intermediate” between amylose and amylopectin (Boyer et al, 1980, Takeda et al, 1993b). In contrast, the mutant in maize plants in the SBEIIa gene due to a modifying insertion element (Mu) and consequently the missing SBEIIa protein expression was imperceptible in wild type plants in the endosperm starch branch (Blauth et al, 2001 ), despite having been altered in the starch in the leaf. In both maize and rice, the SBEIIa and SBEIIb genes are not linked in the genome.
SBEIIa, SBEIIb and SBEI can also be noted for their patterns of expression, both temporal and spatial, in the endosperm and other tissues. SBEI is expressed from the development of the middle endosperm ahead of wheat and corn (Morell et al, 1997). In contrast, SBEIIa and SBEIIb are expressed from an early stage of endosperm development. In maize, SBEIIb is the predominant form in the endosperm while SBEIIa is present in high levels of expression in the leaf (Gao et al, 1997). In rice, SBEIIa and SBEIIb are found in the endosperm in approximately equal amounts. However, there are differences in time and tissue expression. SBEIIa is expressed in an earlier stage of seed development, being detected in 3 days after flowering, and was expressed in the leaves, while SBEIIb was not detectable in 3 days after flowering and was more abundant in seed development in 7 to 10 days after flowering and was not expressed in the leaves. In the wheat endosperm, SBEI (Morell et al, 1997) is found exclusively in the soluble fraction, while SBEIIa and SBEIIb are found in the soluble fractions and associated with the starch granule (Rahman et al, 1995).
Maize varieties with very high amylose content have been known for some time. Low amylopectin starch corn that contains a very high amylose content (> 90%) was achieved by a considerable reduction in SBEI activity along with an almost complete inactivation of SBEII activity (Sidebottom et al, 1998).
In potatoes, the low regulation of the main SBE in the tubers (SBE B, equivalent to SBEI) by antisense methods resulted in some new starch characteristics, but did not alter the amylose content (Safford et al, 1998). The antisense inhibition of the less abundant form of SBE (SBE A, analogous to SBEII in cereals) resulted in a moderate increase in amylose content to 38% (Jobling et al, 1999). However, the low regulation of SBEII and SBEI yielded much greater increases in the relative amylose content, to 60 to 89%, than the low regulation of SBEII alone (Schwall et al., 2000).
International Publication No. WO 2005/001098 and International Publication No. WO 2006/069422 describe, by the way, transgenic hexaploid wheat comprising exogenous double RNA constructs that reduce the expression of SBEIIa and / or SBEIIb in the endosperm. The grain from transgenic lines did not transport any protein from
SBEIIa and / or SBEIIb or reduced protein levels. A loss of the SBEIIa protein from the endosperm was associated with elevated levels of relative amylose of more than 50%. A loss of SBEIIb protein levels did not appear to substantially alter the proportion of amylose in the starch of the grain. It has been proposed, but not established, that a triple-null mutant of SBEIIa and / or SBEIIb substantially without expression of the proteins of SBEIIa and SBEIIb would result in further elevations in amylose levels. However, it has not been known or predictable from the prior art how many mutant alleles of SBEIIa and / or SBEIIb would be required to provide elevated levels of amylose of at least 50% as a proportion of total starch. It was also unknown whether the grain of triple-null genotypes would be viable or if the wheat plants would be fertile.
There is a need in the art for wheat plants with improved high amylose content and methods of producing them. SUMMARY
Throughout this specification, unless the context requires otherwise, the word "understand", or variations such as "understand" or "understanding", will be understood to imply the inclusion of an indicated element or an integer or group of elements or whole numbers, but not excluding any other element or whole number or group of elements or whole numbers.
As used here, the singular forms "one", "one" and "o" include plural aspects, unless the context clearly indicates otherwise. Thus, for example, reference to “a mutation” includes a single mutation, as well as two or more mutations; reference to "one plant" includes one plant, as well as two or more plants; and so on.
Each achievement in this specification should be applied mutatis mutandis for any other achievement, unless expressly stated otherwise.
Genes and other genetic material (for example, mRNA, constructs, etc.) are represented in italics and their protein expression products are represented in non-italics. Thus, for example, SBEIIa is an expression product of SBEIIa.
Nucleotide and amino acid sequences are identified by a sequence identifier number (SEQ ID NO :). SEQ ID NOs: correspond to sequence identifiers <400> 1 (SEQ ID NO: 1), <400> 2 (SEQ ID NO: 2), etc. A sequence listing is provided after the claims.
Unless otherwise stated, all technical and scientific terms used here have the same meaning as commonly understood by those skilled in the art, to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
The present invention provides a variety of wheat plants having modified starch characteristics.
In one embodiment, the invention provides wheat grain (Triticum aestivum) comprising an endosperm and a low level or activity of the total SBEII protein or SBEIIa protein which is 2% to 30% of the level or activity of the total SBEII or SBEIIa protein in a wild-type wheat grain, and the grain comprises an amylose content of at least 50% (w / w), or at least 60% (w / w), or at least 67% (w / w) as a proportion of the total starch in the grain.
In one embodiment, the invention provides wheat grain comprising a germ and a starch, wherein the germ comprises two identical alleles of a SBEIIa-A gene, two identical alleles of a SBEIIa-B gene and two identical alleles of a gene of SBEIIa-D, wherein each of the SBEIIa genes gives rise to an amount of protein (w / w) or a protein having SBEIIa activity that is less than the corresponding wild-type gene, and at least one of said genes comprises a point mutation, where the starch comprises amylose so that the grain has an amylose content of at least 50% (w / w) as a proportion of the extractable starch from the grain.
In one embodiment, the invention provides wheat grain comprising a germ, starch and one, two or three SBEIIa-B proteins, said germ comprising two identical alleles of a SBEIIa-A gene, two identical alleles of a SBEIIa-B gene and two identical alleles of an SBEIIa-D gene, in which the starch has an amylose content of at least 50% (w / w) as a proportion of the extractable starch from the grain, and in which at least one of the SBEIIa proteins it is produced in the endosperm of developing wheat and has enzyme activity of starch branching.
In some embodiments, the amount and activity of the SBEIIa protein is reduced.
Thus, for example, a grain of the invention may comprise a reduced amount of the SBEIIa protein (w / w) which has reduced SBEIIa activity.
In various embodiments, the protein level or activity of total SBEII or SBEIIa in the grain is less than 2% or 2% to 15%, or 3% to 10%, or 2% to 20% or 2% to 25% of the level or activity of the SBEII or total SBEIIa protein in the wild type grain.
In some embodiments, the amount or activity of the SBEIIa protein in the grain is less than 2% of the amount or activity of the SBEIIa protein in a wild type wheat grain.
In another aspect, the grain is hexaploid wheat.
In one embodiment, the hexaploid wheat grain comprises a germ, in which the germ comprises a loss of function mutation in each of 5 to 12 alleles of endogenous SBEII genes selected from the group consisting of SBEIIa-A, SBEIIa-B, SBEIIa-D, SBEIIb-A, SBEIIb-B and SBEIIb-D. In particular, said 5 to 12 alleles including 4, 5 or 6 alleles of SBEIIa each comprise a loss of function mutation. In another particular, when the number of SBEIIa alleles comprising a loss of function mutation is only 4, then the number of SBEIIb alleles comprising a loss of function mutation is 6. In another embodiment, when the number of SBEIIa alleles comprising a loss of function mutation is 6, so at least two SBEIIb alleles comprise a partial loss of function mutation. In another embodiment, the hexaploid wheat germ has no null alleles of SBEIIb genes, or only 1, only 2, only 3, only 4, only 5 or 6 null alleles of SBEIIb genes.
In another embodiment, the hexaploid wheat germ has only 2, only 3, only 4 or only 5 null alleles of SBEIIa genes.
In some embodiments, the hexaploid wheat germ has 6 null alleles of SBEIIa genes.
In some embodiments, the grain or germ has only 1 null SBEIIa gene.
In some embodiments, the grain or germ has only 2 null SBEIIa genes.
In another embodiment, the hexaploid wheat germ has no null alleles of SBEIIb genes, or only 1, only 2, only 3, only 4, only 5 or 6 null alleles of SBEIIb genes.
In yet another embodiment, the null alleles of the SBEIIa or SBEIIb genes are in genome A, genome B, genome D, genomes A and B, genomes A and D, genomes A and D, or all three of genomes A, B and D.
In yet another embodiment, the hexaploid wheat germ comprises 0, 1, 2, 3, 4, 5, or 6 alleles of partial loss of function of the SBEIIa genes. In some cases, the partial loss allele of SBEIIa gene function is in genome A, genome B, genome D, genomes A and B, genomes A and D, genomes A and D, or all three of genomes A, B and D.
Additionally, in some embodiments, the hexaploid wheat germ comprises 0, 1, 2, 3, 4, 5, or 6 alleles of partial loss of function of the SBEIIb genes. In some cases, the partial loss of function allele of the SBEIIb gene is in genome A, genome B, genome D, genomes A and B, genomes A and D, genomes A and D, or all three of genomes A, B and D.
In other embodiments, the partial loss of function alleles of the SBEIIa or SBEIIb genes are in genome A, genome B, genome D, genomes A and B, genomes A and D, genomes A and D, or all three of genomes A , B and D.
In another embodiment, the hexaploid wheat germ comprises 5 alleles of SBEIIa, each comprising a zero or partial loss of function mutation and 1 allele of SBEIIa which is wild type.
In another embodiment, the grain is tetraploid wheat.
In another embodiment, the present invention provides wheat grain from tetraploid wheat in which the grain comprises an endosperm and a low level or activity of the total SBEII protein or SBEIIa protein which is 2% to 30% of the level or activity of the protein of SBEIIa. SBEII or total SBEIIa in a wild-type wheat grain, and where the grain comprises an amylose content of at least 50% (w / w), or at least 60% (w / w), or at least 67% ( w / w) as a proportion of the total starch in the grain.
In some embodiments, in which the germ comprises a loss of function mutation in each of the 5 to 8 alleles of the endogenous SBEII genes selected from the group consisting of SBEIIa-A, SBEIIa-B, SBEIIb-A and SBEIIb-B, the said 5 to 8 alleles including 2, 3 or 4 alleles of SBEIIa each comprising a loss of function mutation, and where when the number of SBEIIa alleles comprising a loss of function mutation is only 2, then the number of alleles of SBEIIb comprising a loss of function mutation is 4, and when the number of SBEIIa alleles comprising a loss of function mutation is 4, then at least one, preferably at least two of such alleles comprise a partial loss of function mutation.
In some embodiments, the germ has only 2, or only 3, null alleles of SBEIIa genes.
In a particular embodiment, the tetraploid wheat germ has no null alleles of SBEIIb genes, or only 1, only 2, only 3, or 4, null alleles of SBEIIb genes.
In some embodiments, an SBEIIb protein is encoded by genome A, genome B or genome D, or the two proteins of SBEIIb are encoded by genomes A and B, genomes A and D, or genomes B and D.
In some embodiments, the null mutation is independently selected from the group consisting of an exclusion mutation, an insertion mutation, a docking site mutation, a premature translational termination mutation and a framing mutation.
In some embodiments, the null alleles of the SBEIIa or SBEIIb genes are in genome A, genome B, or in both genomes A and B.
In other embodiments, the tetraploid wheat germ comprises 0, 1, 2, 3 or 4, 5, or 6 alleles of partial loss of function of the SBEIIa genes.
In other embodiments, the germ comprises 0, 1, 2, 3 or 4 alleles of partial loss of function of the SBEIIb genes.
In yet another embodiment, the partial loss of function alleles of the SBEIIa or SBEIIb genes are in genome A, genome B, or in both genomes A and B.
In some embodiments, the germ is homozygous for mutant alleles in each of the 2 or 3 SBEIIa genes and / or each of the 2 or 3 SBEIIb genes.
In other embodiments, the germ is heterozygous among the 2 or 3 SBEIIa genes and / or each of the 2 or 3 SBEIIb genes.
Beneficially, in various embodiments of the present invention, the grain comprises null alleles and alleles of partial loss of function of SBEIIa and / or SBEIIb, in which each of the null alleles is located in a different genome of each of the partial loss alleles of occupation.
In some embodiments with respect to null alleles, each null mutation is independently selected from the group consisting of an exclusion mutation, an insertion mutation, a docking site mutation, a premature translational termination mutation and a framing mutation. In one embodiment, one or more of the null mutations are non-conservative amino acid substitution mutations or a null mutation has a combination of two or more non-conservative amino acid substitutions. In this context, non-conservative amino acid substitutions are as defined here. The grain can comprise mutations in one of the two SBEIIa genes, each of which are null mutations, and an amino acid substitution mutation in a third SBEIIa gene, where each of the null mutations is preferably premature translational termination mutations or exclusion mutations, or a premature translational termination mutation and an exclusion mutation, and the amino acid substitution mutation is a conservative amino acid substitution or preferably a non-conservative amino acid substitution.
In some broad embodiments, the grain of the present invention includes one or more null mutations or partial loss of function mutations which are amino acid substitution mutations, which are independently non-conservative or conservative amino acid substitutions.
In some embodiments, the grain of the present invention comprises a point mutation, which is an amino acid substitution mutation.
In some embodiments of the invention, one of the SBEIIa-A, SBEIIa-B or SBEIIa-D genes comprises a point mutation so that the protein encoded by said gene has no starch branching enzyme activity.
In some embodiments, the grain of the present invention has null alleles that are exclusion mutations in the B and D genomes that exclude at least part of the SBEIIa-B and SBEIIa-D genes, respectively and in which the SBEIIa-A gene comprises the point mutation; or having null alleles that are exclusion mutations in genomes A and D that exclude at least part of the SBEIIa-A and SBEIIa-D genes, respectively and in which the SBEIIa-B gene comprises the point mutation; or having null alleles that are exclusion mutations in genomes A and B that exclude at least part of the SBEIIa-A and SBEIIa-B genes, respectively and in which the SBEIIa-D gene comprises the point mutation.
In some embodiments of the invention, the germ comprises 6 SBEIIb alleles of which at least one has a loss of function mutation.
In some embodiments of the invention, the germ has no null alleles of the SBEIIb genes, or only 2, only 4 or 6 null alleles of the SBEIIb genes.
In some embodiments, the grain comprises a null mutation which is an exclusion mutation in genome A, B or D, which excludes at least part of an SBEIIa gene and at least part of an SBEIIb gene, preferably excluding all of the SBEIIa gene and / or the SBEIIb gene.
In some embodiments, the grain of the invention comprises a null mutation which is an exclusion mutation in the B genome that excludes at least part of the SBEIIa-B gene and at least a part of the SBEIIb-B gene, preferably which excludes all of the SBEIIa-B gene and / or the SBEIIb-B gene; or comprising a null mutation which is an exclusion mutation in the D genome that excludes at least part of the SBEIIa-D gene and at least part of a SBEIIb-D gene, preferably which excludes the entire SBEIIa-D gene and / or the SBEIIb-D gene; or comprising a null mutation which is an exclusion mutation in the B genome that excludes at least part of the SBEIIa-A gene and at least part of the SBEIIb-A gene, preferably which excludes the entire SBEIIa-A gene and / or the SBEIIb-A gene.
In the illustrative examples, the grain is provided in which the alleles comprising a partial loss of function mutation each express an SBEIIa or SBEIIb enzyme which in quantity and / or activity corresponds to 2% to 60%, or 10% to 50%, the amount or activity of the corresponding wild type allele.
In some embodiments, the grain comprises at least one SBEIIa protein that has starch branching activity when expressed in the development of the endosperm, the protein being present in an amount or having starch branching enzyme activity between 2% to 60%, or between 10% to 50%, or between 2% to 30%, or between 2% to 15%, or between 3% to 10%, or between 2% to 20% or between 2% to 25% of the quantity or activity of the corresponding protein in a wild-type wheat grain.
In some embodiments of the invention, the amount or activity of the total SBEII protein in the grain is less than 60%, preferably less than 2%, of the amount or activity of the total SBEII protein in a wild type wheat grain.
In some embodiments of the invention, there is no SBEIIa protein activity in the grain.
Specifically, in some embodiments, the grain is non-transgenic, that is, it does not comprise any transgene, or in a more specific embodiment it does not comprise an exogenous nucleic acid that encodes an RNA that reduces the expression of a SBEIIa gene, that is, if comprises a transgene, that transgene encodes an RNA other than an RNA that reduces the expression of a SBEIIa gene. Such RNAs include RNAs that encode proteins that confer herbicide tolerance, disease tolerance, increased efficiency of nutrient use or drought or other stress tolerance, for example.
In some embodiments, the grain has only one SBEIIa protein as determined by Western blot analysis, and in which the protein is encoded by one of the SBEIIa-A, SBEIIa-B and SBEIIa-D genes and has reduced branching enzyme activity starch when produced in the developing endosperm when compared to a SBEIIa protein encoded by the corresponding wild type gene.
In some embodiments, the SBEIIa protein has an altered mobility in relation to its corresponding wild type SBEIIa protein, as determined by affinity gel electrophoresis on starch-containing gels.
In some embodiments, the grain has no detectable SBEIIa protein as determined by Western blot analysis.
In some embodiments, the germ comprises only one or only two SBEIIb proteins that have starch branching enzyme activity when produced in the developing endosperm, or only one or only two SBEIIb proteins that are detectable by Western blot analysis.
Regarding loss of function mutations, in some embodiments, at least one, more than one, or all loss of function mutations are i) introduced mutations, ii) were induced in a parental wheat or seed plant by mutagenesis with a mutagenic agent such as a chemical agent, biological agent or irradiation, or iii) were introduced in order to modify the plant genome.
In another illustrative embodiment, the grain comprises an exogenous nucleic acid that encodes an RNA that reduces the expression of an SBEIIa gene, an SBEIIb gene, or both.
As determined here, the grain is provided in some particular embodiments where the grain has a germination rate of about 70% to about 90%, or about 90% to about 100% in relation to the germination rate of a control or wild type grain under standard conditions. Standard conditions are preferably as defined here.
In a particular embodiment, the SBEII activity or the SBEIIa activity is determined by testing the enzyme activity in the grain while it is developing in a wheat plant, or by testing the amount of SBEII protein such as SBEIIa grain protein harvested by immunological or other means.
In another aspect, the present invention provides grain, wherein the starch in the grain is at least 50% (w / w), or at least 60% (w / w), or at least 67% (w / w) amylose as a proportion of the total starch and is characterized by one or more of: (i) comprising 2% to 30% of the amount of SBEII or SBEIIa in relation to granules of wild-type wheat starch or starch; (ii) comprising at least 2% resistant starch; (iii) comprising a low relative glycemic index (GI); (iv) comprising low relative amylopectin levels; (v) distorted starch granules; (vi) reduced granule birefringence; (vii) reduced swelling volume; (viii) distribution of modified chain length and / or branching frequency; (ix) delayed end of gelatinization temperature and higher peak temperature; (x) reduced viscosity (peak viscosity, bonding temperature, etc.); (xi) increased molecular weight of amylopectin; and / or (xii)% modified% crystallinity of type A or type B starch, in relation to granules of wild-type wheat starch or starch.
In some embodiments, the grain is comprised of a wheat plant.
In other embodiments, the grain is a developing grain, or a mature, harvested grain. Preferably, the amount of grain is at least the weight of 1 kg, or at least the weight of 1 ton.
Conveniently, the grain is processed so that it is no longer able to germinate, such as broken, cracked, parboiled, laminated, pearly, ground or crushed grain.
In another aspect, the present invention provides a wheat plant that is capable of producing the grain as defined herein including the grain comprising an endosperm and a low level or activity of the SBEII protein or total SBEIIa protein which is 2% to 30% the level or activity of the SBEII or total SBEIIa protein in a wild-type wheat grain, and where the grain comprises an amylose content of at least 50% (w / w), or at least 60% (w / w) , or at least 67% (w / w) as a proportion of the total starch in the grain.
In particular, the wheat plant is both male and female fertile.
In one embodiment, the wheat plant is common wheat such as Triticum aestivum L. ssp. aestivum or durum wheat. In other embodiments, the wheat plant is characterized by one or more aspects of the grain as described here, preferably including the numbers and types of mutations of SBEIIa and SBEIIb as described here. All combinations of such aspects are provided.
In another embodiment, the invention provides whole flour or flour or other food ingredient such as purified starch produced from the grain as defined herein including the grain comprising an endosperm and a low level or activity of the SBEII protein or total SBEIIa protein which is 2% to 30% of the level or activity of the total protein of SBEII or SBEIIa in a wild type wheat grain, and where the grain comprises an amylose content of at least 50% (w / w), or at least 60% (w / w), or at least 67% (w / w) as a proportion of the total starch in the grain. Wholemeal flour, flour or other food ingredient can be refined by fractionation, whitening, heat treatment to stabilize the ingredient, treated with enzymes or mixed with other food ingredients such as whole wheat flour or wild type flour. The flour is preferably white flour, having specifications as known in the baking technique. In a preferred embodiment, wholemeal flour, flour or other food is packaged ready for sale as a food ingredient, the packaging of which may include recipe instructions for its use.
The present invention further contemplates granules of wheat starch or wheat starch produced from the grain in question. In some embodiments, the starch granules or wheat starch comprise at least 50% (w / w), or at least 60% (w / w), or at least 67% (w / w) amylose as a proportion of starch, and are further characterized by one or more of the aspects: (i) comprising 2% to 30% of the amount of SBEII or SBEIIa in relation to granules of wild-type wheat starch or starch; (ii) comprising at least 2% resistant starch; (iii) comprising a low relative glycemic index (GI); (iv) comprising low relative amylopectin levels; (v) distorted starch granules; (vi) reduced granule birefringence; (vii) reduced swelling volume; (viii) distribution of modified chain length and / or branching frequency; (ix) delayed end of gelatinization temperature and higher peak temperature; (x) reduced viscosity (peak viscosity, bonding temperature, etc.); (xi) increased molecular weight of amylopectin; and / or (xii)% modified% crystallinity of type A or type B amide, in relation to granules of wild-type wheat starch or starch.
The present invention further provides a food ingredient comprising grain, whole flour, flour, starch granules, or starch as defined herein, for use in food production, for consumption by non-human or preferably human animals.
In some embodiments, the food ingredient comprises the grain in which the grain is broken, cracked, parboiled, laminated, pearly, ground or crushed or any combination thereof.
The invention also provides food products or beverages that comprise a food ingredient or beverage at a level of at least 10% on a dry weight basis, where the food ingredient is or comprises the grain, whole flour, flour, starch granules, or starch as defined here. Preferably, the food product or beverage is packaged ready for sale.
In another embodiment, the invention provides a composition or mixture comprising the grain, whole flour, flour, granules of wheat starch or wheat starch as defined herein, at a level of at least 10% by weight, and grain of the wheat having a amylose level less than about 50% (w / w) or flour, wholemeal flour, granules of starch or starch obtained from it. Preferably, the wheat grain having an amylose level of less than 50% (w / w) is wild type wheat grain.
The methods are provided to obtain are or identify or select or produce a wheat plant that produces grain comprising an amylose content of at least 50% (w / w), or at least 60% (w / w), or at least 67% (w / w) as a proportion of the total starch in the grain. The wheat plant can be identified or selected from a population of multiple candidate plants, such as a mutagenized population or a plant population resulting from a crossing process or a backcrossing / reproduction process.
In some embodiments, the method comprises: (i) crossing two parental wheat plants each comprising a loss of function mutation in each of one, two or three SBEIIa or SBEIIb genes selected from the group consisting of SBEIIa-A, SBEIIa- B, SBEIIa-D, SBEIIb-A, SBEIIb-B and SBEIIb-D, or of mutagenesis of a parental plant comprising said loss of function mutations; and (ii) screening of plants or grain obtained from crossing or mutagenesis, or progeny or grain plants obtained from them, by analyzing DNA, RNA, protein, starch granules or starch from the plants or grain, and (iii) selecting a fertile plant that exhibits a level or activity of SBEII or SBEIIa in its grain that is 2% to 30% of the level or activity of the respective protein in a wild type grain. Alternatively, the method comprises steps (ii) and (iii) above, with step (i) being optional, such as when selecting or identifying a plant from a population of multiple candidate plants.
In some embodiments of the method, step (ii) includes screening the first, second, and / or subsequent generation of the progeny or grain plants for a loss of function mutation in 5 to 12 alleles of 6 endogenous genes encoding the protein of SBEII including 4, 5 or 6 alleles of SBEIIa, and where when the number of mutant alleles of SBEIIa by 4, then the number of mutant alleles of SBEIIb is 6, and when the number of mutant alleles of SBEIIa by 6, then at at least two such mutants are partial mutations.
In some embodiments, the grain of the selected fertile wheat plant is characterized by one or more aspects as defined here.
The invention further provides methods of obtaining a hexaploid or tetraploid wheat plant that produces grain comprising an amylose content of at least 50% (w / w), or at least 60% (w / w) or at least 67% (w / p) as a proportion of the total starch in the grain. In some embodiments, the method comprises (i) introducing into an wheat cell an exogenous nucleic acid that encodes an RNA that reduces the expression of one or more genes encoding the SBEII protein or total SBEIIa protein, (ii) regenerating an transgenic wheat plant comprising the exogenous nucleic acid from the cell of step (i), and (iii) screen for and select the first, second or subsequent generation of progeny from the transgenic wheat plant that produces the grain having 2% to 30% % of the level or activity of the SBEII or total SBEIIa protein in a wild type plant. Preferably, the RNA molecule is a double-stranded RNA molecule or a micro-RNA precursor molecule, which is preferably expressed from a chimeric DNA comprising a DNA region that, when transcribed, produces the RNA molecule, operably linked to a heterologous promoter such as an endosperm-specific promoter. Chimeric DNA can be introduced into a wheat cell that comprises one or more mutations of SBEIIa or SBEIIb, so that the total SBEII activity is reduced in the transgenic plant by a combination of mutation (s) and RNA molecule (s) inhibitory (s).
In some embodiments, the grain having 2% to 30% of the level or activity of the total SBEII or SBEIIa protein in a wild type plant is indicative that at least 3 SBEIIa genes or 2 SBEIIa genes and 3 SBEIIb genes of the plant comprises a loss of function mutation and therefore the plant grain comprises more than 50% (w / w), or at least 60% (w / w), or at least 67% (w / w) amylose as a proportion of total starch in the grain.
In some embodiments, the presence of at least a low level of SBEIIa protein is indicative that the plant is fertile.
In another embodiment, the invention provides a method of screening a wheat or grain plant, the method comprising screening a plant or grain for mutations in an SBEIIa or SBEIIa gene and SBEIIb genes in each of the A, B genomes and D of hexaploid wheat or genomes A and B of tetraploid wheat using one or more of the primers selected from the group consisting of SEQ ID NO: 36 to 149.
In another embodiment, the invention provides a method of screening a wheat or grain plant, the method comprising (i) determining the level or activity of SBEIIa and / or SBEIIb in relation to the level or activity in a wild type or control plant or grain and select the plant or grain having 2% to 30% of the SBEII or total SBEIIa protein level or activity in a wild type plant.
In yet another embodiment, the invention provides a method of producing a food or drink comprising (i) obtaining the grain of the invention, (ii) processing the grain to produce a food ingredient or drink and (iii) adding the food ingredient or (ii) drink for another food ingredient or drink, thereby producing the food or drink.
In another aspect, the invention provides a method for improving one or more parameters of metabolic health, intestinal health or cardiovascular health in an individual, or for preventing or reducing the severity or incidence of a metabolic disease such as diabetes, intestinal disease or cardiovascular disease , comprising providing the individual with the grain, food or drink as defined herein. The invention also provides for the use of grain, or products derived therefrom, for use in therapy or prophylaxis of metabolic disease, intestinal disease or cardiovascular disease.
Consequently, similar aspects of the invention provide the grain, food or drink in question for use to improve one or more parameters of metabolic health, intestinal health or cardiovascular health in an individual, or to prevent or reduce the severity or incidence of such a metabolic disease such as diabetes, intestinal disease or cardiovascular disease.
Consequently, similar aspects of the invention provide for the use of the grain, food or drink in question to improve one or more parameters of metabolic health, intestinal health or cardiovascular health in an individual, or to prevent or reduce the severity or incidence of such a metabolic disease such as diabetes, intestinal disease or cardiovascular disease.
Consequently, in some embodiments, the invention provides the food or drink product as defined herein for use to improve one or more parameters of metabolic health, intestinal health or cardiovascular health, or to prevent or reduce the severity or incidence of metabolic, intestinal or cardiovascular in an individual.
In another embodiment, the invention provides a method of producing the grain, comprising the steps of i) obtaining a wheat plant that is capable of producing the grain as defined here comprising an endosperm and a low level or activity of the SBEII protein or total SBEIIa protein which is 2% to 30% of the total SBEII or SBEIIa protein level or activity in a wild type wheat grain, and where the grain comprises an amylose content of at least 50% (w / w) , or at least 60% (w / w), or at least 67% (w / w) as a proportion of the total starch in the grain, and ii) harvesting the wheat grain from the plant, and iii) optionally, grain processing .
In another embodiment, the invention provides a method of producing starch, comprising the steps of i) obtaining the wheat grain as defined herein including comprising an endosperm and a low level or activity of the SBEII protein or total SBEIIa protein which is 2 % to 30% of the level or activity of the total SBEII or SBEIIa protein in a wild-type wheat grain, and where the grain comprises an amylose content of at least 50% (w / w), or at least 60% ( w / w), or at least 67% (w / w) as a proportion of the total starch in the grain, and ii) extracting the starch from the grain, thereby producing the starch.
The present invention also provides a method of marketing the wheat grain, comprising obtaining the wheat grain of the invention, and commercializing the wheat grain obtained for pecuniary gain.
In some embodiments, obtaining the grain of the wheat comprises cultivating or harvesting the grain of the wheat.
In some embodiments, obtaining the grain of the wheat comprises harvesting the grain of the wheat.
In some embodiments, obtaining the grain of the wheat still comprises storing the grain of the wheat.
In some embodiments, obtaining the grain of the wheat still involves transporting the grain of the wheat to a different location.
The above summary is not and should not be viewed in any way as an exhaustive citation of all the achievements of the present invention. BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a representation showing an alignment of the SBEIIa protein alignment (AAK26821.1 is from genome D, CAR95900.1 from genome B and CAA72154 from genome A). Dots in the alignment indicate that the identical amino acid is present as in the highest sequence. Figure 2 is a representation showing an alignment of SBEIIb amino acid sequences encoded by exons 1 to 3 of the wheat genomes A, B and D. Dashes indicate that amino acids are present in the protein, but the sequence is not known, dots in the alignment indicate that the identical amino acid is present as in the highest sequence. Figure 3 is a representation of an alignment of the SBEIIb amino acid sequences. Figure 4 is a graphical representation showing a graph of amylose content dispersion of mutant transgenic strains (see Example 5). Figure 5 is a graphical representation of data showing an amylose model derived from the behavior of the SBEII transgenic strains. Figure 6 is a graphical representation of data showing an amylose model derived from the behavior of the SBEII transgenic lineage. Figure 7 is a representation showing an alignment of the DNA sequences in the exon 12 to 14 region of the SBEIIa homologous genes obtained from the Chara wheat variety. The nucleotide sequence of the B Chara genome fragment is shown in its entirety, while the corresponding nucleotides for the homologous fragments of genome A and D are shown only where there are polymorphisms. Dots indicate that the corresponding nucleotides are identical to the B Chara genome fragment. Dashes indicate that the corresponding nucleotide is missing from the sequence. Figure 8 is a representation showing an alignment of the DNA sequences of the intron 3 region of the SBEIIa genes obtained from the wheat varieties Sunco and Tasman. The nucleotide sequence for the D Tasman genome fragment is shown in its entirety, while the corresponding nucleotides for the homologous fragments are shown only where there are polymorphisms. Dots indicate that the corresponding nucleotides are identical to the D Tasman genome fragment. Dashes indicate that the corresponding nucleotide is missing from the sequence. Figure 9 is a representation showing an alignment of the DNA sequences of the exon 3 region of the SBEIIa homologous genes obtained from the Chinese Spring wheat variety. The nucleotide sequence of the Chinese Spring D genome fragment is shown in its entirety, while the corresponding nucleotides for the homologous genome fragments A and B are shown only where there are polymorphisms. Dots indicate that the corresponding nucleotides are identical to the Chinese Spring D genome fragment. Figure 10 is a representation showing a DNA sequence from the exon 1 region of the SBEIIa gene of the Chinese Spring hexaploid wheat variety. Figure 11 is a representation showing a PCR amplification of the region encompassing exons 12 to 14 of the SBEIIa genes of the CS nulliomic-tetrasomic strains. The projected line BDD is null for genome A, ADD is null for genome B and AAB is null for genome D. Figure 12 is a photographic representation of a Western blot showing the expression of SBEIIa protein in the developing endosperm of lineage S28. The protein extracts from the endosperm were assayed by Western blot analysis as described in Example 1, using SBEIIa specific antibodies. The last strip on the right side shows the bands that appear from the wild type endosperm (variety NB1). The positions of the SBEIIa proteins encoded by genomes A, B and D are indicated. Figure 13 is a graph of SBEIIa interaction mobility ratio in the absence (m0) and presence (m) of β-limit dextrin in Native 1-D PAGE against the β-limit (S) dextrin concentration. The dissociation constant (kd) is derived from the equation m0 / m = 1 + [S] / Kd. Figure 14 shows the relationship of amylose content and enzyme resistant starch in clustered wheat starch samples derived from transgenic wheat strains described in Example 2. Figure 15 provides a scatter plot of biochemical and predicted reference values of NIRS for apparent amylose content in simple wheat seeds. Figure 16 is a graphical representation showing distribution of apparent amylose content in WM and WMC populations as determined by NIRS. Figure 17 (a) and (b) are graphical representations of data showing the effect of adding increasing amounts of wheat strains on water absorption (a) and mixing times in the Mixographer (b). Figure 18 (a) and (b) are graphical representations of data illustrating the effect of adding increasing amounts of wheat flour with high amylose content in resistant starch (a) and expected GI (b) (HI%) of breads small-scale. BRIEF DESCRIPTION OF THE TABLES
Table 1 provides starch branching enzyme genes characterized from cereals. Table 2 provides an amino acid subclassification. Table 3 provides exemplary amino acid substitutions. Table 4 provides genome specific primers for wheat SBEIIa people. Table 5 provides nucleotide sequences of the genome-specific primers for SBEIIa. Table 6 provides primers designed to amplify parts of the SBEIIa gene specifically from the wheat A genome. Table 7 provides primers designed to amplify parts of the SBEIIa gene specifically from the wheat B genome. Table 8 provides primers designed to amplify parts of the SBEIIa gene specifically from the wheat D genome. Table 9 provides genome-specific primers for the wheat SBEIIb gene. Table 10 provides nucleotide sequences of the genome-specific primers for SBEIIb. Table 11 provides total SBEII and SBEIIa and SBEIIb expression and amylose content of the RNAi lines of wheat as described in Example 4. Table 12 provides a list of microsatellite markers tested on the mutants as described in Example 5. Table 13 provides identified mutants from the HIB population and microsatellite mapping data as described in Example 5. Table 14 provides a description of SBEII double-mutants identified as described in Example 5. Table 15 provides a description of the crosses performed between single mutants and double-null as described in Example 5. Table 16 provides cataloging of the amylose content in the starch of the grain of triple-null mutants as described in Example 5. Table 17 provides fertility observations in F2 progeny plants. Table 18 provides SBEII allelic composition and amylose proportion data for identified double-nulls. Table 19 provides details of other crosses between single and double null mutants. Table 20 provides the observed frequency of grain genotypes normally germinating from an A2B2D2 cross. The numbers in parentheses indicate the expected frequency based on Mendelian segregation. Table 21 provides other crosses between single and double-null mutants. Table 22 provides putative double and triple-null mutants in the SBEIIa genes identified in an initial screening using dominant markers. Table 23 provides starch characterization of grain starch from transgenic wheat lines. Table 24 provides molecular weight distribution of starch fractions from transgenic wheat lines. Table 25 provides RVA parameters for hp5'-SBEIIa transgenic wheat starch. Table 26 provides DSC parameters for peak gelatinization of hp5'-SBEIIa transgenic wheat starch compared to the NB1 control. Table 27 provides RS content in rolled and flaked grain products. Table 28 provides resistant starch content in food products in the variation of the level of incorporation of wheat with high amylose content (HAW). Table 29 provides genome specific primers mentioned in Example 18. DETAILED DESCRIPTION
The present invention is based in part on the Observations made in the experiments described here that wheat plants completely without SBEIIa activity in the whole plant could not be recovered at the crossroads designed to produce them, in fact the complete lack of SBEIIa has been completed as being lethal to seed development and / or fertility. This was surprising since previous studies showed that simple null mutants in SBEIIa could be quickly obtained in wheat and be fertile. In addition, it was observed that the minimum level of SBEIIa activity that needed to be retained in the wheat plant to produce normal, viable seed, was around 2% of the wild type level.
It was also observed that mutant and grain plants comprising at least one point mutation in an SBEIIa gene were favored over plants and grain that had exclusions in each of the SBEIIa genes to combine the mutant SBEIIa genes, in particular, to obtain fertile male and female plants and phenotypically normal grain that germinated at rates similar to wild type grain. One possible explanation for this observation was that the exclusions tend to remove important elements adjacent to the SBEIIa genes.
It was also observed that in order to obtain an amylose content of at least 50% (w / w) in the starch of the grain, at which level the amount of resistant starch and associated healthy benefits were increased substantially, the total SBEII activity and particularly the activity of SBEIIa in the grain had to be reduced to below 30% of the wild type level.
In addition, it has been determined that, in hexaploid wheat, the reduction of the SBEII protein level and / or activity of each of the three SBEIIa homologous genes or of at least two SBEIIa homologous genes and two or three SBEIIb homologous genes leads to to a substantial non-linear increase in the proportion of amylose to starch in the wheat endosperm compared to plants that have zero mutation in two homologous SBEIIa genes. This non-linear relationship between amylose content and SBEII levels in a hexaploid wheat grain is illustrated graphically in Figures 5 and 6.
By studying the partial and complete loss of function mutations in combinations of SBEIIa and / or SBEIIb alleles of genomes A, B and D, the role of multiple SBEII genes in modulating starch characteristics has been established. Specifically, the number of mutant alleles and combinations of mutant alleles required to obtain fertile wheat plants having very high levels of amylose have been investigated and determined.
The synthesis of starch in the endosperm of taller plants including wheat is accomplished by a set of enzymes that catalyze four main steps. Firstly, ADP-glucose pyrophosphorylase (EC 2.7.7.27) activates the precursor monomer of starch through the synthesis of ADP-glucose from G-1-P and ATP. Second, the activated glycosyl donor, ADP-glucose, is transferred to the unreduced end of a pre-existing α (1-4) bond by starch synthases (EC 2.4.1.24). Third, starch branching enzymes introduce points by cleaving an α-linked glycan region (1-4) followed by transferring the cleaved chain to an acceptor chain, forming a new α (1-6) link. Starch branching enzymes are the only enzymes that can introduce α (1-6) bonds into α-polyglycans and therefore play an essential role in the formation of amylopectin. Fourth, starch de-branching enzymes (EC 2.4.4.18) remove some of the branch bonds.
Starch is the main reserve carbohydrate in plants such as cereals, including wheat. Starch is synthesized in amyloplasts and formed and stored in granules in the developing storage organ such as grain; it is referred to here as “storage starch” or “grain starch”. In cereal grains, most of the storage starch is deposited in the endosperm. "Starch" is defined here as a polysaccharide composed of units of polymerized glycopyranose through a combination of α (1-4) and α (1-6) bonds. Polydispersed starch molecules are classified as belonging to two component fractions, known as amylose and amylopectin, on the basis of their degree of polymerization (GP) and the ratio of α (1-6) to α (1-4) bonds. Grain starch from wild type cereal plants, including wheat, comprises about 20% to 30% amylose and about 70% to 80% amylopectin. “Amylose” is defined here as including essentially linear molecules of glycosidic units (glycophananosis) linked to α (1,4), sometimes referred to as “true amylose”, and amylose-like long-chain starch which is sometimes referred to as “Intermediate material” or “amylose-like amylopectin” that looks like iodine-binding material in an iodometric assay with true amylose (Takeda et al., 1993b; Fergason, 1994).
Typically, linear molecules in true amylose have a GP of between 500 and 5000 and contain less than 1% of α (1-6) bonds. Recent studies have shown that about 0.1% of α (1-6) -glycosidic branching sites can occur in amylose, so it is described as "essentially linear". On the other hand, amylopectin is a much larger molecule with a GP ranging from 5000 to 50,000 and contains 4 to 5% of α (1-6) bonds. Amylopectin molecules are therefore more highly branched. Amylose has a helical conformation with a molecular weight of about 104 to about 106 Daltons while amylopectin has a, molecular weight of about 107 to about 108 Daltons. These two types of starch can be easily distinguished or separated by methods well known in the art.
The proportion of amylose in starch as defined here is on a weight / weight (w / w) basis, that is, the weight of amylose as a percentage of the weight of the total extractable starch from the grain, with respect to the starch before any fractionation amylose and amylopectin fractions. The terms "amylose to starch ratio" and "amylose content" when used here in the context of grain, flour or other product of the invention are essentially interchangeable terms. The amylose content can be determined by any of the methods known in the art including size exclusion high performance liquid chromatography (HPLC), for example, in 90% (w / v) DMSO, concanavalin A methods (Megazyme Int , Ireland), or preferably by an iodometric method, for example, as described in Example 1. The HPLC method may involve de-branching the starch (Batey and Curtin, 1996) or may not involve de-branching. It will be appreciated that methods such as the HPLC method by Batey and Curtin, 1996 that test only "true amylose" can underestimate the amylose content as defined here. Methods such as HPLC or gel permeation chromatography depend on the fractionation of starch in the amylose and amylopectin fractions, while iodometric methods depend on differential iodine binding and therefore do not require fractionation.
From the grain weight and amylose content, the amount of amylose deposited per grain can be calculated and compared for test and control lines.
Starch is initially synthesized and accumulated in the leaves and other green tissues of a plant as a product of photosynthesis. This starch is referred to here as "transient starch" or similar because, in contrast to the seed or starch in the tuber, it accumulates in the plastids of photosynthetic tissues during the day and is degraded at least during the night. At night, transient starch is hydrolyzed to sugars that are transported, primarily as sucrose, from source tissues to sink tissues for use in plant growth, as an energy source for metabolism or to store in tissues as storage starch .
As used here, "starch synthase" means an enzyme that transfers ADP-glucose to the unreduced end of pre-existing α1-4 bonds. Four classes of starch synthase are found in the cereal endosperm, an isoform exclusively located within the starch granule, granule boundary starch synthase (GBSS), two forms that are divided between the granule and the soluble fraction (SSI, Li et al, 1999a; SSII, Li et al., 1999b) and a fourth form that is totally located in the soluble fraction, SSIII (Cao et al, 2000; Li et al, 1999b; Li et al, 2000). GBSS has been shown to be essential for amylose synthesis (Shure et al, 1983), and mutations in SSII and SSIII have been shown to alter the structure of amylopectin (Gao et al, 1998; Craig et al, 1998). Mutants in cereals that do not have GBSS also do not have true amylose and thus accumulate only amylopectin; these are commonly referred to as “wax” mutants. The mutations that define a role for SSI activity have not been described. The synthesis of amylopectin is more complex than the synthesis of amylose, requiring a combination of starch synthases other than GBSS, multiple starch branching enzymes and debranching enzyme.
As used here, "debranching enzyme" means an enzyme that removes some of the branches of the amylopectin formed by the starch branching enzymes. Two types of branching enzymes are present in higher plants and are defined based on their substrate specificities, isoamylase-type branching enzymes, and pullulanase-type branching enzymes (Myers et al, 2000). Sugary-1 mutations in maize and rice are associated with deficiency of de-branching enzymes (James et al, 1995; Kubo et al, 1999); isoamylase type.
Examples of genes encoding starch branching enzymes for cereals including wheat are given in Table 1. As used here, “starch branching enzyme” means an enzyme that introduces α-1,6 glycosidic bonds between glucose residue chains ( EC 2.4.1.18). Three forms of starch branching enzyme are expressed in cereals such as rice, maize, barley and wheat, including in the endosperm of the developing cereal, namely starch branching enzyme I (SBEI), starch branching enzyme IIa ( SBEIIa) and starch branching enzyme IIb (SBEIIb) (Hedman and Boyer, 1982; Boyer and Preiss, 1978; Mizuno et al, 1992, Sun et al, 1997). The genomic and cDNA sequences for genes encoding these enzymes were characterized for rice, barley and wheat (Table 1). Sequence alignment reveals a high degree of sequence similarity in nucleotide and amino acid levels, but also in sequence differences and allows for grouping into classes SBEI, SBEIIa and SBEIIb. SBEIIa and SBEIIb of any of the species generally exhibit about 80% identity of the amino acid sequence to each other, in the central regions of the genes. SBEIIa and SBEIIb can also be distinguished by their expression patterns, but this differs in different species. In maize, SBEIIb is more highly expressed in the endosperm while SBEIIa is present in each plant tissue. In barley, SBEIIa and SBEIIb are present in equal amounts in the endosperm, while in the wheat endosperm, SBEIIa is expressed about 4 times more highly than SBEIIb. Therefore, cereal species show significant differences in the expression of SBEIIa and SBEIIb, and conclusions drawn from one species cannot be easily applied to another species. In wheat, the proteins of SBEIIa and SBEIIb are different in size (see below) and this is a convenient way to distinguish them. Specific antibodies can also be used to distinguish them.
In maize, phenotypes with a high amylose content have been shown to result from lesions in the SBEIIb gene, also known as the amylose extender gene (ea) (Boyer and Preiss, 1981, Mizuno et al., 1993; Nishi et al, 2001) . In these SBEIIb mutants, the endosperm starch grains showed an abnormal morphology, the amylose content was significantly high, the frequency of the residual amylopectin branch was reduced and the proportion of short chains (<DP17, especially DP8-12) was lower . In addition, the gelatinization temperature of the starch has been increased. In addition, there was a significant set of material that was defined as an “intermediate” between amylose and amylopectin (Boyer et al, 1980; Takeda, et al, 1993b). On the other hand, the mutant corn plants in the SBEIIa gene due to a modifier insertion element (Mu) and consequently without the SBEIIa expression protein were indistinguishable from the wild type plants in the endosperm starch branch (Blauth et al., 2001), although they have been altered in the leaf starch.
Similarly, rice plants deficient in SBEIIa activity did not exhibit significant change in the profile of the amylopectin chain in the endosperm (Nakamura, 2002), while mutants in SBEIIb showed a modest increase in amylose levels, up to about 35% in the past. indicators and up to 25 to 30% in a history of Japanese (Mizuno et al, 1993; Nishi et al, 2001). In maize and rice, the SBEIIa and SBEIIb genes are not linked in the genome. In barley, a gene silencing construct that reduced the expression of SBEIIa and SBEIIb in the endosperm was used to generate barley grain with a high amylose content (Regina et al., 2010).
In the endosperm of developing wheat, SBEI (Morell et al., 1997) is found exclusively in the soluble fraction (amyloplast stroma), while SBEIIa and SBEIIb are found in the soluble fractions and associated with the starch granule in the endosperm (Rahman et al. , 1995). In wheat, apparent gene duplication events increased the number of SBEI genes in each genome (Rahman et al, 1999). The elimination of more than 97% of SBEI activity by combining mutations in the higher forms of expression of SBEI genes from genomes A, B and D had no measurable impact on starch structure or functionality (Regina et al, 2004). In contrast, the reduction in SBEIIa expression by a gene silencing construct in wheat resulted in high levels of amylose (> 70%), while a corresponding construct that reduced SBEIIb expression but not SBEIIa had minimal effect (Regina et al, 2006).
The enzymatic activity of starch branching (SBE) can be measured by the enzyme assay, for example, by the phosphorylase stimulation assay (Boyer and Preiss, 1978). This assay measures SBE stimulation of the incorporation of glucose 1-phosphate into methanol-insoluble polymer (α-D-glycan) by phosphorylase A. SBE activity can be measured by the iodine stain assay, which measures the decrease in absorbance of a glycan-poly-iodine complex resulting from the branching of glycan polymers. SBE activity can also be tested by the branch-binding assay that measures the generation of the reduced ends of the reduced amylose as a substrate, following the digestion of isoamylose (Takeda et al, 1993a). Preferably, activity is measured in the absence of SBEI activity. SBE isoforms show different substrate specificities, for example, SBEI exhibits higher activity on amylose branching, while SBEIIa and SBEIIb show higher branching rates with an amylopectin substrate. Isoforms can also be distinguished based on the length of the glycan chain that is transferred. SBE protein can also be measured by using specific antibodies such as those described here. SBEII activity can be measured during grain development in the developing endosperm. Alternatively, SBEII levels are measured in the mature grain where the protein is still present and can be tested by immunological methods.
In some embodiments, the level or activity of SBEII or SBEIIa can be assessed by assessing transcription levels such as by Northern or RT-PCR analysis. In a preferred method, the amount of SBEIIa protein in grain or developing endosperm is measured by separating the proteins in grain / endosperm extracts into gels by electrophoresis, then transferring the proteins to a membrane by Western blotting, followed by quantitative detection of the membrane protein using specific antibodies (“Western blot analysis”). This is exemplified in Example 11.
As shown here, the developing hexaploid wheat endosperm expresses SBEIIa and SBEIIb from each of genomes A, B and D. Tetraploid wheat expresses SBEIIa and SBEIIb from each of genomes A and B. As used here, “SBEIIa expressed from genome A ”or“ SBEIIA-A ”means a starch branching enzyme whose amino acid sequence is shown in SEQ ID NO: 1 or which is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or comprising such sequence. The amino acid sequence of SEQ ID NO: 1 (Genbank Accession Number CAA72154) corresponds to an expressed SBEIIa of the wheat genome A, which is used here as a reference sequence for wild type SBEIIa-A. The protein of SEQ ID NO: 1 is 823 amino acids in length. The active variants of this enzyme exist in wheat, for example, in Cheyenne cultivars, see Accession number AF286319 which is 99.88% (822/823) identical to SEQ ID NO.1. Such variants are included in "SBEIIa-A" as long as they have essentially wild-type starch branching enzyme activity as for SEQ ID NO: 1.
As used here, “BSEIA expressed from genome B” or “BEIIA-B” means a starch branching enzyme whose amino acid sequence is shown in SEQ ID NO: 2 or which is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 2 or comprising such a sequence. The amino acid sequence of SEQ ID NO: 2 (Genbank Accession Number CAR95900) corresponds to the expressed SBEIIa of the Chinese Spring wheat variety B genome, which is used here as the reference sequence for wild type SBEIIa-B. The protein of SEQ ID NO: 2 is 823 amino acids in length. Active variants of this enzyme may exist in wheat and are included in SBEIIa-B as long as they have essentially wild-type starch branching enzyme activity as for SEQ ID NO: 2. SEQ ID NO: 2 is 98.42% ( 811/824) identical to SEQ ID NO: 1. The alignment of the amino acid sequences in figure 1 shows the amino acid differences that can be used to distinguish proteins or to classify variants as SBEIIa-A or SBEIIa-B.
As used here, “SBEIIa expressed in the D genome” or “SBEIIA-D” means a starch branching enzyme whose amino acid sequence is shown in SEQ ID NO: 3 or which is at least 98% identical to the amino acid sequence shown in SEQ ID NO: 3 or comprising such a sequence. The amino acid sequence of SEQ ID NO: 3 (Genbank Accession Number AAK26821) corresponds to the expressed SBEIIa of the D genome in A. tauschii, a probable progenitor of the hexaploid wheat D genome, which is used as the reference sequence for SBEIIa -D wild type. The protein of SEQ ID NO: 3 is 819 amino acids long. Active variants of this enzyme can exist in wheat and are included in SBEIIa-D as long as they have essentially wild-type starch branching enzyme activity as for SEQ ID NO: 3. SEQ ID NO: 3 is 97.57% (803 / 823) identical to SEQ ID NO: 1 and 97.81% (805/823) identical to SEQ ID NO: 2. The alignment of the amino acid sequences in figure 1 shows amino acid differences that can be used to distinguish proteins or to classify the variants as SBEIIa- A, SBEIIa-B or SBEIIa-D.
When comparing the amino acid sequences to determine the percentage identity in this context, for example, by Blastn, the full length sequences should be compared, and the spaces in a sequence counted as amino acid differences.
As used herein, a "SBEIIa protein" includes protein variants that have reduced or been left without starch branching enzymatic activity, as well as proteins having essentially wild-type enzymatic activity. It is also understood that SBEIIa proteins may be present in the grain, particularly dormant grain as commonly harvested commercially, but in an inactive state because of the physiological conditions in the grain. Such proteins are included in the "SBEIIa proteins" as used here. SBEIIa proteins can be enzymatically active during only part of the grain's development, particularly in the developing endosperm when the storage starch is typically deposited, but in an inactive state otherwise. Such a SBEIIa protein can be easily detected and quantified using immunological methods such as Western blot analysis. An "SBEIIb protein" as used here has an analogous meaning.
As used herein, "SBEIIb expressed from genome A" or "SBEIIb-A" means a starch branching enzyme comprising the amino acid sequence shown in SEQ ID NO: 4 or that is at least 98% identical to the amino acid sequence shown in SEQ ID NO: 4 or comprising such a sequence. The amino acid sequence of SEQ ID NO: 4 corresponds to the SBEIIb amino terminal sequence expressed from the wheat genome A, which is used as the reference sequence for wild type SBEIIb-A.
As used herein, “SBEIIb expressed from genome B” or “SBEIIb-B” means a starch branching enzyme comprising the amino acid sequence shown in SEQ ID NO: 5 or that is at least 98% identical to the amino acid sequence shown in SEQ ID NO: 5 or comprising such a sequence. The amino acid sequence of SEQ ID NO: 5, which is used here as the reference sequence for wild type SBEIIb-B, is a partial amino acid sequence encoded by exons 2 to 3 of the SBEIIb-B gene in wheat. A variant sequence of SBEIIb-B is the amino acid sequence encoded by the nucleotide sequence of Accession Number AK335378 isolated from cv. Chinese Spring.
As used here, “SBEIIb expressed in the D genome” or “SBEIIb-D” means a starch branching enzyme whose amino acid sequence is shown in SEQ ID NO: 6 or which is at least 98% identical to the amino acid sequence shown in SEQ ID NO: 6 or comprising such a sequence. The amino acid sequence of SEQ ID NO: 6 (Genbank Accession Number AAW80631) corresponds to the SBEIIb expressed in the D genome of A. tauschii, a probable progenitor of the D genome of hexaploid wheat, and is used here as a reference sequence for SBEIIa -D wild type. Active variants of this enzyme exist in wheat and are included in SBEIIb-D as long as they have essentially wild-type starch branching enzyme activity as for SEQ ID NO: 6. For example, SEQ ID NO: 4 of the publication of the application for US Patent No. 20050074891, starting with the first methionine, shows the amino acid sequence of a SBEIIb-D protein that is 99.5% identical to SEQ ID NO: 6 in that patent application. The alignment of the amino acid sequences in figure 2 shows the differences in the amino acid that can be used to distinguish proteins from SBEIIb or to classify variants as SBEIIb-A, SBEIIb-B or SBEIIb-D.
Thus, "wild type" as used here when referring to SBEIIa-A means a starch branching enzyme whose amino acid sequence is shown in SEQ ID NO: 1; “Wild type” as used here when referring to SBEIIa-B means a starch branching enzyme whose amino acid sequence is shown in SEQ ID NO: 2; "Wild type" as used here when referring to SBEIIa-D means a starch branching enzyme whose amino acid sequence is shown in SEQ ID NO: 3; "Wild type" as used here when referring to SBEIIb-A means a starch branching enzyme whose amino acid sequence is shown in SEQ ID NO: 4; "Wild type" as used here when referring to SBEIIb-B means a starch branching enzyme whose amino acid sequence is shown in SEQ ID NO: 5; and, "wild type" as used here when referring to SBEIIb-D means a starch branching enzyme whose amino acid sequence is shown in SEQ ID NO: 6.
As used here, the terms "wheat SBEIIa gene" and "wheat SBEIIb gene" refer to the genes encoding functional SBEIIa or SBEIIb enzymes, respectively, in wheat, including homologous genes present in other wheat varieties , and also mutant forms of the genes that encode enzymes with reduced activity or undetectable activity. These include, but are not limited to, the wheat SBEII genes that have been cloned, including the genomic and cDNA sequences listed in Table 1. The genes as used here encompass mutant forms that do not encode any proteins, in which case the mutant forms represent null alleles of the genes.
An "endogenous SBEII gene" refers to an SBEII gene that is at its site of origin in the wheat genome, including wild-type and mutant forms. On the other hand, the terms "isolated SBEII gene" and "exogenous SBEII gene" refer to a SBEII gene that is not in its place of origin, for example, having been cloned, synthesized, comprised in a vector or in the form of a transgene in a cell, preferably as a transgene in a transgenic wheat plant. The SBEII gene, in this context, can be any of the specific forms as described below.
As used herein, “the SBEIIa gene in the wheat genome A” or “SBEIIa-A gene” means any polynucleotide that encodes SBEIIa-A as defined here or that is derived from a polynucleotide that encodes SBEIIa-A, including polynucleotides from naturally occurring, sequence variants or synthetic polynucleotides, including “SBEIIa-A wild-type gene (s)” encoding an SBEIIa-A with essentially wild-type activity, and mutant SBEIIa gene (s) -A ”that does not (m) encode an SBEIIa-A with essentially wild-type activity but is known to be derived from a wild-type SBEIIa-A gene. Comparison of the nucleotide sequence of a mutant form of an SBEII gene with a set of wild-type SBEII genes is used to determine which of the SBEII genes is derived in order to classify it. For example, a mutant SBEIIa A gene is considered a mutant SBEIIa-A gene if its nucleotide sequence is more closely related, that is, having a higher degree of sequence identity, for a wild-type SBEIIa-A gene than than for any other SBEII gene. A SBEIIa-A mutant gene encodes an SBE with reduced starch branching enzyme activity (partial mutant), or a protein that has no SBE activity or no protein (null mutant gene). An exemplary nucleotide sequence of a corresponding cDNA for an SBEIIa-A gene is given in Genbank Accession Number Y11282. The sequences of parts of the SBEIIa-A genes are also given herein as figures 7, 8, 9 and 10 and SEQ ID NOs 13, 14 and 15.
As used here, the terms "SBEIIa expressed from genome B" or "SBEIIa-B", "SBEIIa expressed from genome D" or "SBEIIa-D", "SBEIIb expressed from genome A" or "SBEIIb-A", "SBEIIb expressed from genome B "or" SBEIIb-B "and" expressed SBEIIb from genome D "or" SBEIIb-D "have corresponding meanings from those for SBEIIa-A in the previous paragraph.
The illustrative partial protein sequences of SBEIIb-A, SBEIIb-B and SBEIIb-D are provided in figure 2. The illustrative amino acid sequences of SBEIIb-A are shown in SEQ ID NO: 1 and SEQ ID NO: 4 (amino sequence terminal coded by exon 1 to 3). The illustrative amino acid sequences of SBEIIb-B are shown in SEQ ID NO: 2 and SEQ ID NO: 5. The illustrative amino acid sequences of SBEIIb-D are shown in SEQ ID NO: 3 and SEQ ID NO: 6 and SEQ ID NO: 9.
The SBEII genes as defined above include any regulatory sequences that are 5 'or 3' from the transcribed region, including the promoter region, which regulates the expression of the associated transcribed region, and introns within the transcribed regions.
It would be understood that there is natural variation in the sequences of the SBEIIa and SBEIIb genes of the different varieties of wheat. Homologous genes are easily recognizable to the person skilled in the art based on the identity of the sequence. The degree of sequence identity between homologous SBEIIa genes or proteins is considered to be at least 90%, similarly for SBEIIb genes or proteins. The SBEIIa wheat genes are about 80% identical in sequence to SBEIIb wheat genes. The encoded proteins are also about 80% identical in sequence.
An allele is a variant of a gene at a simple genetic site. A diploid organism has two sets of chromosomes. Each chromosome has a copy of each gene (an allele). If both alleles are the same, the organism is homozygous with respect to that gene, if the alleles are different, the organism is heterozygous with respect to that gene. The interaction between alleles at one location is generally described as either dominant or recessive. A loss of function mutation is a mutation in an allele leading to none or a reduced detectable level or enzymatic activity of SBEII, SBEIIa or SBEIIb in the grain. The mutation may mean, for example, that no or little RNA is transcribed from the one comprising the mutation or that the protein produced has none or reduced activity. Alleles that do not code or are not able to lead to the production of any active enzyme are null alleles. A loss of function mutation, which includes a partial loss of function mutation in an allele, means a mutation in the allele leading to a reduced level or enzymatic activity of SBEII, SBEIIa or SBEIIb in the grain. Mutation in the allele may mean, for example, that less protein having reduced or wild-type activity is translated or that reduced or wild-type transcription levels are followed by the translation of an enzyme with reduced enzyme activity. A "reduced" amount or level of protein means reduced in relation to the amount or level produced by the corresponding wild type allele. A "reduced" activity means reduced in relation to the corresponding SBEII, SBEIIa or wild type SBEIIb enzyme. The different alleles in the germ can have the same or a different mutation, and the different alleles can be combined using methods known in the art. In some embodiments, the amount of SBEIIa protein or SBEIIb protein is reduced because there is less transcription or translation of the SBEIIa gene or SBEIIb gene, respectively. In some embodiments, the amount by weight of the SBEIIa protein or SBEIIb protein is reduced even if there is a wild-type number of SBEIIa protein molecules or SBEIIb protein molecules in the grain, because some of the proteins produced are shorter than the SBEIIa protein or wild-type SBEIIb protein, for example, the SBEIIa protein or mutant SBEIIb protein is truncated due to a premature translation termination signal.
The representative starch biosynthesis genes that have been cloned from the cereals are listed in Table 1.
As used here, "two identical alleles of a SBEIIa-A gene", means that the two alleles of the SBEIIa-A gene are identical to each other; “Two identical alleles of a SBEIIa-B gene”, means that the two alleles of the SBEIIa-B gene are identical to each other; “Two identical alleles of a SBEIIa-D gene”, means that the two alleles of the SBEIIa-D gene are identical to each other; “Two identical alleles of a SBEIIb-A gene”, means that the two alleles of the SBEIIb-A gene are identical to each other; “Two identical alleles of a SBEIIb-B gene”, means that the two alleles of the SBEIIb-B gene are identical to each other; and, "two identical alleles of a SBEIIb-D gene", means that the two alleles of the SBEIIb-D gene are identical to each other.
The wheat plants of the invention can be produced and identified after mutagenesis. This can provide a wheat plant that is non-transgenic, that is desirable in some markers, or that is free of any exogenous nucleic acid molecule that reduces the expression of a SBEIIa gene. Mutant wheat plants having a mutation in a simple SBEII gene that can be combined by crossing and selecting with other SBEII mutations to generate the wheat plants of the invention can be synthetic, for example, by carrying out site-directed mutagenesis in the nucleic acid or induced by mutagenic treatment, or may be naturally occurring, that is, isolated from a natural source. Generally, a parent plant cell, tissue, seed or plant can undergo mutagenesis to produce single or multiple mutations, such as nucleotide substitutions, exclusions, additions and / or codon modification. The preferred wheat and grain plants of the invention comprise at least one introduced SBEII mutation, more preferably two or more introduced SBEII mutations, and may not comprise any mutation from a natural source, i.e., all of the mutant SBEIIa and SBEIIb alleles in the plant were obtained by synthetic means or by mutagenic treatment.
Mutagenesis can be achieved by chemical or radiation, for example, EMS or sodium azide (Zwar and Chandler, 1995) seed treatment, or gamma irradiation, well known in the art. Chemical mutagenesis tends to favor nucleotide substitutions other than exclusions. Heavy ion beam (HIB) irradiation is known to be an effective technique for creating mutations to produce new plant cultivars, see, for example, Hayashi et al, 2007 and Kazama et al, 2008. Ion beam irradiation has two physical factors, the dose (gy) and LET (linear energy transfer, keV / um) for biological effects that determine the amount of DNA damage and the size of DNA exclusion, and this can be adjusted according to desired extent of mutagenesis. The HIB generates a collection of mutants, many of them comprising exclusions that can be examined for mutations in specific SBEII genes as shown in the Examples. The mutants that are identified can be backcrossed with non-mutated wheat plants as recurrent pairs in order to remove and therefore reduce the effect of unbound mutations in the mutagenized genome, see Example 9.
Biological agents useful in the production of site-specific mutants include enzymes that include double breaks of DNA strands that stimulate endogenous repair mechanisms. These include endonucleases, zinc finger nucleases, transposases and site-specific recombinases. Zinc finger nucleases (ZFNs), for example, facilitate site-specific cleavage within a genome by allowing endogenous or other end-joint repair mechanisms to introduce exclusions or insertions to repair the space. The zinc finger nuclease technology is reviewed in Le Provost et al, 2009, see also Durai et al, 2005 and Liu et al, 2010.
Isolation of mutants can be achieved by screening mutagenized plants or seeds. For example, a mutagenized wheat population can be examined directly for the SBEIIa and / or SBEIIb genotype or indirectly by screening for a phenotype that results from mutations in the SBEII genes. Screening directly for the genotype preferably includes assays for the presence of mutations in the SBEII genes, which can be observed in the PCR assays by the absence of specific SBEIIa or SBEIIb markers as expected when some of the genes are excluded, or heteroduplex based assays such as in Tilling. Screening for the phenotype may comprise screening for a loss or reduction in the amount of one or more proteins of SBEIIa or SBEIIb by ELISA or affinity chromatography, or high amylose content in the starch of the grain. In hexaploid wheat, screening is preferably done on a genotype that is already without one or two of the SBEII activities, for example, on a wheat plant already mutated in the SBEIIa or SBEIIb genes in two of the three genomes, so that a mutant still without functional activity it is sought. In tetraploid wheat, screening is preferably done on a genotype that is already without SBEII activity, in genome A or B, and identification of a mutant that is reduced in SBEII from the second genome. Affinity chromatography can be performed as shown in Example 11. Large populations of mutagenized seeds (thousands or tens of thousands of seeds) can be examined for phenotypes with a high amylose content using near infrared (NIR) spectroscopy as shown in Example 10 Using the NIR, an enriched subpopulation for candidates with a high amylose content was obtainable. By these means, high-throughput screening is easily achievable and allows for the isolation of mutants at a frequency of approximately one per several hundred seeds.
The plants and seeds of the invention can be produced using the process known as TILLING (Targeting Locally Induced Lesions In Genomes), in which one or more of the mutations in the wheat or grain plants can be produced by this method. In a first step, introduced mutations such as new simple base pair changes are induced in a plant population by treating seeds or pollen with a chemical or radiation mutagen, and then advancing plants to a generation where the mutations will be stably inherited, typically an M2 generation where homozygotes can be identified. DNA is extracted, and seeds are stored from all members of the population to create a resource that can be accessed repeatedly over time. For a TILLING assay, PCR primers are designed to specifically amplify a single gene target of interest. Then, the dye-labeled primers can be used to amplify the PCR products of the pooled DNA of multiple individuals. These PCR products are denatured and annealed to allow the formation of incompatible base pairs. Incompatibilities, or heteroduplexes, represent naturally occurring simple nucleotide polymorphisms (SNPs) (that is, several plants in the population are likely to carry the same polymorphism) and induced SNPs (that is, only individual rare plants are likely to exhibit the mutation) . After heteroduplex formation, the use of an endonuclease, such as Cel I, that recognizes and cleaves incompatible DNA is the key to discovering new SNPs within a TILLING population.
Using this approach, thousands of plants can be examined to identify any individual with a simple base change as well as small insertions or exclusions (1-30 bp) in any specific gene or region of the genome. The genomic fragments that are tested can vary in size anywhere from 0.3 to 1.6 kb. In an 8-fold cluster and amplification of 1.4 kb fragments with 96 tracks per assay, this combination allows up to one million base pairs of genomic DNA to be examined by simple assay, making TILLING a high-yielding technique. TILLING is further described in Slade and Knauf, 2005, and Henikoff et al, 2004.
In addition to allowing efficient detection of mutations, high-performance Tilling technology is ideal for detecting natural polymorphisms. Therefore, interrogating an unknown homologous DNA by heteroduplexing to a known sequence reveals the number and position of polymorphic sites. Nucleotide changes and small insertions and deletions are identified, including at least some repeated number polymorphisms. This was called Ecotilling (Comai et al, 2004). Plates containing the tested ecotypic DNA can be examined instead of DNA pools of mutagenized plants. Because detection is in gels with close base pair resolution and background patterns are uniform across bands, bands that are of identical size can be combined, thus discovering and genotyping mutations in one simple step. Thus, the sequential mutant gene is simple and efficient.
The identified mutations can be introduced into desirable genetic origins by crossing the mutant with a plant of the desired genetic origin and performing an adequate number of backcrosses to cross the background of originally unwanted origin.
In the context of this patent application, an "induced mutation" or "introduced mutation" is an artificially induced genetic variation that may be the result of chemical, radiation or biological based mutagenesis, for example, transposon or T-DNA insertion. Preferred mutations are null mutations such as absurd mutations, framing mutations, exclusions, insertion mutations or snapshot site variants that completely inactivate the gene. Other preferred mutations are partial mutations that retain some SBEII activity, but less than the wild-type levels of the enzyme. Derivatives of nucleotide insertion include 5 'and 3' terminal fusions as well as single or multiple nucleotide intra-sequence insertions. Insertion nucleotide sequence variants are those in which one or more nucleotides are introduced at a location in the nucleotide sequence, at a predetermined location as is possible with zinc finger nucleases (ZFN) or other homologous recombination methods, or by random insertion with appropriate screening of the resulting product. Exclusion variants are characterized by the removal of one or more nucleotides from the sequence. Preferably, a mutant gene has only a simple insertion or exclusion of a nucleotide sequence in relation to the wild type gene. The exclusion can be extensive enough to include one or more exons or introns, both exons and introns, an intron-exon boundary, a part of the promoter, the site of initiation of translation, or even the entire gene. The exclusions may extend far enough to include at least part of, or the total, of the SBEIIa and SBEIIb genes in the A, B, or D genome, based on the close genetic linkage of the two genes. The insertions or exclusions within the exons of the protein coding region of a gene that inserts or excludes a number of nucleotides that is not an exact multiple of three, thus causing a change in the reading frame during translation, almost always revokes the activity of the mutant gene comprising such insertion or exclusion.
Substitution nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place. The preferred number of nucleotides affected by substitutions in a mutant gene over the wild-type gene is a maximum of ten nucleotides, more preferably a maximum of 9, 8, 7, 6, 5, 4, 3, or 2, or more preferably just a nucleotide. Substitutions can be "silent" in that the substitution does not change the amino acid defined by the codon. Nucleotide substitutions can reduce the efficiency of translation and thus reduce the level of SBEII expression, for example, by reducing the stability of the mRNA or, if close to an exon-intron fitting limit, altering the efficiency of fitting. Silent substitutions that do not alter the translation efficiency of an SBEIIa or SBEIIb gene are not expected to alter the activity of the genes and are therefore considered here as non-mutants, that is, such genes are active variants and are not included in the “ mutant alleles ”. Alternatively, the nucleotide substitution (s) can (s) change the encoded amino acid sequence and thus alter the encoded enzyme activity, particularly if the conserved amino acids are substituted for another amino acid that is quite different, ie , a non-conservative replacement. Typical conservative substitutions are those made according to Table 3.
The term "mutation" as used here does not include silent nucleotide substitutions that do not affect the activity of the gene, and therefore includes only changes in the gene sequence that affect the activity of the gene. The term "polymorphism" refers to any change in the nucleotide sequence including such silent nucleotide substitutions. Screening methods can first involve screening for polymorphisms and secondly for mutations within a group of polymorphic variants.
As is understood in the art, hexaploid wheat such as common wheat comprises three genomes that are commonly designed genomes A, B and D, while tetraploid wheat, such as durum wheat, comprises two genomes commonly designed genomes A and B. Each genome comprises 7 pairs of chromosomes that can be observed by cytological methods during meiosis and thus identified, as is well known in the art.
The terms "plant (s)" and "wheat plant (s)" as used here as a noun generally refer to whole plants, but when "plant" or "wheat" is used as an adjective, the terms refer to to any substance that is present in it, obtained from, derived from or related to a wheat plant or plant, such as, for example, plant organs (for example leaves, stems, roots, flowers), single cells (for example, pollen), seeds, plant cells including, for example, tissue culture cells, products produced from the plant such as "wheat flour", "wheat grain", "wheat starch", "wheat starch granules" and similar. Seedlings and germinated seeds from which the roots and shoots emerged are also included within the "plant". The term "plant parts" as used herein refers to one or more plant tissues or organs that are obtained from an entire plant, preferably a wheat plant. Plant parts include plant structures (e.g., leaves, stems), roots, floral organs / structures, seed (including germ, endosperm, and seed coat), plant tissue (for example, vascular tissue, soil tissue, and similar), cells and progeny thereof. The term “plant cell” as used here refers to a cell obtained from a plant or plant, preferably a wheat plant, and includes protoplasts or other plant-derived cells, gamete-producing cells, and cells that regenerate in whole plants. Plant cells can be cultured cells. “Plant tissue” means tissue differentiated in a plant or obtained from a plant (“explant”) or non-differentiated tissue derived from mature and immature germs, seeds, roots, sprouts, fruits, pollen, and various forms of aggregation of plant cells in the culture, such as corns. The plant tissues in or on seeds such as wheat seeds are seed coat, endosperm, scutellum, aleurone layer and germ.
The cereals as used here means plants or grain from the Poaceae or Graminae monocot families that are grown for the edible components of their seeds, and includes wheat, barley, corn, oats, rye, rice, sorghum, triticale, millet, buckwheat. Preferably, the cereal plant or grain is a wheat or barley plant or grain, more preferably wheat plant or grain. In another preferred embodiment, the cereal plant is not rice or corn or both.
As used here, the term “wheat” refers to any species of the genus Triticum, including its parents, as well as its progeny produced by crosses with another species. Wheat includes "hexaploid wheat" which has AABBDD genome organization, comprised of 42 chromosomes, and "tetraploid wheat" which has AABB genome organization, comprised of 28 chromosomes. Hexaploid wheat includes T. aestivum, T. spelta, T. macha, T. compactum, T. sphaerococcum, T. vavilovii, and interspecies crossing. Tetraploid wheat includes T. durum (also referred to as durum wheat or Triticum turgidum ssp. Durum), T. dicoccoides, T. dicoccum, T. polonicum, and interspecies crossing. In addition, the term "wheat" includes possible parents of hexaploid or Triticum sp. tetraploid such as T. uartu, T. monococcum or T. boeoticum for genome A, Aegilops speltoides for genome B, and T. tauschii (also known as Aegilops squarrosa or Aegilops tauschii) for genome D. A wheat cultivar for use in the present invention may belong to, among others, any species listed above. Also included are plants that are produced by conventional techniques using Triticum sp. as a precursor in a sexual cross with a non-Triticum species, such as Secale rye cereale, including, among others, Triticale. Preferably, the wheat plant is suitable for commercial grain production, such as commercial varieties of hexaploid wheat or durum wheat, having suitable agronomic characteristics that are known to those skilled in the art. Most preferably, the wheat is Triticum aestivum ssp. aestivum or Triticum turgidum ssp. durum, and more preferably, the wheat is Triticum aestivum ssp. aestivum, here also referred to as “common wheat”.
As used here, the term "barley" refers to any species of the Hordeum genus, including its parents, as well as its progeny produced by crosses with another species. It is preferred that the plant be of a Hordeum species that is commercially cultivated such as, for example, a strain or cultivar or variety of Hordeum vulgare suitable for commercial production of the grain.
The wheat plants of the invention can have many different uses than those for food or animal feed, for example, research or breeding uses. In seed-propagated crops, such as wheat, plants can be self-crossed to produce a plant that is homozygous for the desired genes, or haploid tissues, such as developing germ cells, can be induced to duplicate the chromosome complement to produce a homozygous plant. The innate wheat plant of the invention thus produces seed containing the combination of mutant SBEII alleles that can be homozygous. These seeds can be grown to produce plants that would have the selected phenotype such as, for example, high amylose content in their starch.
The wheat plants of the invention can be crossed with plants containing a more desirable genetic background, and therefore the invention includes the transfer of the low SBEII trait to other genetic funds. After the initial crossover, an adequate number of backcrosses can be performed to remove a less desirable background. SBEII allele-specific PCR-based markers such as those described here can be used to screen or identify plants or progeny grains with the desired combination of alleles, thereby tracking the presence of alleles in the breeding program. The desired genetic background may include an appropriate combination of genes providing commercial yield and other characteristics such as agronomic performance or resistance to abiotic stress. The genetic background can also include other altered starch biosynthesis or modification genes, for example, genes from other wheat strains. The genetic background may comprise one or more transgenes, such as, for example, a gene that confers tolerance to an herbicide such as glyphosate.
The desired genetic background of the wheat plant will include considerations of agronomic yield and other characteristics. Such characteristics may include, if it is desired to have a winter or spring type, agronomic performance, resistance to disease and resistance to abiotic stress. For Australian use, one may want to cross the modified starch trace of the invented wheat plant in wheat cultivars such as Baxter, Kennedy, Janz, Frame, Rosella, Cadoux, Diamondbird or other commonly grown varieties. Other varieties will be suitable for other growing regions. It is preferred that the wheat plant of the invention provides a grain yield of at least 80% in relation to the yield of the corresponding wild type variety in at least some growing regions, more preferably at least 85% or at least 90%, and still more preferably at least 95% with respect to a wild type variety having about the same genetic background, grown under the same conditions. More preferably, the grain yield of the wheat plant of the invention is at least as great as the yield of the wild type wheat plant having about the same genetic background, grown under the same conditions. Yield can be easily measured in controlled field tests, or in simulated field tests in the greenhouse, preferably in the field.
Marker-assisted selection is a well-recognized method of selecting heterozygous plants obtained by backcrossing with the recurring precursor in a classical breeding program. The plant population in each backcross generation will be heterozygous for the gene (s) of interest normally present in a 1: 1 ratio in a backcross population, and the molecular marker can be used to distinguish the two alleles of the gene . To extract DNA from, for example, new shoots and test with a specific marker for the desirable introgression trait, initial selection of plants for another backcross is done while energy and resources are concentrated on fewer plants.
Procedures such as crossing wheat plants, self-fertilizing wheat plants or marker-assisted selection are standard procedures and are well known in the art. Transferring the alleles of tetraploid wheat such as durum wheat to a hexaploid, or other forms of hybridization, is more difficult, but is also known in the art.
To identify the desired phenotypic characteristic, wheat plants that contain a combination of mutant SBEIIa and SBEIIb alleles or other desired genes are typically compared to control plants. When evaluating a phenotypic characteristic associated with enzymatic activity such as amylose content in the starch in the grain, the plants to be tested and control plants are grown in a culture chamber, greenhouse, upper opening chamber and / or field conditions. Identification of a particular phenotypic trait and comparison for controls are based on routine statistical analysis and scoring. The statistical differences between plant strains can be assessed by comparing - enzymatic activity between plant strains within each type of tissue that expresses the enzyme. Expression and activity are compared to cultivation, development and yield parameters that include plant part morphology, color, number, size, dimensions, dry and wet weight, ripening, above and below ground biomass proportions, and time, rate and duration of various stages of growth through senescence, including vegetative growth, fruiting, flowering, and soluble carbohydrate content including sucrose, glucose, fructose and starch levels as well as levels of endogenous starch. Preferably, the wheat plants of the invention differ from wild type plants in one or more of these parameters by less than 50%, more preferably less than 40%, less than 30%, less than 20%, less than 15%, less than 10 %, less than 5%, less than 2% or less than 1% when grown under the same conditions.
As used here, the term "linked" refers to a placeholder and a second place being close enough to a chromosome where they will be inherited together in more than 50% of meiosis, for example, not randomly. This definition includes the situation where the placeholder and second place are part of the same gene. In addition, this definition includes the situation where the placeholder comprises a polymorphism that is responsible for the trait of interest (in other words, PE placeholder directly "linked" to the phenotype). The term “genetically linked” as used here is narrower, only used in relation to where a placeholder and a second place being close enough to a chromosome in which they will be inherited together in more than 50% of meiosis. Thus, the percentage of recombination observed between places per generation (centimorgans (cM)), will be less than 50. In particular embodiments of the invention, genetically linked places can be 45, 35, 25, 15, 10, 5, 4, 3 , 2, or 1 or less cM in addition to a chromosome. Preferably, the markers are less than 5 cM or 2 cM in addition to and more preferably about 0 cM separated. As described in Example 5 here, the SBEIIa and SBEIIb genes are genetically linked on a long arm on chromosome 2 of each of the wheat genomes, with about 0.5 cM separated, which corresponds to about 100 to 200 kb in the physical distance.
As used herein, the "other genetic markers" can be any molecules that are linked to a desired trait in the wheat plants of the invention. Such markers are well known in the art and include molecular markers linked to gene determining traits such as disease resistance, yield, plant morphology, grain quality, other dormancy traits such as grain color, gibberellic acid content in the seed, plant height, color of flour and similar. Examples of such genes are Sr2 or Sr38 genes with stem rust resistance, the Yr10 or Yr17 genes with rust resistance of the band, the genes with nematode resistance such as Cre1 and Cre3, alleles in the glutenin sites that determine the strength of the mass such as Ax, Bx, Dx, Ay, By and Dy alleles, the Rht genes that determine a semiannual cultivation habit and, therefore, deposit resistance (Eagles et al, 2001; Langridge et al., 2001; Sharp et al , 2001).
Wheat plants, wheat plant parts and products thereof are preferably non-transgenic for genes that inhibit SBEIIa expression, that is, they do not comprise a transgene encoding an RNA molecule that reduces the expression of endogenous genes from SBEIIa, although in this realization they may compromise other transgenes, for example, herbicide tolerance genes. More preferably, the wheat plant, the grain and the products thereof are non-transgenic, that is, they do not contain any transgene, which is preferred in some markets. Such non-transgenic plants and grain comprise the multiple mutant alleles of SBEII as described here, such as those produced after mutagenesis.
The terms "transgenic plant" and "transgenic wheat plant" as used here refer to a plant that contains a gene construct ("transgene") not found in a wild type plant of the same species, variety or cultivar. That is, transgenic plants (transformed plants) contain genetic material that they do not contain before transformation. A "transgene" as referred to herein has normal significance in the biotechnology technique and refers to a genetic sequence that has been produced or altered by recombinant DNA or RNA technology and that has been introduced into the plant cell. The transgene can include genetic sequences obtained or derived from a plant cell, or another plant cell, or a non-plant source or synthetic sequence. Typically, the transgene has been introduced into the plant by human manipulation, such as, for example, by transformation, but any method can be used as recognized by a person skilled in the art. The genetic material is typically stable and integrated into the plant's genome. The genetic material introduced may comprise sequences that occur naturally in the same species, but in a reorganized order or in a different arrangement of the elements, for example, an antisense sequence. Plants containing such sequences are included here in "transgenic plants". Transgenic plants as defined here include the entire progeny of an early transformed and regenerated plant (TO plant) that has been genetically modified using recombinant techniques, where the progeny comprise the transgene. Such progeny can be obtained by self-fertilization of the primary transgenic plant or by crossing such plants with another plant of the same species. In one embodiment, transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny do not secrete the desired phenotype. The parts of the transgenic plant include all parts and cells of said plants that comprise the transgene such as, for example, seeds, cultured tissues, callus and protoplasts. A "non-transgenic plant", preferably a non-transgenic wheat plant, is one that is not genetically modified by the introduction of genetic material by recombinant DNA techniques.
As used here, the term "corresponding non-transgenic plant" refers to a plant that is the same or similar in most characteristics, preferably isogenic or almost isogenic in relation to the transgenic plant, but without the transgene of interest. Preferably, the corresponding non-transgenic plant is of the same cultivar or variety as the parent of the transgenic plant of interest, or a lineage of sister plant that does not have the construct, often called a “segregant”, or a plant of the same cultivar or transformed variety with an “empty vector” construct, and can be a non-GM plant. "Wild type", as used herein, refers to a cell, tissue or plant that has not been modified according to the invention. Wild-type cells, tissue or plants, known in the art and can be used as controls to compare the levels of expression of an exogenous nucleic acid or the extent and nature of the trait modification with modified cells, tissue or plants as described herein. As used here, "wild type wheat grain" means a corresponding non-mutagenized, non-transgenic wheat grain. The specific wild-type wheat grains as used here include, but are not limited to, Sunstate and Cadoux.
Any of several methods can be used to determine the presence of a transgene in a transformed plant. For example, polymerase chain reaction (PCR) can be used to amplify sequences that are unique to the transformed plant, with detection of products amplified by gel electrophoresis or other methods. DNA can be extracted from plants using conventional methods and the PCR reaction performed using primers that will distinguish between transformed and untransformed plants. An alternative method for forming a positive transformant is by Southern blot hybridization, well known in the art. Wheat plants that are transformed can also be identified, that is, distinguished from unprocessed wheat plants or wild type by their phenotype, for example, conferred by the presence of a selectable marker gene, or by immunoassays that detect or quantify the expression of an enzyme encodes by the transgene, or any other phenotype conferred by the transgene.
The wheat plants of the present invention can be grown or harvested for the grain, primarily for use as food for human consumption or as animal feed, or for fermentation or production of industrial raw material such as ethanol production, among other uses. Alternatively, wheat plants can be used directly as feed. The plant of the present invention is preferably useful for food production and in particular for commercial food production. Such food production may include the manufacture of flour, dough, semolina or other products from the grain which can be an ingredient in the production of commercial food.
As used here, the term “grain” generally refers to the mature seed, harvested from a plant, but it can also refer to the grain after imbibition or germination, according to the context. The mature cereal grain such as wheat commonly has a moisture content of less than about 18 to 20%. As used here, the term “seed” includes seed harvested, but it also includes seed that is developing in the plant after anthesis and mature seed comprised in the plant before harvest.
As used here, “germination” refers to the appearance of the root tip of the seed coat after imbibition. “The germination rate” refers to the percentage of seeds in a population that has germinated for a period of time, for example, 7 or 10 days after imbibition. Germination rates can be calculated using techniques known in the art. For example, a seed population can be evaluated daily for several days to determine the percentage of germination over time. With respect to the grain of the present invention, as used herein, the term "germination rate which is substantially the same" means that the germination rate of the grain is at least 90% of that of the corresponding wild type grain.
Starch is easily isolated from wheat grain using standard methods, for example, the method by Schulman and Kammiovirta, 1991. On an industrial scale, wet or dry milling can be used. The size of the starch granule is important in the starch processing industry where there is separation of the larger A granules from the smaller B granules.
Commercially grown wild-type wheat has a starch content in the grain that is normally in the range of 55 to 65%, depending somewhat on the growth of the cultivar. In comparison, the seed or grain of the invention has a starch content of at least 90% with respect to that of the wild type grain, and preferably at least 93%, at least 95%, or at least 98% with respect to the starch content of the wild type grain when the plants are grown under the same conditions. In other embodiments, the starch content of the grain is at least about 25%, at least about 35%, at least about 45%, or at least about 55% to about 65% depending on the weight percentage of the grain (w / w). Other desirable characteristics include the ability to grind the grain, in particular the hardness of the grain. Another aspect that can make a wheat plant of higher value is the degree of starch extraction from the grain, the higher extraction rates being more useful. The shape of the grain is also another aspect that can impact the commercial utility of a plant, so the shape of the grain can have an impact on the ease or otherwise with which the grain can be ground.
In another aspect, the invention provides starch granules or starch obtained from the plant grain as described above, having an increased amylose ratio and a reduced amylopectin ratio. The purified starch can be obtained from the grain through a milling process, for example, a wet milling process, which involves the separation of starch from protein, oil and fiber. The initial product of the milling process is a mixture or composition of the starch granules, and the invention therefore encompasses such granules. Wheat starch granules comprise proteins linked to the starch granule including GBSS, SBEIIa and SBEIIb among other proteins and, therefore, the presence of these proteins distinguish wheat starch granules from the starch granules of other cereals. The starch from the starch granules can be purified by removing the proteins after breaking and dispersing the starch granules by heat and / or chemical treatment. The wheat grain starch granules of the invention are typically distorted in surface shape and morphology when viewed under light microscopy, as exemplified here, particularly for wheat grain having an amylose content of at least 50% as a percentage of total grain starch. In one embodiment, at least 50%, preferably at least 60% or at least 70% of the starch granules obtained from the grain show distorted shapes or surface morphology. Starch granules also show a loss of birefringence when viewed in polarized light.
Grain starch, starch granule starch, and the purified starch of the invention can still be characterized by one or more of the following properties: (i) at least 50% (w / w), or at least 60% ( w / w), or at least 67% (w / w) amylose as a proportion of the total starch; (ii) modified swelling volume; (iii) modified chain length distribution and / or frequency branching; (iv) modified gelatinization temperature; (v) modified viscosity (peak viscosity, bonding temperature, etc.); (vi) modified molecular weight of amylopectin and or amylose; (vii)% modified crystallinity (viii) comprising at least 2% resistant starch; and / or (ix) comprising a low relative glycemic index (GI).
Starch can also be characterized by its swelling volume in excess heated water compared to wild type starch. The swelling volume is typically measured by mixing a starch or flour with excess water and heating to elevated temperatures, typically greater than 90 ° C. The sample is then collected by centrifugation and the swelling volume is expressed as the mass of the sedimented material divided by the dry weight of the sample. A low swelling feature is useful where desired to increase the starch content of a food preparation, in particular, a hydrated food preparation.
A measurement of an altered amylopectin structure is the distribution of chain lengths, or the degree of polymerization of the starch. The chain length distribution can be determined by the use of fluorophore-assisted carbohydrate electrophoresis (FACE) following the isoamylose debranching. The amylopectin of the starch of the invention can have a chain length distribution in the range of 5 to 60 which is greater than the distribution of starch of wild-type plants in debranching. Starch with longer chain lengths will also have a decrease consistent with the frequency of branching. Thus, starch can also have a distribution of longer amylopectin chain lengths in the amylopectin still present. The amylopectin of the grain can be characterized by understanding a reduced proportion of the chain length fraction from 4 to 12 dp in relation to the amylopectin of the wild type grain, as measured after amylopectin isoamylase de-branching.
In another aspect of the invention, wheat starch can have an altered gelatinization temperature, which can be easily measured by differential scanning calorimetry (DSC). Gelatinization is the collapse triggered by heat (disruption) of a molecular order within the starch granule in excess of water, with concomitant and irreversible changes in properties such as granular swelling, crystallite melting, loss of birefringence, development of viscosity and solubilization of starch. The gelatinization temperature can be increased or decreased compared to starch in wild-type plants, depending on the chain length of the remaining amylopectin. The high amylose starch of the corn amylose extender (ea) mutants showed a higher gelatinization temperature than normal corn (Fuwa et al., 1999; Krueger et al, 1987). On the other hand, sex6 mutants of barley starch that do not have Ila starch synthase activity had lower gelatinization temperatures and the enthalpy for the gelatinization peak was reduced when compared to that of control plants (Morell et al, 2003).
The gelatinization temperature, in particular the temperature at the beginning of the first peak or the temperature at the apex of the first peak, can be increased by at least 3 ° C, preferably at least 5 ° C or more preferably at least 7 ° C as measured by DSC compared to starch extracted from a similar but unchanged grain. The starch may comprise a high level of resistant starch, with an altered structure indicated by specific physical characteristics including one or more of the group consisting of physical inaccessibility to digest the enzymes which may be due to having an altered starch granule morphology, the presence of appreciable starch-associated lipid, altered crystallinity, and altered amylopectin chain length distribution. The high proportion of amylose also contributes to the level of resistant starch.
The structure of the wheat starch of the present invention may also differ in that the degree of crystallinity is reduced compared to the normal starch isolated from wheat. The reduced crystallinity of a starch is also considered to be associated with increased organoleptic properties and contributes to a smoother mouth feel. Thus, the starch can additionally exhibit reduced crystallinity resulting from reduced levels of activity of one or more amylopectin synthesis enzymes. Crystallinity is typically investigated by X-ray crystallography.
In some embodiments, the present starch provides modified digestive properties such as increased resistant starch including between 1% to 20%, 2% to 18%, 3% to 18% or 5% to 15% of resistant starch and a Glycemic Index (GI ) reduced.
The invention also provides flour, bran or other products produced from the grain. These can be processed or not processed, for example, by fractionation or whitening.
The invention also provides starch from the grain of the exemplified wheat plants comprising high amounts of dietary fiber, preferably in combination with a high level of resistant starch. This increase is also, at least in part, a result of the high level of relative amylose.
The term "dietary fiber" as used here includes carbohydrate and carbohydrate digestion products that are not absorbed in the small intestine of healthy humans, but that enter the large intestine. This includes resistant starch and other soluble and insoluble carbohydrate polymers. It is intended to understand that portion of carbohydrates that are fermentable, at least partially, in the large intestine by the resident microflora. The starch of the invention contains relatively high levels of dietary fiber, more particularly amylose. The dietary fiber content of the grain of the present invention results at least in part from the increased amylose content in the grain starch, and also, or in combination with an increased content of the resistant starch as a percentage of the total starch. "Resistant starch" is defined here as the sum of starch and starch digestion products not absorbed in the small intestine of healthy humans, but entering the large intestine. This is defined in terms of a percentage of the total starch in the grain, or a percentage of the total starch content in the food, depending on the context. Thus, resistant starch excludes products digested and absorbed in the small intestine. Resistant starches include physically inaccessible starch (form RS1), granules of resistant native starch (RS2), retrograded starches (RS3), and chemically modified starches (RS4). The altered starch structure and in particular the high levels of amylose content of the starch of the invention gives rise to an increase in the resistant starch when consumed in the food. Starch may be in RS1 form, being somewhat inaccessible to digestion. The association of starch lipid as measured by complex V crystallinity is also likely to contribute to the level of resistant starch.
While the invention may be particularly useful in the treatment or prophylaxis of humans, it should be understood that the invention is also applicable to non-human individuals, including, but not limited to, agricultural animals such as cows, sheep, pigs and the like, domestic animals such as dogs or cats, laboratory animals such as rabbits or rodents such as mice, rats, hamsters, or animals that can be used for sports such as horses. The method can be particularly applicable to non-ruminant mammals or animals such as monogastric mammals. The invention can also be applicable to other agricultural animals, for example, poultry including, for example, chicken, geese, ducks, turkeys, or quails, or fish.
The method of treatment of the individual, particularly humans, may comprise the step of administering the altered wheat grain, flour, starch or a food or drink product as defined here for the individual, in one or more doses, in an amount and for a period of time where the level of one or more of the intestinal or metabolic health indicators improves. The indicator may change in relation to the consumption of unmodified wheat or wheat starch or its product, within a time period of hours, as in the case of some indicators, such as pH, elevation of SCFA levels, glucose fluctuation postprandial, or it may take days such as in the case of increased fecal volume or improved laxation, or perhaps more in the order of weeks or months such as in the case where increased butyrate proliferation of normal colonocytes is measured. It may be desirable that the administration of the starch or wheat or altered wheat product is for life. However, there are good prospects for observance by the individual to be treated given the relative ease with which the altered starch can be administered.
Dosages may vary depending on the condition to be treated or prevented, but are predicted for humans to be at least 1 g of the wheat grain or starch of the invention per day, more preferably at least 2 g per day, preferably at least 10 or at least minus 20 g per day. Administration of more than about 100 grams per day may require considerable release volumes and reduce compliance. Most preferably the dosage for a human is between 5 and 60 g of wheat grain or starch per day, or for adults between 5 and 100 g per day.
The Glycemic Index (GI) refers to the rate of digestion of foods comprising starch, and is a comparison of the effect of a test food with the effect of white bread or glucose on excursions in the concentration of glucose in the blood. The Glycemic Index is a measure of the likely effect of food in relation to the concentration of postprandial serum glucose and demand for insulin for blood glucose homeostasis. An important feature provided by the foods of the invention is a reduced Glycemic Index. Serum glucose levels were lower 30 min after ingestion of wheat products with a high amylose content by human volunteers compared to wheat with a low amylose content (Goddard et al, 1984). In addition, foods may have a low level of final digestion and therefore be relatively low calorie. A product with a low calorific value can be based on the inclusion of flour produced from the ground wheat grain. Such foods can have a filling effect, increasing intestinal health, reducing postprandial serum glucose and lipid concentration, as well as providing a food product with a low calorific value.
Indicators of improved intestinal health may include, among others: i) decreased pH of intestinal contents, ii) increased total SCFA concentration or amount of total SCFA in intestinal contents, iii) increased concentration or quantity of one or more SCFAs in intestinal contents , iv) increased fecal volume, v) increase in the total water volume of the intestine or feces, without diarrhea, vi) improved laxation, vii) increase in the number or activity of one or more species of probiotic bacteria, viii) increase in the excretion of the fecal bile acid, ix) reduced urinary levels of putrefactive products, x) reduced fecal levels of putrefactive products, xi) increased proliferation of normal colonocytes, xii) reduced inflammation in the intestine of individuals with an inflamed intestine, xiii) reduced fecal or intestinal levels bulk of any of urea, creatinine and phosphate in uremic patients, and xiv) any combination of the above.
Indicators of improved metabolic health can include, among others: i) stabilization of postprandial glucose oscillation, ii) improved (decreased) glycemic response, iii) reduced insulin concentration in prandial plasma, iv) lipid profile in the improved blood, v) decreased plasma LDL cholesterol, vi) reduced plasma levels of one or more of urea, creatinine and phosphate in uremic patients, vii) an improvement in a dysglycemic response, or viii) any combination of the above.
It will be understood that a benefit of the present invention is one that provides products such as bread that are of particular nutritional benefit, and, in addition, does so without having to modify the starch or other constituents of the wheat grain after harvest.
However, it may be desired to make modifications to the starch or other constituent of the grain, and the invention encompasses such a modified constituent. Modification methods are well known and include extraction of starch or another constituent by conventional methods and modification of the starches to increase the resistant form. Starch can be modified through heat and / or moisture treatment, physically (for example, grinding), enzymatically (using, for example, α- or β-amylase, pulalanase or similar), chemical hydrolysis (wet or using liquid reagents or gaseous), oxidation, cross-linking with bifunctional reagents (for example, sodium trimetaphosphate, phosphorus oxychloride), or carboxymethylation.
The wheat starch of the present invention will be a suitable substrate for fermentation for ethanol (biofuel) or beverages containing ethanol and the grain of wheat or wheat starch for other fermentation products such as food, nutraceuticals (soluble or insoluble fiber), enzymes and industrial materials. The methods for fermentation using plant-derived starch are well known to those skilled in the art, with processes established for various fermentation products (see, for example, Vogel et al., 1996 and references cited here). In one embodiment, the starch carbohydrates can be extracted by grinding parts of the wheat plant of the invention such as grain, or by diffusing the plant tissues in water or another suitable solvent. The wheat or starch tissues of the invention can be used directly as a substrate for fermentation or bioconversion in a batch, continuous, or immobilized cell process.
The terms "polypeptide" and "protein" are generally used interchangeably here. The terms "proteins" and "polypeptides" as used herein also include variants, mutants, modifications and / or derivatives of the polypeptides of the invention as described herein. As used herein, "substantially purified polypeptide" refers to a polypeptide that has been separated from lipids, nucleic acids, other peptides and other molecules with which it is associated in its natural state. Preferably, the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and most preferably at least 90% free from the other components with which it is naturally associated. By "recombinant polypeptide" is meant a polypeptide made using recombinant techniques, that is, through the expression of a recombinant polynucleotide in a cell, preferably a plant cell and more preferably a wheat cell. In one embodiment, the polypeptide has starch branching enzymatic activity, particularly SBEII activity, and is at least 90% identical to an SBEII described here.
As used here, a "biologically active" fragment is a portion of a polypeptide of the invention that maintains a defined activity of the full length polypeptide. In a particularly preferred embodiment, the biologically active fragment has starch branching enzymatic activity. The biologically active fragments can be of any size as long as they maintain the defined activity, but preferably have at least 700 or 800 amino acid residues in length.
The% identity of a polypeptide in relation to another polypeptide can be determined by GAP analysis (Needleman and Wunsch, 1970) (GCG program) with a gap creation penalty = 5, and a gap extension penalty = 0, 3. The search sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. Most preferably, the search sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the search sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. More preferably, two SBEII polypeptides are aligned over their full length amino acid sequences.
With respect to a defined polypeptide, it will be appreciated that the% of the figures of identity greater than those provided above will encompass preferred embodiments. Thus, where applicable, taking into account the minimum% of the identity figures, it is preferred that the polypeptide comprises an amino acid sequence that is at least 75%, more preferably at least 80%, more preferably at least 85%, most preferably at less 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, most preferably at least 95%, most preferably at least 96%, most preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, most preferably at least 99.3%, most preferably at least 99, 4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, most preferably at least 99.8%, and even more preferably pe it minus 99.9% identical to the relevant named SEQ ID NO.
Mutants of the amino acid sequence of the polypeptides of the present invention can be prepared by appropriately introducing the nucleotide changes into a nucleic acid of the present invention or by mutagenesis in vivo such as by chemical treatment or radiation. Such mutants include, for example, exclusions, insertions or substitutions of residues within the amino acid sequence. The polynucleotides of the invention can be subjected to DNA shuffling techniques as described by Harayama, 1998 or other in vitro methods to produce altered polynucleotides that encode the polypeptide variants. Such DNA shuffling techniques can use genetic sequences related to those of the present invention, such as SBE genes from a plant species other than wheat. Products derived from mutated / altered DNA can be easily examined using techniques described here to determine whether they have, for example, SBE activity.
Exclusions from the amino acid sequence generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
The substitution mutants have at least one amino acid residue in the removed polypeptide molecule and a different residue inserted in its place. Sites of greatest interest for substitutional mutagenesis include sites identified as the active site (s). Other places of interest are those in which the particular residues obtained from various strains or species are identical, that is, conserved amino acids. These positions can be important for biological activity. These amino acids, especially those that span a contiguous sequence of at least three other identically conserved amino acids, are preferably substituted in a relatively conservative manner in order to retain function such as SBEII activity. Such conservative substitutions are shown in Table 1 under the heading “exemplary substitutions”. "Non-conservative amino acid substitutions" are defined here as substitutions other than those listed in Table 3 (exemplary conservative substitutions). Non-conservative substitutions in an SBEII are expected to reduce enzyme activity and many will correspond to an SBEII encoded by a "mutant SBEII gene with partial loss of function".
Also included within the scope of the invention are the polypeptides of the present invention that are differentially modified during or after synthesis, for example, by phosphorylation, as has been shown for SBEI, SBEIIa and SBEIIb in wheat amyloplasts (Tetlow et al, 2004). These modifications can serve to regulate the enzymatic activity, for example, by regulating the formation of protein complexes in amyloplasts during the synthesis of starch (Tetlow et al, 2008), or to increase the stability and / or bioactivity of the polypeptide of the invention, or serve as a binder to bind to another molecule.
In some embodiments, the present invention involves modification of gene activity, particularly of SBEII gene activity, combinations of mutant genes, and the construction and use of chimeric genes. As used herein, the term "gene" includes any deoxyribonucleotide sequence that includes a protein coding region or that is transcribed in a cell, but not translated, along with the associated regulatory and non-coding regions. Such associated regions are typically located adjacent to the coding region at both the 5 'and 3' ends for a distance of about 2 kb on either side. In this regard, the gene includes control signals such as promoters, enhancers, transcription termination and / or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals, in which case the gene is referred to as a “Chimeric gene”. Sequences that are located 5 'from the protein coding region and that are present in the mRNA are referred to as 5' untranslated sequences. Sequences that are located 3 'or downstream of the protein coding region and that are present in the mRNA are referred to as 3' untranslated sequences. The term "gene" encompasses cDNA and genomic forms of a gene. The term "gene" includes synthetic or fusion molecules encoding the proteins of the invention described here. The genes are commonly present in the wheat genome as double-stranded DNA. A chimeric gene can be introduced into an appropriate vector for extrachromosomal maintenance in a cell or for integration into the host genome. The genes or genotypes as referred to herein in italic form (eg, SBEIIa) while proteins, enzymes or phenotypes are indicated in non-italic form (SBEIIa).
A genomic form or clone of a gene containing the coding region can be interrupted with non-coding sequences called "introns" or "intervention regions" or "intervention sequences". An "intron" as used here is a segment of a gene that is transcribed as part of a primary RNA transcript, but is not present in the mature mRNA molecule. Introns are removed or "embedded" from the nuclear or primary transcript; introns, therefore, are absent in messenger RNA (mRNA). Introns can contain regulatory elements such as enhancers. "Exons" as used here refer to the DNA regions corresponding to the RNA sequences that are present in the mature RNAm or in the mature RNA molecule in cases where the RNA molecule is not translated. The functions of mRNA during translation to specify the sequence or order of amino acids in a nascent polypeptide.
The present invention relates to several polynucleotides. As used here, a “polynucleotide” or “nucleic acid” or “nucleic acid molecule” means a polymer of nucleotides, which can be DNA or RNA or a combination of these, for example, a DNA and RNA heteroduplex, and includes, for example, mRNA, cRNA, cDNA, tRNA, siRNA, shRNA, hpRNA, and single or double stranded DNA. It can be DNA or RNA of cellular, genomic or synthetic origin, for example, made from an automatic synthesizer, and can be combined with carbohydrate, lipids, protein or other materials, labeled with fluorescent or other groups, or attached to a solid support for perform a particular activity defined here. Preferably, the polynucleotide is just DNA or just RNA as it does in a cell, and some bases can be methylated or otherwise modified as it is in a wheat cell. The polymer can be single-stranded, essentially double-stranded or partially double-stranded. An example of a partially double-stranded RNA molecule is a hairpin RNA (hpRNA), short hairpin RNA (shRNA), or self-complementary RNA that includes a double-stranded stem formed by the base pairing between the nucleotide sequence and its complement and a sequence cycle that covalently unites the nucleotide sequence and its complement. Base pairing as used here refers to the standard base pairing between nucleotides, including G: U base pairs in an RNA molecule. "Complementary" means two polynucleotides that are capable of base pairing along part of their lengths, or along the total length of one or both.
By "isolated" is meant the material that is substantially or essentially free of the components that normally accompany it in its natural state. As used herein, an "isolated polynucleotide" or "isolated nucleic acid molecule" means a polynucleotide that is at least partially separated from, preferably substantially or essentially free of, the polynucleotide sequences of the same type with which it is associated or linked in its natural state. For example, an "isolated polynucleotide" includes a polynucleotide that has been purified or separated from the sequences that flank it in a naturally occurring state, for example, a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment. Preferably, the isolated polynucleotide is also at least 90% free of other components such as proteins, carbohydrates, lipids, etc. The term "recombinant polynucleotide" as used here refers to a polynucleotide formed in vitro by manipulating nucleic acid in a form not normally found in nature. For example, the recombinant polynucleotide can be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory nucleic acid operationally connected to the nucleotide sequence to be transcribed in the cell.
The present invention relates to the use of oligonucleotides that can be used as "probes" or "primers". As used here, "oligonucleotides" are polynucleotides up to 50 nucleotides in length. They can be RNA, DNA, or combinations or derivatives of them. Oligonucleotides are typically relatively short single-chain molecules of 10 to 30 nucleotides, commonly 15 to 25 nucleotides in length, typically comprised of 10 to 30 or 15 to 25 nucleotides that are identical to, or complementary to, part of a SBEIIa gene or SBEIIb or cDNA corresponding to an SBEIIa or SBEIIb gene. When used as a probe or as a primer in an amplification reaction, the minimum size of such an oligonucleotide is the size required for the formation of a stable hybrid between the oligonucleotide and a complementary sequence in a target nucleic acid molecule. Preferably, the oligonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, even more preferably at least 25 nucleotides in length. Polynucleotides used as a probe are typically conjugated to a detectable label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule. The oligonucleotides and probes of the invention are useful in methods of detecting an allele of an SBEIIa, SBEIIb or another gene associated with a trait of interest, for example, modified starch. Such methods employ nucleic acid hybridization and in many instances include extending the oligonucleotide primer by a suitable polymerase, for example, as used in PCR for detection or identification of mutant or wild-type alleles. Preferred oligonucleotides and probes hybridize to a wheat SBEIIa or SBEIIb gene sequence, including any of the sequences disclosed here, for example, SEQ ID NOs: 36 to 149. The preferred oligonucleotide pairs are those that reach one or more introns, or a part of an intron, and therefore can be used to amplify an intron sequence in a PCR reaction. Numerous examples are provided in the Examples here.
The terms "polynucleotide variant" and "variant" and similar refer to polynucleotides that exhibit substantial sequence identity with a reference polynucleotide sequence and that are capable of functioning in an analogous manner, or with the same activity as the sequence of reference. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, exclusion or substitution of at least one nucleotide, or that, when compared to naturally occurring molecules, have one or more mutations. Consequently, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions and substitutions can be made to a reference polynucleotide, where the altered polynucleotide retains the biological function or activity of the reference polynucleotide. Consequently, these terms encompass polynucleotides that encode polypeptides that exhibit enzymatic or other regulatory activity, or polynucleotides capable of serving as selective probes or other hybridizing agents. The terms "polynucleotide variant" and "variant" also include naturally occurring allelic variants. The mutants can be naturally occurring (that is, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis at the nucleic acid). Preferably, a polynucleotide variant of the invention that encodes a polypeptide with enzymatic activity is greater than 400, more preferably greater than 500, more preferably greater than 600, more preferably greater than 700, more preferably greater than 800, more preferably greater than 900, and even more preferably greater than 1,000 nucleotides in length, up to the total length of the gene.
A variant of an oligonucleotide of the invention includes molecules of varying sizes that are capable of hybridizing, for example, the wheat genome in a position close to that of the specific oligonucleotide molecules defined here. For example, variants can comprise additional nucleotides (such as 1, 2, 3, 4, or more), or fewer nucleotides as long as they still hybridize to the target region. In addition, some nucleotides can be replaced without influencing the ability of the oligonucleotide to hybridize to the target region. In addition, variants can be easily designed that hybridize close (for example, among others, within 50 nucleotides) to the region of the plant genome where the specific oligonucleotides defined here hybridize.
By "corresponds to" or "corresponds to" in the context of polynucleotides or polypeptides is meant a polynucleotide (a) that has a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or ( b) which encodes an amino acid sequence identical to an amino acid sequence in a peptide or protein. This expression also includes, within its scope, a peptide or polypeptide that has an amino acid sequence that is substantially identical to an amino acid sequence in a reference peptide or protein. Terms used to describe the sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", "substantial identity" and "identical ”, And are defined with respect to a defined minimum number of nucleotides or amino acid residues or preferably along the total length. The terms "sequence identity" and "identity" are used interchangeably here to refer to the extent that the sequences are identical on a nucleotide basis per nucleotide or an amino acid basis per amino acid over a comparison window. Thus, a “percentage of sequence identity” is calculated by comparing two perfectly aligned sequences on the comparison window, determining the number of positions in which the identical nucleic acid base (for example, A, T, C, G, U) or the identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occur in both sequences to yield the number of combined positions, dividing the number of combined positions by the total number of positions in the comparison window (that is, the size of the window), and multiplying the result by 100 to yield the percentage sequence identity.
The% identity of a polynucleotide can be determined by GAP analysis (Needleman and Wunsch, 1970) (GCG program) with a gap creation penalty = 5, and a gap extension penalty = 0.3. Unless otherwise indicated, the search sequence is at least 45 nucleotides in length, and GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the search sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the search sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides, or at least 400, 500 or 600 nucleotides in each case. Reference can also be made to the BLAST program family, as, for example, revealed by Altschul et al, 1997. A detailed discussion of the sequence analysis can be found in Unit 19.3 of Ausubel et al., 1994-1998, chapter 15.
Nucleotide or amino acid sequences are indicated as "essentially similar" when such sequences have a sequence identity of at least about 95%, particularly at least about 98%, more particularly at least about 98.5%, quite particularly about 99 %, especially about 99.5%, more especially about 100%, quite especially are identical. It is clear that when RNA sequences are described as essentially similar to, or have a certain degree of sequence identity with, DNA sequences, thymine (T) in the DNA sequence is considered to be equal to uracil (U) in the RNA sequence.
With respect to the defined polynucleotides, it will be appreciated that the% of the figures of identity greater than those provided above will encompass the preferred embodiments. Thus, where applicable, taking into account the minimum% of the identity figures, it is preferred that the polynucleotide comprises a polynucleotide sequence that is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least less 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, most preferably at least 95%, most preferably at least 96%, most preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, most preferably at least 99.3%, most preferably at least 99, 4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, most preferably at least 99.8%, and even more preferable at least 99.9% identical to the relevant named SEQ ID NO.
In some embodiments, the present invention relates to the rigidity of the hybridization conditions to define the extent of complementarity of two polynucleotides. “Rigidity” as used here, refers to the temperature and conditions of ionic resistance, and the presence or absence of certain organic solvents, during hybridization. The greater the stiffness, the greater the degree of complementarity between the target nucleotide sequence and the labeled polynucleotide sequence. "Strict conditions" refers to the temperature and ionic conditions under which only nucleotide sequences that have a high frequency of complementary bases will hybridize. As used here, the term "hybridizes at low rigidity, medium rigidity, high rigidity, or conditions with very high rigidity" describes conditions for hybridization and washing. Guidance for conducting hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated here by reference. The specific hybridization conditions referred to here are as follows: 1) low rigidity hybridization conditions in 6 X sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by two washes in 0.2 X SSC, 0.1% SDS at 50-55 ° C; 2) medium rigidity hybridization conditions in 6 X SSC at about 45 ° C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 60 ° C; 3) high rigidity hybridization conditions in 6 X SSC at about 45 ° C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 65 ° C; and 4) very high rigidity hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65 ° C, followed by one or more washes at 0.2 X SSC, 1% SDS at 65 ° C .
As used here, a "chimeric gene" or "genetic construct" refers to any gene that is not a native gene at its place of origin, that is, it has been artificially manipulated, including a chimeric gene or genetic construct that is integrated into the wheat genome. Typically a chimeric gene or genetic construct comprises regulatory and transcribed or protein coding sequences that are not found together in nature. Consequently, a chimeric gene or genetic construct can comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged differently than that found in nature. The term "endogenous" is used here to refer to a substance that is normally produced in an unmodified plant at the same stage of development as the plant under investigation, preferably a wheat plant, such as starch or an SBEIIa or SBEIIb. An "endogenous gene" refers to a gene native to its natural location in the genome of an organism, preferably an SBEIIa or SBEIIb gene in a wheat plant. As used herein, "recombinant nucleic acid molecule" refers to a nucleic acid molecule that has been constructed or modified by recombinant DNA technology. The terms "external polynucleotide" or "exogenous polynucleotide" or "heterologous polynucleotide" and the like refer to any nucleic acid that is introduced into the genome of a cell by experimental manipulations, preferably the wheat genome, but which do not occur naturally in the cell. These include modified forms of gene sequences found in that cell as long as the introduced gene contains some modification, for example, an introduced mutation or the presence of a selectable marker gene, in relation to the naturally occurring gene. The foreign or exogenous genes can be genes found in nature that are inserted into a non-native organism, native genes introduced in a new location within the native host, or chimeric genes or genetic constructs. A "transgene" is a gene that was introduced into the genome by a transformation procedure. The term "genetically modified" includes introducing genes into cells, mutating genes into cells and altering or modifying the regulation of a gene in a cell or organisms in which these acts were done or their progeny.
The present invention relates to elements that are operationally connected or connected. "Operationally connected" or "operationally connected" and the like refer to a connection of polynucleotide elements in a functional relationship. Typically, the operably connected nucleic acid sequences are linked together and, where necessary, join the two protein coding regions, contiguous and in the reading frame. A coding sequence is "operationally connected to" another coding sequence when RNA polymerase will describe the two coding sequences in a single RNA, which if translated is then translated into a simple polypeptide having amino acids derived from both coding sequences. The coding sequences do not need to be contiguous with each other, as long as the expressed sequences are finally processed to produce the desired protein.
As used here, the term "cis-regulatory sequence", "cis-regulatory element" or "cis-regulatory region" or "regulatory region" or similar term should be understood as any sequence of nucleotides that regulate the expression of the genetic sequence. This can be a naturally occurring cis-regulatory sequence in its context of origin, for example, regulating the SBEIIa or SBEIIb gene in wheat, or a sequence in a genetic construct that when properly positioned in relation to an expressible genetic sequence, regulates your expression. Such cis-regulatory region may be able to activate, silence, increase, repress or otherwise alter the level of expression and / or cell type specificity and / or specificity of development of a gene sequence at the transcriptional or post-transcriptional level. In the preferred embodiments of the present invention, the cis-regulatory sequence is an activator sequence that enhances or stimulates the expression of an expressible genetic sequence, such as a promoter. The presence of an intron in the 5 'leader (RTU) of the genes has been shown to increase gene expression in monocot plants such as wheat (Tanaka et al, 1990). Another type of cis-regulatory sequence is a matrix association region (MAR) that can influence gene expression by anchoring the active chromatin domains to the nuclear matrix.
"Operationally connecting" a promoter or enhancer element to a transcript polynucleotide means placing the transcript polynucleotide (e.g., protein encoding polynucleotide or other transcription) under the regulatory control of a promoter, which then controls transcription that polynucleotide. In the construction of heterologous promoter / structural gene combinations, it is generally preferred to position a promoter or its variant at a distance from the transcription initiation site of the polynucleotide that can be transcribed, which is approximately the same distance between that promoter and the gene it controls in its natural configuration; that is, the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function.
The present invention makes use of vectors for the production, manipulation or transfer of chimeric genes or genetic constructs. By "vector" is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage or plant virus, into which a nucleic acid sequence can be inserted. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or its tissue, or integrable into the genome of the defined host so that the sequence cloned is reproducible. Consequently, the vector can be an autonomous replication vector, that is, a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, for example, a closed linear or circular plasmid, an extrachromosomal element, a minichromosome , or an artificial chromosome. The vector can contain any means to guarantee self-replication. Alternatively, the vector can be one that, when introduced into a cell, is integrated into the genome of the recipient cell and replicated next to the chromosome (s) within which it has been integrated. The vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the host cell's genome, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the cell in which the vector is to be introduced. The vector can also include a selection marker such as an antibiotic-resistant gene that can be used to select suitable transformants, or sequences that enhance the transformation of prokaryotic or eukaryotic cells (especially wheat) such as T-DNA sequences or P-DNA. Examples of such resistance genes and sequences are well known to those skilled in the art.
By "marker gene" is meant a gene that confers a distinct phenotype on cells that express the marker gene and thus allows such transformed cells to be distinguished from cells that do not have the marker. A “selectable marker gene” gives a trait that one can 'select' based on resistance to a selective agent (eg, a herbicide, antibiotic, radiation, heat, or other harmful treatment to untransformed cells) or based on in a growth advantage in the presence of a metabolized substrate. A marker gene capable of being examined (or reporter gene) gives a trait that someone can identify through observation or testing, that is, by 'screening' (for example, β-glucuronidase, luciferase, GFP or other enzyme activity not present) in untransformed cells). The marker gene and the nucleotide sequence of interest do not need to be linked.
Examples of selectable bacterial markers are markers that confer resistance to antibiotics such as resistance to ampicillin, kanamycin, erythromycin, chloramphenicol or tetracycline. Exemplary selectable markers for selecting plant transformants include, among others, a hyg gene that confers resistance to hygromycin B; a neomycin phosphotransferase (npt) gene conferring resistance to kanamycin, paromomycin, G418 and the like as, for example, described by Potrykus et al, 1985; a glutathione-S-transferase gene from the rat liver conferring resistance to glutathione-derived herbicides as, for example, described in EP-A-256223; a glutamine synthetase gene conferring, by overexpression, resistance to glutamine synthetase inhibitors such as phosphinothricin as, for example, described in WO87 / 05327, an acetyl transferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin as, for example, described in EP-A-275957, a gene encoding a 5-enolshikimato-3-phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as, for example, described by Hinchee et al, 1988, a bar gene conferring resistance against bialafos as, for example, described in WO91 / 02071; a nitrilase gene such as Klebsiella ozaenae bxn that confers resistance to bromoxynil (Stalker et al, 1988); a dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate (Thillet et al, 1988); a mutant acetolactate synthase (ALS) gene, which confers resistance to imidazolinone, sulfonylurea or other ALS inhibiting chemicals (EP-A-154204); a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan; or a dalapon dehalogenase gene that confers resistance to the herbicide.
Preferred markers that can be examined include, among others, a uidA gene encoding a β-glucuronidase enzyme (GUS) for which several chromogenic substrates are known, a β-galactosidase gene encoding an enzyme for which chromogenic substrates, a gene aquorine (Prasher et al, 1985), which can be used in calcium-sensitive luminescent detection; a green fluorescent protein gene (GFP, Niedz et al, 1995) or one of its variants; a luciferase (luc) gene (Ow et al, 1986), which allows the detection of bioluminescence, and others known in the art.
In some embodiments, the level of endogenous starch branching activity or other enzyme activity is modulated by decreasing the level of expression of genes that encode the proteins involved in these activities in the wheat plant, or increasing the level of expression of a nucleotide sequence which codes for the enzyme in a wheat plant. Increasing expression can be achieved at the level of transcription by using promoters of different strengths or inducible promoters, which are able to control the expressed level of transcription of the coding sequence. Heterologous sequences can be introduced which encode transcription factors that modulate or increase the expression of genes whose products downward regulate starch branching. The level of expression of the gene can be modulated by changing the number of copies per cell of a construct comprising the coding sequence and a transcriptional control element that is operationally connected to it and that is functional in the cell. Alternatively, a plurality of transformants can be selected, and examined for those with a favorable level and / or specificity of transgene expression resulting from influences of endogenous sequences in the vicinity of the transgene integration site. A favorable level and pattern of transgene expression is one that results in a substantial increase in starch synthesis or amylose content in the wheat plant. This can be detected by a simple test of the transformants.
Reducing gene expression can be achieved by introducing and transcribing a "chimeric gene silencing gene" introduced into the wheat plant. The chimeric gene silencing gene is preferably introduced stably into the wheat genome, preferably into the nuclear wheat genome. As used here, "the gene silencing effect" refers to the reduction of expression of a target nucleic acid in a wheat cell, preferably an endosperm cell, which can be achieved by introducing a silencing RNA. In a preferred embodiment, a chimeric gene silencing gene is introduced which encodes an RNA molecule that reduces the expression of one or more endogenous genes, preferably the SBEIIa and / or SBEIIb genes. The target genes in wheat also include the genes listed in Table 1. Such a reduction may be the result of reduced transcription, including via chromatin methylation remodeling, or post-transcriptional modification of RNA molecules, including via RNA degradation, or both. Gene silencing should not necessarily be interpreted as a suppression of expression of the target nucleic acid or gene. It is sufficient that the level of expression of the target nucleic acid in the presence of the silencing RNA is lower than in the absence of it. The level of expression of the target gene can be reduced to at least about 40% or at least about 45% or at least about 50% or at least about 55% or at least about 60% or at least about 65% or at least about 70% or at least about 75% or at least about 80% or at least about 85% or at least about 90% or at least about 95% or effectively suppressed to a level not detected.
Anti-sense techniques can be used to reduce gene expression in wheat cells. The term "antisense RNA" should be understood as an RNA molecule that is complementary to at least a portion of a specific mRNA molecule and capable of reducing the expression of the gene encoding the mRNA, preferably a SBEIIa and / or SBEIIb gene . Such a reduction typically occurs in a sequence-dependent manner and is likely to occur by interfering with a post-transcriptional event such as mRNA transport from the nucleus to the cytoplasm, mRNA stability or translation inhibition. The use of antisense methods is well known in the art (see, for example, Hartmann and Endres, 1999). Anti-sense methods are now well-established techniques for manipulating gene expression in plants.
The antisense molecules typically include sequences that correspond to the part of the transcribed region of a target gene or to sequences that perform control over gene expression or fitting events. For example, the antisense sequence may correspond to the coding region of the target protein of the genes of the invention, or the 5 'untranslated region (UTR) or the 3-UTR or combination thereof, preferably only for exon sequences of the target gene. In view of the generally greater divergence between related RTU genes, targeting these regions provides greater specificity of gene inhibition. The length of the antisense sequence must be at least 19 contiguous nucleotides, preferably at least 50 nucleotides, and more preferably at least 100, 200, 500 or 1000 nucleotides, for a maximum of the total length of the gene to be inhibited. The full-length sequence complementary to the transcription of the entire gene can be used. The length is more preferably 100 to 2000 nucleotides. The degree of identity of the antisense sequence for the target transcript should be at least 90% and more preferably 95 to 100%. The antisense RNA molecule can certainly comprise unrelated sequences that can work to stabilize the molecule.
The genetic constructs for expressing an antisense RNA can be easily done by joining a promoter sequence in a region of the target gene in an “antisense” orientation, which as used here refers to the reverse orientation in relation to the transcription and translation orientation (if occur) of the sequence in the target gene in the plant cell. Preferably, the antisense RNA is preferentially expressed in the endosperm of a wheat plant through the use of an endosperm-specific promoter.
The term "ribozyme" refers to an RNA molecule that specifically recognizes a distinct RNA substrate and catalyzes its cleavage. Typically, the ribozyme contains an antisense sequence for specific recognition of a target nucleic acid, and an enzyme region referred to herein as the "catalytic domain". The types of ribozymes that are particularly useful in this invention are the hammerhead ribozyme (Haseloff and Gerlach, 1988; Perriman et al, 1992) and the hairpin ribozyme (Shippy et al., 1999).
As used here, "artificially introduced dsRNA molecule" refers to the introduction of a double-stranded RNA (dsRNA) molecule, which is preferably synthesized in the wheat cell by the transcription of a chimeric gene that encodes such a dsRNA molecule. Interference RNA (RNAi) is particularly useful for specifically reducing the expression of a gene or inhibiting the production of a particular protein, also in wheat (Regina et al, 2006). This technology is based on the presence of dsRNA molecules that contain a sequence that is essentially identical to the mRNA of the gene of interest or part of it, and its complement, thereby forming a dsRNA. Conveniently, dsRNA can be produced from a simple promoter in the host cell, where the sense and antisense sequences are transcribed to produce a hairpin RNA in which the sense and antisense sequences hybridize to form the dsRNA region as a related sequence (for an SBEII gene) or unrelated, forming a loop structure, so that the RNA hairpin comprises a hairpin loop structure. The design and production of dsRNA molecules suitable for the present invention is well within the skill of a person skilled in the art, particularly considering Waterhouse et al., 1998; Smith et al., 2000; WO 99/32619; WO 99/53050; WO 99/49029; and WO 01/34815.
The DNA encoding the dsRNA typically comprises both the sense and antisense sequences arranged as an inverted repeat. In a preferred embodiment, the sense and antisense sequences are separated by a spacer region comprising an intron which, when transcribed into RNA, is joined. This arrangement has been shown to result in a higher efficiency of gene silencing (Smith et al, 2000). The double-stranded region can comprise one or two RNA molecules, transcribed from one or two regions of DNA. DsRNA can be classified from hpRNA, having long, sense and antisense regions that can be broadly complementary, but need not be entirely complementary (typically greater than about 200 bp, ranging between 200 and 1000 bp). hpRNA can also be quite small with the double-stranded portion ranging in size from about 30 to about 42 bp, but not much longer than 94 bp (see WO04 / 073390). The presence of the double-stranded RNA region is considered to trigger a response from an endogenous plant system that destroys the double-stranded RNA and also the homologous RNA transcript of the target plant gene, efficiently reducing or eliminating the activity of the target gene.
The length of the sense and antisense sequences that hybridize should each have at least 19 contiguous nucleotides, preferably at least 30 or 50 nucleotides, and more preferably at least 100, 200, 500 or 1000 nucleotides. The full length sequence corresponding to the entire gene transcript can be used. The lengths are more preferably 100 to 2000 nucleotides. The degree of identity of the sense and antisense sequences for the target transcript must be at least 85%, preferably at least 90% and most preferably 95 to 100%. The longer the sequence, the less stringent the requirement for the identity of the total sequence. The RNA molecule can certainly comprise unrelated sequences that can work to stabilize the molecule. The promoter used to express the dsRNA-forming construct can be any type of promoter that is expressed in cells that express the target gene. When the target gene is SBEIIa or SBEIIb or another gene selectively expressed in the endosperm, an endosperm promoter is preferred, so as not to affect the expression of the target gene (s) in other tissues.
Examples of dsRNA molecules that can be used to down-regulate the SBEII gene (s) are provided in Example 4.
Another silencing RNA can be "non-polyadenylated RNA" comprising at least 20 consecutive nucleotides having at least 95% sequence identity to complement a nucleotide sequence of a target gene RNA transcript, as described in WO01 / 12824 or US6423885. Yet another type of silencing RNA is an RNA molecule as described in WO03 / 076619 (incorporated herein by reference) comprising at least 20 consecutive nucleotides having at least 95% sequence identity to the target nucleic acid sequence or its complement , and further comprising a wide double-stranded region as described in WO03 / 076619.
As used here, "silencing RNAs" are RNA molecules that have 21 to 24 contiguous nucleotides that are complementary to a region of the target gene's mRNA transcript, preferably SBEIIa or SBEIIb. The sequence of 21 to 24 nucleotides is preferably completely complementary to a sequence of 21 to 24 contiguous mRNA, that is, identical to the complement of 21 to 24 nucleotides of the mRNA region. However, miRNA sequences that have up to five incompatibilities in the mRNA region can also be used (Palatnik et al, 2003), and base pairing can involve one or two G-U base pairs. When not all 21 to 24 nucleotides of the silencing RNA are able to pair with the mRNA, it is preferred that there are only one or two incompatibilities between the 21 to 24 nucleotides of the silencing RNA and the region of the mRNA. With regard to miRNAs, it is preferred that any incompatibilities, up to a maximum of five, are found towards the 3 'end of the miRNA. In a preferred embodiment, there are no more than one or two incompatibilities between the silencing RNA sequences and their target mRNA.
Silencing RNAs are derived from longer RNA molecules that are encoded by the chimeric DNAs of the invention. The longer RNA molecules, also referred to here as "precursor RNAs", are the initial products produced by the transcription of chimeric DNAs in wheat cells and have a partial double-stranded attribute formed by the intramolecular base pairing between complementary regions. The precursor RNAs are processed by a specialized class of RNAs, commonly called "Dicer (s)", in silencing RNAs, typically 21 to 24 nucleotides in length. Silencing RNAs as used here include short interference RNAs (siRNAs) and microRNAs (miRNAs), which differ in their biosynthesis. SiRNAs are derived from full or partial double-stranded RNAs having at least 21 contiguous base pairs, including possible G-U base pairs, without incompatibilities or unpaired nucleotides pushing out of the double-stranded region. These double-stranded RNAs are a simple, self-complementary transcript that forms by folding over itself and forming a hairpin loop structure, here referred to as a “hairpin RNA”, or of two separate RNAs that are at least partially complementary and that hybridize to form a double stranded RNA region. MiRNAs are produced by processing single-stranded transcripts that include complementary regions that are not completely complementary and thus form an imperfectly matched structure, thus having incompatible or unpaired nucleotides within the partial double-stranded structure. The paired structure can also include G-U base pairs. The processing of precursor RNAs to form miRNAs leads to the preferential accumulation of a distinct, small RNA having a specific sequence, the miRNA. It is derived from a precursor RNA chain, typically the “antisense” chain of the precursor RNA, while processing the long complementary precursor RNA to form siRNAs produces a population of siRNAs that are not uniform in sequence, but correspond to many portions and both strands of the precursor.
MiRNAs were first discovered as a small regulatory RNA that controls the lin-4 gene in C. elegans (Lee et al., 1993). Since then, large numbers of other naturally occurring miRNAs have been reported to be involved in regulating gene function in animals and plants. MiRNA precursor RNAs of the invention, also referred to herein as "artificial miRNA precursors", are typically derived from naturally occurring Mirna precursors by altering the nucleotide sequence of the naturally occurring precursor miRNA portion, so that it is complementary , preferably completely complementary, to the 21 to 24 nucleotide region of the target mRNA, and alter the nucleotide sequence of the complementary region of the miRNA precursor that matches the miRNA sequence to maintain base pairing. The rest of the miRNA precursor RNA can be unchanged and thus have the same sequence as the naturally occurring Mirna precursor, or it can also be altered in the sequence by nucleotide substitutions, nucleotide insertions, or preferably nucleotide exclusions, or any combination of them. The remainder of the miRNA precursor RNA is considered involved in the recognition of the structure by the Dicer enzyme called Dicer-type 1 (DCL1), and therefore it is preferred that little or no change is made to the rest of the structure. For example, paired nucleotides can be substituted for other paired nucleotides without further change in the total structure. The naturally occurring miRNA precursor from which the artificial miRNA precursor of the invention is derived may be from wheat, another plant such as another cereal plant, or from non-vegetable sources.
Examples of such precursor RNAs are the rice precursor mi395, the precursor to Arabidopsis mi 159b, or the precursor mi 172. Artificial miRNAs have been demonstrated in plants, for example Alvarez et al, 2006; Parizotto et al, 2004; Schwab et al, 2006.
Another molecular biological approach that can be used is co-suppression. The co-suppression mechanism is not well understood, but it is considered to involve post-transcriptional gene silencing (PTGS) and in this respect it can be very similar to many examples of antisense suppression. This involves introducing an extra copy of a gene or its fragment into a plant in "sense orientation" with respect to a promoter for its expression, which as used here refers to the same orientation as transcription and translation (if it occurs) of the sequence in regarding the sequence in the target gene. The size of the sense fragment, its correspondence to the target gene regions, and its degree of homology to the target gene are as for the antisense sequences as described above. At times, the additional copy of the gene sequence interferes with the expression of the target plant gene. Reference is made to the WO 97/20936 specification and European patent specification 0465572 for methods of implementing co-suppression approaches. The antisense, co-suppressed or double-stranded RNA molecules can also comprise a wide double-stranded RNA region, preferably comprising a nuclear localization signal, as described in WO 03/076619.
Any of these technologies to reduce gene expression can be used to coordinately reduce the activity of multiple genes. For example, an RNA molecule can be directed against a family of related genes to target a region of genes that is in common.
Alternatively, unrelated genes can be targeted by including multiple regions on an RNA molecule, each region targeting a different gene. This can easily be done by merging multiple regions under the control of a single promoter.
Various techniques are available for introducing nucleic acid molecules into a wheat cell, well known to those skilled in the art. The term "transformation" as used here means changing the genotype of a cell, for example, a bacterium or a plant, particularly a wheat plant, by the introduction of an exogenous or external nucleic acid. “Transformer” means an organism thus altered. Introduction of DNA into a wheat plant by crossing parent plants or by mutagenesis per se is not included in the transformation. As used here, the term "transgenic" refers to a genetically modified plant in which the endogenous genome is supplemented or modified by random or site-directed integration, or stable maintenance in an unintegrated, replicable form, of an exogenous gene or sequence or external key introduced. By "transgene" is meant an external or exogenous gene or sequence that is introduced into a plant. The nucleic acid molecule can be replicated as an extrachromosomal element or is preferably stably integrated into the plant's genome. “Genome” means the total genetic complement inherited from the cell, plant or part of the plant, and includes molecules of chromosomal DNA, plasmid DNA, mitochondrial DNA and extrachromosomal DNA. In one embodiment, a transgene is integrated into the nuclear genome of wheat which in hexaploid wheat includes subgenomes A, B and D, here referred to as “genomes” A, B and D.
The methods most commonly used to produce transgenic, fertile wheat plants comprise two stages: the distribution of DNA in regenerable wheat cells and plant regeneration through an in vitro tissue culture. Two methods are commonly used to deliver DNA: transfer of T-DNA using Agrobacterium tumefaciens or related bacteria and direct introduction of DNA through particle bombardment, although other methods have been used to integrate DNA sequences into wheat or other cereals . It will be apparent to the person skilled in the art that the particular choice of a transformation system to introduce a nucleic acid construct into plant cells is not essential to or a limitation of the invention, as long as it achieves an acceptable level of nucleic acid transfer. Such techniques for wheat are well known in the art.
The transformed wheat plants can be produced by introducing a nucleic acid construct according to the invention into a recipient cell and cultivating a new plant that comprises and expresses a polynucleotide according to the invention. The process of growing a new plant from a transformed cell that is in cell culture is referred to here as "regeneration". Regenerable wheat cells include cells from mature germs, meristematic tissue such as mesophilic cells from the base of the leaf, or preferably from the scutella of immature germs, obtained 12 to 20 days post-anthesis, or calluses derived from any of them. The most commonly used pathway to recover regenerated wheat plants is somatic embryogenesis using means such as MS-agar supplemented with an auxin such as 2,4-D and a low level of cytokinin, (see Sparks and Jones, 2004).
The transformation of wheat mediated by Agrobacterium can be carried out by the methods of Cheng et al, 1997; Weir et al., 2001; Kanna and Daggard, 2003 or Wu et al, 2003. Any strain of Agrobacterium with sufficient virulence can be used, preferably strains that have additional virulence such as LBA4404, AGL0 or AGL1 (Lazo et al, 1991) or versions of C58. Agrobacterium-related bacteria can also be used. The DNA that is transferred (T-DNA) from Agrobacterium to the recipient wheat cells is comprised of a genetic construct (chimeric plasmid) that contains one or two boundary regions of a T-DNA region of a flanking wild-type Ti plasmid the nucleic acid to be transferred. The genetic construct can contain two or more T-DNAs, for example, where one T-DNA contains the gene of interest and a second T-DNA contains a selectable marker gene, providing independent insertion of the two T-DNAs and possible segregation of the gene selectable marker away from the transgene of interest.
Any type of wheat that is regenerable can be used; Bob White, Fielder, Veery-5, Cadenza and Florida varieties have been reported successfully. Transformation events into one of these more easily regenerable varieties can be transferred to any other wheat cultivars including elite varieties by standard backcrossing. An alternative method using Agrobacterium makes use of an in vivo inoculation method followed by the regeneration and selection of transformed plants using tissue culture and has proved to be efficient, see WO00 / 63398. Other methods involving the use of Agrobacterium include: co-cultivation of Agrobacterium with cultured isolated protoplasts; transformation of seeds, apexes or meristems with Agrobacterium, or plant inoculation such as the floral immersion method for Arabidopsis as described by Bechtold et al., 1993. The latter approach is based on the vacuum infiltration of a suspension of Agrobacterium cells. Alternatively, the chimeric construct can be introduced using Agrobacterium root (Ri) induction plasmids as vectors.
Another commonly used method for introducing the nucleic acid construct into a plant cell is high-speed ballistic penetration by small particles (also known as particle bombardment or microprojectile bombardment) with the nucleic acid to be introduced contained within the matrix of beads. or small particles, or on its surface, as, for example, described by Klein et al, 1987. This method was adapted for wheat (Vasil, 1990). Microprojectile bombardment to induce injury followed by co-culture with Agrobacterium can be used (EP-A-486233). The genetic construct can also be introduced into plant cells by electroporation as, for example, described by Fromm et al, 1985 and Shimamoto et al, 1989. Alternatively, the nucleic acid construct can be introduced into a wheat cell such as a cell pollen by contact of the cell with the nucleic acid using mechanical or chemical means.
Preferred selectable marker genes for use in wheat processing include the Streptomyces hygroscopicus bar gene or pat gene in conjunction with selection using the herbicide glyphosinate ammonium, the hpt gene in conjunction with the antibiotic hygromycin, or the nptII gene with kanamycin or G418. Alternatively, positively selectable markers such as the manA gene encoding phosphomanosis isomerase (PMI) with sugar mannose-6-phosphate since the single C source can be used. The present invention is further described by the following non-limiting Examples.
EXAMPLE 1: METHODS AND MATERIALS Determination and analysis of carbohydrates. Starch was isolated on a small scale from the grain of developing or ripe wheat using the method of Regina et al, (2006). The extraction of starch on a large scale was carried out following the method of Regina et al, (2004). The starch content was determined using the total starch analysis kit provided by Megazyme (Bray, Co Wicklow, Republic of Ireland) and calculated on a weight basis as a percentage of the weight of the mature, unground grain. The starch content was then compared to control the plants. Subtracting the weight of the starch from the total weight of the grain to give a total non-starch content of the grain determined whether the reduction in the total weight was due to a reduction in the starch content.
The amylose content of starch samples was determined by the colorimetric (iodometric) method of Morrison and Laignelet (1983) with slight modifications as follows. Approximately 2 mg of starch was weighed precisely (to the nearest 0.1 mg) in a 2 ml screw cap tube equipped with a rubber ring on the cap. To remove the lipid, 1 ml of 85% (v / v) methanol was mixed with the starch and the tube heated in a 65 ° C water bath for 1 hour with an occasional vortex. After centrifugation at 13,000g for 5 min, the supernatant was carefully removed and the extraction steps repeated. The starch was then dried at 65 ° C for 1 hour and dissolved in a solution of urea-dimethyl sulfoxide (UDMSO; 9 volumes of dimethyl sulfoxide for 1 volume of 6M urea), using 1 ml of UDMSO per 2 mg of starch ( heavy as above). The mixture was immediately stirred vigorously and incubated in a 95 ° C water bath for 1 hour with an intermittent vortex to completely dissolve the starch. An aliquot of the UDMSO starch solution (50 μl) was treated with 20 μl of I2-KI reagent that contained 2 mg of iodine and 20 mg of potassium iodine per ml of water. A mixture was made up to 1 ml with water. The absorbance of the mixture at 620 nm was measured by transferring 200 μl to microplates and reading the absorbance using an Emax Precision Microplate Reader (Molecular Devices, USA). Standard samples containing 0 to 100% amylose and 100% to 0% amylopectin were made from potato amylose and corn (or potato) amylopectin (Sigma) and treated as for the test samples. The amylose content (percentage of amylose) was determined from the absorbance values using a regression equation derived from the absorbances for the standard samples. The analysis of the amylose / amylopectin ratio of non-branched starches can also be performed according to Case et al, (1998) or by an HPLC method using 90% DMSO to separate the branched starches as described by Batey and Curtin, ( 1996).
Statistical analysis of amylose data was performed using the 8th edition of Genstat by Windows (VSN International Ltd, Herts, UK).
The distribution of chain lengths in starch was analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) using a capillary electrophoresis unit according to Morell et al, (1998) after debranching the starch samples. The gelatinization temperature profiles of the starch samples were measured on a Pyris 1 differential scanning calorimeter (Perkin Elmer, Norwalk CT, USA). The viscosity of the starch solutions was measured on a Rapid Viscosity Analyzer (RVA, Newport Scientific Pty Ltd, Warriewood, Sydney), for example, using conditions as reported by Batey et al, (1997). The measured parameters included peak viscosity (the maximum viscosity of hot paste), holding strength, final viscosity and bonding temperature. The swelling volume of the flour or starch was determined according to a method by Konik-Rose et al, (2001). Water absorption was measured by weighing the sample before and after mixing the flour or starch sample in the water at defined temperatures and following the collection of the gelatinized material.
The morphology of the starch granule was analyzed by microscopy. Purified starch granule suspensions in water were examined under normal and polarized light using a Leica-DMR compound microscope to determine the starch granule morphology. Scanning electron microscopy was performed using a Joel JSM 35C instrument. The purified starches were coated with gold spray and digitalized at 15kV at room temperature.
Β-Glycan levels were determined using the kit provided by Megazyme (Bray, Co, Wicklow, Republic of Ireland).
Analysis of protein expression in the endosperm. The specific expression of the SBEI, SBEIIa and SBEIIb proteins in the endosperm, in particular the level of expression or accumulation of these proteins, was analyzed by Western blot procedures. The endosperm was dissected away from all maternal tissues and samples of approximately 0.2 mg were homogenized in 600 μl of 50 mM Kphosphate buffer (42 mM K2HP0O4 and 8 mM KH2PO4), pH 7.5, containing EDTA at 5 mM, 20% glycerol, 5 mM DTT and 1 mM Pefabloc. The ground samples were centrifuged for 10 min at 13,000g and the supernatant was aliquoted and frozen at -80 ° C until use. For total protein estimation, a standard BSA curve was established using 0, 20, 40, 60, 80 and 100 μl of 0.25 mg / ml aliquots of the standard BSA. Samples (3 μl) were made up to 100 μl with distilled water and 1 ml of Coomassie Plus Protein reagent was added to each. The absorbance was read after 5 min at 595 nm, using the BSA zero sample from the standard curve as a void, and the protein levels in the determined samples. Samples containing 20 μg of total protein from each endosperm were run on 8% non-denaturing polyacrylamide gel containing 0.34 M Tris-HCl (pH 8.8), acrylamide (8.0%), ammonium persulfate ( 0.06%) and TEMED (0.1%). Following electrophoresis, the proteins were transferred to a nitrocellulose membrane according to Morell et al, 1997 and immunoreacted with specific antibodies from SBEIIa, SBEIIb or SBEI. An antiserum against wheat SBEIIa protein (anti-wBEIIa) was generated using a synthetic peptide having the amino acid sequence of the N-terminal sequence of mature wheat SBEIIa, AASPGKVLVPDGESDDL (SEQ ID NO: 16) (Rahman et al, 2001) . An antiserum against wheat SBEIIb (anti-wBEIIb) was generated in an analogous manner using the synthetic N-terminal peptide, AGGPSGEVMI (SEQ ID NO: 17) (Regina et al, (2005). This peptide was considered to represent the N- terminal of the mature SBEIIb peptide and in addition was identical to the N-terminus of the SBEIIb barley protein (Sun et al, 1998). A polyclonal antibody against wheat SBEI was synthesized in an analogous manner using the synthetic N-terminal peptide VSAPRDYTMATAEDGV (SEQ ID NO: 18) (Morell et al., 1997). Such antisera were obtained from rabbits immunized with synthetic peptides according to standard methods.
Enzymatic assay for SBE. The enzymatic activity assays of branching enzymes to detect the activity of all three isoforms, SBEI, SBEIIa and SBEIIb were based on the method of Nishi et al, 2001 with less modification. After electrophoresis, the gel was washed twice in 50 mM HEPES, pH 7.0 containing 10% glycerol and incubated at room temperature in a reaction mixture consisting of 50 mM HEPES, pH 7.4, glucose-1- phosphate at 50 mM, AMP at 2.5 mM, 10% glycerol, 50 U of phosphorylase 1 DTT at 1 mM and 0.08% maltotriose for 16 h. The bands were visualized with a solution of 0.2% (P / V) of I2 and 2% of KI. The specific activities of the SBEI, SBEIIa and SBEIIb isoforms were separated under these electrophoresis conditions. This was confirmed by immunoblotting using anti-SBEI, anti-SBEIIa and anti-SBEIIb antibodies. Densitometric analysis of immunoblots using TotalLab software package (Nonlinear Dynamics Ltd, Newcastle, UK) that measures the intensity of each band was conducted to determine the level of enzyme activity for each isoform.
The enzymatic activity of starch branching (SBE) can be measured by the enzymatic assay, for example, by the phosphorylase stimulation assay (Boyer and Preiss, 1978). This assay measures SBE stimulation of glucose-1-phosphate incorporation into methanol-insoluble polymer (α-D-glycan) by phosphorylase A. The activity of SBE-specific isoforms can be measured by this assay following the purification of individual isoforms as described in Regina et al., 2004. The total soluble protein extracts were applied to a 3 ml β-cyclodextrin (β-CD) affinity column pre-equilibrated with the extraction buffer described above. The column was prepared by coupling β-CD to Sepharose activated by Epoxy 6B (Amersham Biosciences, Uppsala, Sweden) following the manufacturer's instructions. Bound proteins (containing SBEs) were eluted using 1% β-CD in the Phosphate buffer and then dialyzed against buffer A (20 mM phosphate buffer, pH 8.0, 1 mM EDTA and 1 mM DTT). The dialyzed samples were subjected to anion exchange chromatography using a 1 ml MonoQ column (Amersham Pharmacia), pre-equilibrated with buffer A. After eluting the unbound proteins, a linear gradient of 30 min was applied by introducing buffer B (500 mM Phosphate buffer, pH 8.0, 1 mM EDTA, 1 mM DTT) in buffer A to elute bound proteins.
SBE activity can also be measured by the iodine stain assay, which measures the decrease in the absorbance of a glycan-poly-iodine complex resulting from the branching of glycan polymers. SBE activity can also be tested by the branch-binding assay that measures the generation of reduced amylose reducing ends as a substrate, following isoamylase digestion (Takeda et al, 1993a). Preferably, activity is measured in the absence of SBEI activity. The SBE isoforms show different substrate specificities, for example, SBEI exhibits greater activity in branching amylose, while SBEIIa and SBEIIb show higher branching rates with an amylopectin substrate. Isoforms can also be distinguished based on the length of the glycan chain that is transferred. SBE protein can also be measured by using specific antibodies such as those described here. Preferably, SBEII activity is measured during grain development in the developing endosperm. The protein levels of SBEII are preferably measured in the mature grain where the protein is still present through immunological methods such as Western blot analysis.
DNA analysis of wheat plants. PCR analysis of transformed wheat plants or plants to be tested for the presence of transgenes was performed on genomic DNA extracted from 1-2 cm2 of fresh leaf material using the mini-prep method described by Stacey and Isaac, (1994 ). PCR assays to determine the presence of hairpin RNA constructs used the primers SBEIIa-Por: 5 '-CCCGCTGCTTTCGCTCATTTTG-3' (SEQ ID NO: 19) and SBEIIa-Rev: 5'-GACTACCGGAGCTCCCACCTTC-3 '(SEQ ID NO : 20) designed to amplify a fragment (462bp) of the SBEIIa gene. The reaction conditions were as follows: “hot start” (94 ° C, 3 min) followed by 30 cycles of denaturation (95 ° C, 30 sec), annealing (55 ° C, 30 sec), extension (73 ° C , 2 min) followed by cycling at 73 ° C (5 min). The reaction products were analyzed by agarose or polyacrylamide gel electrophoresis.
The Southern blot hybridization analysis was performed on DNA from a larger scale extraction (9 ml) from the lyophilized ground tissue (Stacey and Isaac, 1994). The DNA samples were adjusted to 0.2 mg / ml and digested with restriction enzymes such as Hindlll, EcoRl and Kpnl. The restriction enzyme digestion, gel electrophoresis and empty blotting is performed as described by Stacey and Isaac, (1994). The probes labeled digoxigenin including region 3 of the intron of the ds-SBEII constructs are produced by PCR according to a method by McCreery and Helentjaris, (1994). Hybridization of the probes for Southern blot and detection by chemiluminescence are performed according to a method by McCreery and Helentjaris, (1994).
Transformation of wheat by Agrobactaerium. The genetic constructs for transformation of wheat were introduced by electroporation into the LBA4404 strain of Agrobacterium tumefaciens disarmed carrying the plasmid vir pAL4404 and pSBl, with subsequent selection in the media with spectinomycin. The transformed Agrobacterium strains were incubated in solidified YEP media at 27 ° C for 2 days. The bacteria were then collected and resuspended in TSIM1 (MS media with 100 mg / 1 myo-inositol, 10 g / 1 glucose, 50 mg / 1 MES buffer at pH 5.5) containing 400 mM acetosyringone for one optical density from 2.4 to 650 nm for inoculation of wheat.
The wheat plants (variety NB1, a variety of Spring wheat obtained from Nickerson Seeds Ltd, Rothwell, Strains.) Were grown in a greenhouse at 22/15 ° C day / night temperature with supplemented light to give a 16-hour day. . Tillers were harvested approximately 14 days post-anthesis (germs approximately 1 mm long) to include 50 cm of stem of the tiller. All leaves were then removed from the tillers except the flag leaf, which was cleaned to remove contaminating fungal spores. The glumella of each spikelet and the motto of the first two florets were then carefully removed to expose the immature seed. Generally, only these two seeds in each spikelet have been discovered. This procedure was carried out along the total length of the inflorescence. The ears were then sprayed with 70% IMS as a brief surface sterilization.
Agrobacterium suspensions (1 μl) were inoculated using a 10 μl Hamilton syringe into the immature seed at approximately the position of the scutellum: endosperm interface so that all exposed seeds were inoculated. The tillers were then placed in the water, covered with a translucent plastic bag to prevent dehydration of the seed, and placed in an illuminated incubator for 3 days at 23 ° C, 16 hours a day, 45 μEM-2s-1 PAR. After 3 days of co-cultivation, the immature inoculated seed was removed and the surface sterilized with 70% ethanol (30 sec), then 20% bleach (Domestos, 20 min), followed by thorough washing in sterile distilled water. Immature germs were isolated aseptically and placed in W3 media (MS supplemented with 20 g / l of sucrose and 2 mg / l of 2,4-D and solidified with 6 g / l of Type I agarose, Sigma) with the addition of 150mg / l of Timentin (medium W3T) and with the highest scutellum (20 germs per plate). The cultures were placed at 25 ° C in the light (16 hours a day, 80 μEM-2s-1PAR). The development of the embryonic axis in germs was evaluated about 5 days after isolation and the axis was removed where necessary to improve callus production. The germs were kept in W3T for 4 weeks, with a transfer to fresh media in 2 weeks post-isolation and evaluated for embryogenic capacity.
After 4 weeks of growth, the callus derived from the inoculated germs was very similar to the control of the callus obtained from the non-inoculated germs placed on a plate in the W3T medium. The presence of the bacteria did not appear to have a substantially reduced embryogenic capacity of the callus derived from inoculated germs. The embryogenic calluses were transferred to W3 media with 2 mg / 1 Asulam or geneticin at 25 mg / l and 150 mg / l of Timentin (W32AT medium). The calluses were kept in these media for a further 2 weeks and then each callus was divided into 2 mm pieces and replaced on the W32AT plates. The control germs derived from inoculations with LBA4404 without binary vector constructs did not produce callus transformed in the selection media.
After another 2 weeks of culture, all tissue was evaluated for the development of embryogenic callus: any callus showing signs of continuous development after 4 weeks in the selection was transferred to the regeneration media (RMT - MS with 40 g / l of maltose and 150 mg / l Timentin, pH 5.8, solidified with 6 g / l agarose, Sigma type 1). The shoots were regenerated within 4 weeks in these media and then transferred to MS30 with 150 mg / l of Timentin for elongation and rooting of the shoot. The juvenile plants were then transferred to the soil mixture and kept in a mixing bank for two weeks and finally transferred to a greenhouse.
Alternative strains of Agrobacterium such as strain AGL1 or selectable markers such as genes encoding hygromycin resistance can also be used in the method.
EXAMPLE 2: INHIBITION OF SBEIIA GENES IN WHEAT USING FOUR HAIRPIN RNA CONSTRUCTS Four constructs of RNA hairpin (dsRNA) were made to reduce the expression of i) to SBEIIa, or ii) genes of SBEIIa, SBEIIb and SBEI of wheat. In each construct, the DNA encoding the hairpin RNA was linked to a high molecular weight promoter sequence glutenin (HMWG) obtained from a Dx5 wheat gene to provide the specific expression of the hairpin RNA endosperm, and a transcription terminator sequence. of the nopaline synthase gene from Agrobacterium (nos3 '). This promoter provided for specific expression of the endosperm of the synthetic genes that encode the hairpin RNAs. hp5'-SBEIIa. The construction and use of the first of the constructs, designed as hp5'-SBEIIa, is described in Regina et al, (2006). The hp5'-SBEIIa construct contained 1536 bp of the PCR amplified nucleotide sequence of the SBEIIa wheat gene (GenBank accession number AF338431). This included a 468 bp sequence comprising set of exons 1 and 2 and part of exon 3 (nucleotide positions 1058 to 1336, 1664 to 1761 and 2038 to 2219 (which includes nucleotide positions 1 to 578 of Aegilops tauschii cDNA encoding SBEIIa, GenBank accession number AF338431.1) with EcoKl and Kpnl restriction sites on either side (fragment 1), a 512 bp sequence consisting of part of exons 3 and 4 and the intron 3 set of SBEIIa ( nucleotide positions 2220 to 2731) with Kpnl and SacI sites on either side (fragment 2) and a 528bp fragment consisting of the complete exons 1, 2 and 3 of SBEIIa (nucleotide positions 1058 to 1336, 1664 to 1761 and 2038 to 2279 in AF338431, which includes nucleotide positions 1 to 638 of SBEIIa Aegilops tauschii cDNA, GenBank accession number AF338431.1) with BamHI and SacI sites on either side (fragment 3). so that the sequence of fragment 3 was linked to fragment 2 in the orientation antisense in relation to fragment 1. The RNA constructs hairpin were initially generated in the vector pDVO3000 which contains the HMWG promoter sequence and terminator nos3 '. hpc-SBEIIa. The hpc-SBEIIa designed SBEIIa construct comprised a 293 base pair DNA fragment corresponding to nucleotides 1255 to 1547 of the SBEIIa cDNA (Genbank Accession Number AF338432.1), which corresponds to the part of exon 12, exons 13 and 14 and part of exon 15 of the SBEIIa gene. This SBEIIa region was chosen because it had only about 81% identity for the nucleotide sequence of the corresponding SBEIIb cDNA region, thus increasing the chance of specific SBEIIa silencing, but not SBEIIb. hp3'-SBEIIa. The projected SBEIIa hp3 '- SBEIIa construct comprises a 130-base pair DNA fragment corresponding to nucleotides 2305 to 2434 of the SBEIIa cDNA, which corresponds to part of exon 21, exon 22 and part of the 3' untranslated region (3 'UTR) of the SBEIIa gene. hp-combo. The hp-combo designed RNA hairpin construct comprises the regions of the SBEI wheat gene, in addition to parts of the SBEIIa gene, and contained i) a 417 base pair sequence corresponding to nucleotides 1756 to 2172 of the SBEIIa cDNA, corresponding to the part of exon 16, exons 17 to 19, and part of exon 20, and ii) a sequence of 357 base pairs corresponding to nucleotides 267 to 623 of an SBEI cDNA (Genbank Accession Number AF076679), corresponding to part of exon 3, exon 4, and part of exon 5 of the SBEI gene. The SBEIIa gene fragment had about 86% identity to the corresponding region of the SBEIIb gene, including several regions of 23 consecutive nucleotides with 100% identity to its corresponding SBEIIb regions, and therefore the combination construct was designed with the expectation that it would reduce the expression of genes encoding SBEIIb as well as genes encoding SBEIIa and SBEI in wheat.
Two copies of each of the fragments described above were inserted, one in the sense orientation and the other in the antisense, in a suitable vector, so that a rice tubulin gene intron was present between the two copies. The synthetic gene was inserted into a binary vector and used to transform wheat.
These constructs were used to transform the wheat as described in Example 1. The numbers of independent transgenic wheat strains that were PCR positive for the respective constructs were as follows: hp5 * -SBEIIa, 27; hpc-SBEIIa, 10; hp3'-SBEIIa, 10; and hp-combo, 63.
Analysis of transgenic plants: DNA analysis. PCR analysis was performed to detect one or more of the transgenes in the regenerated plants using genomic DNA extracted from 1-2 cm2 of fresh leaf material using the mini-prep method described by Stacey and Isaac, (1994). PCR reactions were performed for plants transformed with the hp5'-SBEIIa transgene, for example, using the SBEIIa-Por: 5'-CCCGCTGCTTTCGCTCATTTTG-3 '(SEQ ID NO: 19) and SBEIIa-Rev: 5'- GACTACCGGAGCTCCCACCTTC-3 '(SEQ ID NO: 20). These PCR reactions were designed to amplify an approximately 462bp fragment of the SBEIIa gene. The reaction conditions were as follows: “hot start” (94 ° C, 3 min) followed by 30 cycles of denaturation (95 ° C, 30 sec), annealing (55 ° C, 30 sec) and extension (73 ° C, 2 min), followed by 1 cycle at 73 ° C (5 min).
Morphology of the starch granule. The morphology of the mature T1 seed starch granules obtained from the transformed T0 wheat plants was observed by light microscopy. Ten individual grains from each of 25 T0 hp5'-SBEIIa plants were analyzed. Each endosperm was gently crushed to release the starch granules that were dispersed in water and visualized under a light microscope. Of the 25 strains analyzed, 12 had grains with distorted granules, although visual observation revealed varying levels of distortion in different seeds. Nine seeds from each of the plants transformed with the hpc-SBEIIa, hp3'-SBEIIa and hp-combo transgenes were similarly analyzed for morphological changes in the starch granules. In this case, half of the seeds were analyzed so that each remaining half of the seed could be grown on a T1 plant, thus preserving each strain. Fifty-five out of the 63 hp-combo strains had seeds with altered granule morphology with varying levels of distortion. All ten strains of hp5'-SBEIIa had seeds with altered starch granule morphology, again with varying levels of distortion. No significant changes in starch granule morphology were observed in any of the 3 'SBEIIa strains. Distorted starch granules are an indicator of high amylose levels in endosperm starch, typically above 50% amylose, or above 70% amylose for highly distorted starch granules. This indicated that a range in the extent of the phenotype was observed for each of the effective silencing constructs.
Protein expression by Western blotting in the developing endosperm. Four to seven endosperm in T2 development of transgenic T1 lines were analyzed for the protein level of SBEIIa and SBEIIb by Western blotting using anti-SBEIIa and anti-SBEIIb antibodies, respectively. In the case of hp-combo strains, SBEI expression was also analyzed using anti-SBEI antibody. The total protein levels of SBEII (SBEIIa and SBEIIb) of selected transgenic strains were calculated as a percentage of the level in the wild type (variety NB1) and are shown in Table 11. The levels of amylose in the mature grain of the transgenic strains, calculated as a percentage of the total starch in the grain, were also determined (Table 11) using an iodometric method as described in Example 1. This is plotted in Figure 5.
A range of expression levels of SBEIIa and SBEIIb was obtained in the grain of transgenic plants of independent strains. Such a range is normally expected in transgenic lines obtained with any one construct, due to the variation in the locations of integration of the transgene in different transgenic events, commonly referred to as “position effect”. The range of expression levels seen in these experiments was extended because it was observed that the four constructs were not equally efficient in reducing the expression of the SBEIIa and SBEIIb genes. In particular, the extent of the reduction in SBEIIb expression caused by the hp-combo construct in some transformed strains did not correlate with the extent of the reduction in SBEIIa expression, for example, lines 679.5.3 and 672.2.3. However, all constructs reduced the expression of the corresponding genes in a majority of transformed strains.
When the percentage of amylose was plotted against the SBEII total protein level and a best fit curve generated from the data points (see Figure 5), it was observed that the reduction in total SBEII to at least 75% in relation to the wild type yield an amylose content of 50% (w / w) or more in the endosperm starch. The reduction of the total SBEII activity to at least 40% in relation to the wild type tended to have an amylose content of at least 40% (w / w).
When the percentage of amylose was plotted against the remaining SBEIIa protein level, a very similar curve was obtained (see Figure 6), leading to the conclusion that the SBEIIa level in the wheat endosperm was the primary determinant of the amylose level in the starch, and that SBEIIb and SBEI levels were secondary determinants.
The amylose model was further developed based on three input sets (Figure 6): (1) theoretical data based on the relative expression levels of SBEIIa and SBEIIb and amylose data from transgenics (2) amylose data for simple and double-null and theoretical data based on the relative expression levels of SBEIIa and SBEIIb (3) measured amylose data and SBEIIa and SBEIIb levels measured from the “additional construct” transgenics
In Figure 6, a power curve was adjusted for these data. Bringing these three sets of data together generated a model that was highly consistent across input types, reinforcing the model as a predictive tool. The predicted model of the importance of generating multiple mutations in the SBEII genes in order to generate high amylose content in common wheat or tetraploid wheat.
EXAMPLE 3: CLONING AND SEARCH COMPARISON OF SEA WHEEL GENES Isolation of SBEII genes from an Aegilops tauschii genomic library and their PCR characterization are described in WO99 / 14314 and WO200162934-A. The DNA sequences of the intron 5 region of the SBEIIa gene of genomes A, B and D are described in WO200162934-A. Another research led to obtaining sequences from other regions of the wheat SBEIIa genes of different wheat genotypes and also characterization of homologous genes, for example, as follows. The exe region 12 to 14 of SBEIIa was amplified from the Chara hexaploid wheat variety using the AR2aE12F07 (5'-CATTCGTCAAATAATACCCTTGACGG-3 '(SEQ ID NO: 21)) and AR2aE14R07 (5'-CTTCACCAATGGATACAGCATCAGCATCAGCATCAGCATCAGCATCAGCATCAGCATCAGC NO: 22)). This yielded a PCR product of about 656bp that was presumed to be a mixture of the amplified fragments from each of the three homologous genes. This product was sequenced according to cloning in a TOPO vector. Three polymorphic sequences were obtained which covered the region between exon 12 to 14 (Figure 7). Based on the PCR analysis of the projected Chinese Spring chromosome strains using amplified polymorphic marker (CAP) cleavage, sequence F1-1 was assigned to genome D, sequence F1-13 was assigned to genome B and sequence F1-15 was assigned to genome A as detailed in Example 4.
The intrinsic 3 region of SBEIIa was amplified from two varieties of hexaploid wheat, Sunco and Tasman, using the starter pair AR2akpnIF (5'- GGTACCGGCAAATATACGAG ATTGACCCG-3 '(SEQ ID NO: 23)) and AR2aSacIR (5'- GAGCTCCCACCTTCATGTT GGTCAATAGC-3 '(SEQ ID NO: 24)). Three polymorphic sequences were obtained from each of Sunco and Tasman (Figure 8). By comparison with the wheat SBEIIa D genome sequence (Genbank Accession Number AF338431.1), Tasman 0257 and Sunco 0242 sequences were assigned to genome D. Tasman 0272 and Sunco 0241 sequences were assigned to genome B based on in polymorphic marker mapping based on a simple nucleotide polymorphism in a segregation population. The strings
Tasman 0264 and Sunco 0243 appeared to be different from the sequences of genome B and D and it was concluded that they must be from genome A. Genotype specific polymorphisms were also observed for this region of SBEIIa between Sunco and Tasman in each of the three genomes.
The Chinese Spring (CS) SBEIIa exon 3 region was amplified using the AR2aexon3F (5'- GATACCTGAAGATATCGAGGAGC-3 '(SEQ ID NO: 25)) and AR2aexon3R (5' -CGGTAGTCAAGATGGCTCCG-3 '(SEQ ID NO:: 26)). The three polymorphic sequences were obtained (Figure 9). The comparison with the wheat SBEIIa gene (Genbank Accession Number AF338431.1) revealed that the CS sequence of exon 3a was of genome D. It was found that the CS sequence of exon 3b is of genome B based on the identity of 100% with Genbank Accession Number FM865435 which has been reported to be a common wheat chromosome 2B. The third CS sequence of exon 3d showed 99% identity with Genbank Access Number Y11282.1, which, in turn, had a high degree of identity (99%) with a reported partial coding sequence of the A genome. Chinese Spring (Genbank Accession Number EU670724). This led to the prediction that the CS sequence of exon 3d was of genome A.
The CSEIA exon 1 region of the CS was amplified using the primers AR2aexon1F (5'-CACACGTTGCTCCCCCTTCTC-3 '(SEQ ID NO: 29)) and AR2aexon1R (S'-GAGAGGAGTCCTTCTTCCTGAGG-3' (SEQ ID NO: 28). The sequences were obtained (Figure 10). The alignment with SBEII of the accesses to GenBank led to the assignment of the CS sequence of exon 1a for genome B (100% homology to FM865435), exon 1b of CS for genome A (99% homology for Y11282.1) and exon 1c CS for the D genome (100% homology for AF338431.1).
The SBEIIa gene sequences were also obtained from diploid or common wheat parents, Triticum urartu who is believed to be the parent of the common wheat genome A, Aegilops speltoides (also known as Triticum speltoides) who is believed to be the parent genome B, and Aegilops tauschii believed to be closely related to the genome D parent. The gene fragments were obtained from these species below: ten primers were designed based on the nucleotide sequence of the D genome SBEIIa gene. (Accession Number AF338432) or its complement and covering all that sequence. These primer sets were used to amplify the fragments of the SBEIIa genes of the diploid species by PCR. Using the 10 primers, 16 combinations were used in PCRs with DNA of the diploid species T. urartu (genome AA), A. speltoides (BB), A. tauschii (DD) and the tetraploid species T. durum (genome AABB). In total, 35 fragments were selected from these amplifications that were of sufficient quality for sequencing, to determine their nucleotide sequences. The sequences will be compared and edited using Contig Express and combined sequences determined for the diploid SBEIIa progenitor genes. Polymorphisms such as SNPs or insertions / deletions will be identified, which can be used to distinguish genes in genomes A, B and D, and specific primers designed using Amplifier to identify mutants.
The nucleotide sequence of exon 11 to 22 of the T. urartu SBEIIa gene is shown in SEQ ID NO: 13, of exons 3 to 8 as SEQ ID NO: 15 and of exons 1 to 3 as SEQ ID NO: 14. The nucleotide sequence of the A. tauschii total SBEIIa gene is provided in WO2005001098 (incorporated herein by reference).
Mapping of genetic linkage of SBEIIa and SBEIIb of SBEIIa and SBEIIb in wheat. The SBEIIa and SBEIIb genes were located on the long arm of wheat chromosome 2 (Regina et al., 2005; Rahman et al., 2001) and based on these reports were considered linked, although it was not exactly known how close the Link. The genetic mapping of the SBEIIa and SBEIIb genes was performed using a population of segregation obtained from a 4-way crossover involving the parent cultivars Baxter, Yitpi, Chara and Westonia. Population analysis for recombinants between genes revealed only one recombinant out of approximately 900 progenies. From these data, it was calculated that the genetic distance between SBEIIa and SBEIIb was only 0.5 cM, which was a very tight link between the two genes.
To determine the physical distance between the two genes, a BAC library of Aegilops tauschii built by Moullet et al, (1999) was selected to identify SBEII containing clones. Hybridization probes labeled with 32P were prepared from the 5 'and 3' regions of each of the SBEIIa and SBEIIb genes and used to examine the BAC library. When examined with a mixture of the four probes, nine clones were identified with positive hybridization signals. The nine clones were then examined separately with each of the probes and three selected clones. One (BAC2) was fully sequenced and shown to contain a full length of the SBEIIb gene. Of the other clones, BAC1 was shown to contain an SBEIIa gene by direct partial sequencing and BAC3 appeared to contain portions of both SBEIIa and SBEIIb genes as shown by PCR. This indicated how closely the two genes are physically linked. BAC1 and BAC3 will be completely sequenced. These physical data confirmed the close genetic link.
Therefore, it was predicted that exclusion mutations created by agents, such as radiation that affected one of the genes, were likely to extend within or through the genes, that is, to be null for both genes. In addition, this suggested the possibility that such exclusion mutants may be viable and have wild type aptitude. At least, the tight binding observed increased the possibility of obtaining mutants with relatively small exclusions that did not extend to other linked genes necessary for viability or fitness. Such mutants were therefore sought after as described below in Examples 5 to 7.
EXAMPLE 4: DISTINGUISHING THE WHEEL GENES OF SBEIIA AND SBEIIB IN THE WHEAT Based on the sequence polymorphisms obtained in Example 2, the PCR assays were designed and prepared to distinguish homologous SBEIIa genes in common wheat. A nested primary pair, AR2aI13genomaF2 (5'-GTAC AATTTTACCTGATGAGATCATGG-3 '(SEQ ID NO: 29)) and AR2aI13genomaR2 (5'-CTTCAGGAATGGATACAGCATCAG-3' (SEQ ID NO: 30)) was designed to amplify a bp of the region between exons 12 to 14 of SBEIIa of wheat. When digested with two restriction enzymes, Ssp1 and Mse1, products amplified using these Chinese Spring (CS) primers yielded four clear bands of sizes 207 bp, 147 bp, 99 bp and 108 bp. The use of this PCR marker assay in the projected strains of the CS chromosome revealed that the 207 bp product came from genome A, the 147 bp product came from genome B and the 99 bp and 108 bp products came from genome D (Figure 11).
Based on the SBEIIa sequences of the diploid wheat ancestors named Triticum urartu for genome A, Aegilops speltoides for genome B and Aegilops tauschii for genome D, the primer pairs were designed that could specifically amplify fragments from different regions of the SBEIIa genes of different genomes and distinguish them (Tables 4 to 8). Tables 6 to 8 list some of the nucleotide polymorphisms (column labeled SNP) and the sizes of the amplified fragments obtained when the designed pairs of primers are used. These same combinations of primers can be used to distinguish SBEIIa homologous genes from genomes A and B of durum wheat.
The development of some sets of PCR primers distinguishing SBEIIb homologous genes from genomes A, B and D of common wheat and the identification of SBEIIb in each of these genomes in hexaploid wheat are described in WO200162934-A. Based on the SBEIIb sequences of the diploid wheat ancestors named Triticum urartu for genome A, Aegilops speltoides for genome B and Aegilops tauschii for genome D, the pairs of primers that could specifically amplify each of the three genomes of different regions of SBEIIb were designed (Tables 9 to 10). These same combinations of primers can be used to distinguish SBEIIb homologous genes from genome A and B of durum wheat.
EXAMPLE 5: GENERATION AND IDENTIFICATION OF SBEII MUTANTS Mutagenesis of wheat by heavy ion bombardment. A population of mutagenized wheat was generated in the wheat variety Chara, a commercial variety commonly used, by heavy ion bombardment (HIB) of wheat seeds. Two sources of heavy ions were used, named carbon and neon, for mutagenesis that was conducted at Riken Nishina Center, Wako, Saitama, Japan. The mutagenized seeds were sown in the greenhouse to obtain the M1 plants. These were self-pollinated to produce the M2 generation. DNA samples isolated from each of the approximately 15,000 M2 plants were individually examined for mutations in each of the SBEIIa and SBEIIb genes using the SBEIIa (ARIIaF2 / ARIIaR2) and SBEIIb (ARA 19F / ARA23R genome-specific PCR primers) ) (Diagnostic PCR). Each of the PCR reactions in the wild-type DNA samples yielded 3 distinct amplification products that correspond to the amplified regions of the SBEIIa or SBEIIb genes in genomes A, B and D, while the absence of one of the fragments in the PCRs of the M2 samples mutagenized indicated the absence of the corresponding region in one of the genomes, that is, the presence of a mutant allele in which at least part of the gene was excluded. Such mutant alleles would almost certainly be null alleles.
Screening the M2 strains using the genome-specific primer pairs identified a total of 34 mutants that were mutants for the SBEIIa and / or SBEIIb genes. The mutants in SBEIIa were then examined for the presence of the SBEIIb genes, and vice versa. The identified mutants were thus classified into three groups: “Type 1” where both the SBEIIa and SBEIIb genes were mutants, that is, without both wild type genes in a genome, “Type 2”, where only the SBEIIa gene it was mutant while the SBEIIb gene was wild type, and "Type 3", where only the SBEIIb gene was mutant and the SBEIIa gene was wild type in the particular genome. Since the SBEIIa genes in genomes A, B and D were distinguished by the diagnostic PCR reactions, and likewise the SBEIIb genes, the mutant alleles could be assigned to one of the genomes according to which the amplification product was absent. . As used here, the designation “A1” refers to the genotype where both the SBEIIa and SBEIIb genes in genome A were mutants, “A2” refers to the genotype where the SBEIIa gene was mutant and the SBEIIb gene in the genome A was wild type, and "A3" refers to the genotype where the SBEIIa gene was wild type and the SBEIIb gene in genome A was mutant. The designations "B1", "B2", "B3", "D1", "D2" and "D3" have the same meanings for genomes B and D. The mutants of each of these nine possible types were identified from the collection of 34 mutants.
The extent of chromosome exclusion in each of the 34 mutants was determined by microsatellite mapping. Microsatellite markers previously mapped to the long arm of chromosomes 2A, 2B and 2D (Table 12) were tested in these mutants to determine the presence or absence of each marker in each mutant. The mutant plants in which all or most of the specific chromosome microsatellite markers were retained, based on the production of the appropriate amplification product in the reactions, were inferred to be relatively small exclusion mutants. Such mutants were preferred, considering that other important genes were less likely to be affected by the mutations. The mutants identified and the results of microsatellite mapping are summarized in Table 13.
Crossover of mutants. The mutant plants that were homozygous for minor exclusions as judged by the analysis of the microsatellite marker were selected for crossing to generate the progeny and grain plants that had SBEII mutant alleles in multiple genomes. The F1 progeny plants from the crosses were self-pollinated, and the F2 seed obtained and analyzed for their SBEII genotype. Screening 12 such F2 populations leads to the identification of 11 different combinations of mutant alleles (“double-null”) (Table 14). The double-null combination of the B1D1 genotype was not obtained in the twelfth cross despite examining more than 1200 F2 progenies from that particular cross. A possible explanation for this may be the presence of a critical gene in the vicinity of the SBEII site in genomes B and D, but not in genome A, and, therefore, the combination of double-zero mutations B1 and D1 can yield the seed not feasible. Twenty-seven combinations of double-null mutants are theoretically possible, and more F2 populations will be examined to identify the other combinations.
EXAMPLE 6: AMYLOSE CONTENT OF SIMPLE AND DOUBLE-NULL WHEAT MUTANTS OF SBEII The percentage of amylose in the starch of the grain of single and double-zero plants described in Example 5 was determined using the iodometric method as described in Example 1. A content of amylose from the plot of the dispersion diagram (Y axis) against the number of the mutant strain (X axis) is shown in Figure 4. The amylose content in the mutant grains ranged from 27.3 to 38.7%. The amylose content of the wild type Chara samples (not mutagenized) ranged from 27.4% to 29.5%. Twenty-six strains recorded an amylose content above 34%. It was observed that of these 26 strains, 20 were double-null, of which some were replicated from the same crossing, of Type 1 or Type 2 combinations. In other words, there was a trend in amylose content increasing significantly in Type 1 and Type 2 combinations. double-null compared to the amylose content in single zero grains.
Importantly, and unexpectedly before this study, none of the double-null mutant grains had starch with more than 40% amylose. This included the genotypes A1B1, A1D1 and B1D1 in which each contained four null alleles of SBEIIa and four of SBEIIb and retained two wild type alleles of SBEIIa and two wild type of SBEIIb. This observation was consistent, however, with the prediction made from the data in Example 2. Therefore, it was concluded that to obtain the wheat grain with more than 40% amylose by combining the mutations, the grain needed to have more than four alleles mutants of SBEIIa, or alternatively, if only four mutant alleles of SBEIIa were present, more than four mutant alleles of SBEIIb in combination with the four alleles of SBEIIa, preferably all six genes of SBEIIb being mutants. It was also suggested from the data that the SBEIIa genes in each of the A, B and D genomes were expressed at similar levels relative to each other, that is, SBEIIa expression in common wheat was not predominantly of any genome.
It was interesting to note that the “A3” and “A3D3” genotypes had low levels of amylose consistent with the data in Example 2, confirming that SBEIIb had a lesser role in determining the amylose content in wheat compared to SBEIIa.
EXAMPLE 7: CROSSING IN ATTEMPTS TO CREATE TRIPLE-NULL MUTANTS In order to create mutant strains with more than four mutant SBEIIa alleles, some of the single and double-null strains were crossed and the F2 progeny of these crosses analyzed using diagnostic PCR assays . The assays tested for the presence of the three genes of SBEIIa and three of SBEIIb were therefore used in an attempt to identify plants that had zero mutations in the genes of SBEIIa and / or SBEIIb in each of the genomes A, B and D (triple strains) -null for SBEIIa and / or SBEIIb). The crosses that were performed in a first experiment and the genotypes of the parental and potential triple-F2 progeny lines are listed in Table 15.
The morphology of the starch granule was analyzed by microscopy of wilted / shrunken F2 seeds and of normal appearance from these crosses. Six wilted / shrunken seeds were selected, 5 from crosses 08 / dd and 1 from crossing 08 / bb, each obtained from crosses between a single parent plant D2 and a double parent plant A1B2. Each of the six seeds showed severe distortion of starch granules, showing abnormal, distorted shapes for most granules in the seeds that were similar to the granules observed in transgenic seeds with high levels of amylose (Example 2). Inspection of several wilted / shrunken seeds and strange-looking seeds selected from the other crosses did not reveal altered morphology of the starch granule, indicating that the phenotype observed in 08 / dd and 08 / bb seeds was a specific genotype and not due to the problems of development during seed development.
The starch isolated from 6 of the seeds that have distorted starch granules was pooled and tested for amylose content using the iodometric method as described in Example 1. The amylose content of the pooled sample was measured to be 67% (Table 16). The levels of amylose in the wild type (control) seeds of cultivars Cadoux and Chara were approximately 35%.
Genotypic analysis of seeds with altered starch granule morphology. The seeds from crosses 08 / dd and 08 / bb with altered morphology of the starch granule were sown and the resulting plants were grown in the greenhouse. The DNA extracted from the plants was analyzed using the genome specific primers for SBEIIa and SBEJIb described in Example 3. The results of the PCR assays indicated that each of these seeds were homozygous double-null mutants with genotype A1B2, B2D2 or A1D2 while the third gene (wild type) was present in the homozygous or heterozygous state. The DNA of these plants was further tested using quantitative PCR (real-time PCR, Rotorgene 6000) using pairs of individual specific genome primers to analyze the presence or absence and the homozygosity or heterozygosity of the 3 SBEIIa genes in the plants. The primer pairs used for SBEIIa were Snp6for / Arev5 (SEQ ID NO: 51 / SEQ ID NO: 61) (genome A, 205 bp amplification product), BSnp4 / Arev5 (SEQ ID NO: 55 / SEQ ID NO: 61) (genome B, 494 bp amplification product) and DSnp7for / Drevl (SEQ ID NO: 58 / SEQ ID NO: 62) (genome D, 278 bp amplification product). In order to normalize the amplification reactions of SBEIIa, a pair of primers (SJ156 / SJ242) that amplified a 190bp product of the CslF6 gene, which is a cell wall biosynthesis gene expected to be equally expressed in all plants and located on Wheat Chromosome 7, it was used in the control amplifications. The DNA of a wild type plant from the mutagenized population, designated 2B2, and Chinese Spring (CS) wild type cv. was used as control standards. The relative concentration values generated in the reactions with the SBEIIa primers were normalized to the value for Cslf6 primers for each standard DNA preparation. The values for the potential triple-null plants and CS were calculated in relation to line 2B2.
Out of these three pairs of primers, the genome D primers produced as a clear single band for a plant designated as S14 that allowed quantification. No bands of the SBEIIa genes were obtained in genomes A and B of S14, indicating that it was homozygous for the mutant alleles in those genomes. Quantification indicated that S14 had approximately 30 to 50% of the complement of allele D compared to 2B2 while CS yielded a value of approximately 95% of 2B2 for the SBEIIa gene of genome D. This showed that S14 that yielded seed with amylose levels about 67% was homozygous for SBEIIa null mutations for two of the genomes (A and B) and heterozygous for the third genome (D), in addition to being homozygous for SBEIIb null mutation in genome A. That is, S14 had a genotype A1 (homozygous), B2 (homozygous), D2 / + (heterozygous). In a similar way, quantitative PCR showed that the plant designated as S24 had a genotype B2 (homozygous), D2 (homozygous) and A1 (heterozygous). PCR analysis showed that the remaining 5 plants had the following genotypes: 08dd9-B4 was homozygous for an A1B2 genotype, that is, homozygous mutant for SBEIIa and SBEIIb in genome A, homozygous mutant of SBEIIa and wild-type SBEIIb in genome B and wild-type homozygote for both genes in the D genome, while 08bb11-D9 was homozygous for the B2D2 genotype and S28 and S22 were homozygous for an A1D2 genotype.
F3 seed analysis. The seeds of lines S28, S22, S14 and S24 were sown in the greenhouse, the resulting plants were self-pollinated, and the seeds (generation F3) obtained from each plant. It was observed that the fertility of the plants was affected, in which the number of seeds per head and the percentage of ears that were fertile were significantly reduced compared to the simple null mutants wild type or other double-null cultivated at the same time and under the same conditions, but not abolished (Table 17).
The starch granule morphology was determined by light microscopy on 100 to 200 seeds of each of the S28, S14 and S22 strains. From the S22 strain, 102 F3 seeds were identified with distorted starch granules out of 200 tested seeds. The data revealed a distortion of segregation proportions far from the expected 1: 2: 1 (homozygous mutant: heterozygous: wild type) with a greater number of normal phenotypes than expected. In order to see if a homozygous plant with a phenotype with a high amylose content could be identified, 102 seeds with distorted granules were placed in the appropriate conditions for germination. Sixty-one out of 102 germinated seeds. The DNA of these 61 plants was analyzed by PCEI specific to the SBEIIa genome and all 61 plants appeared to be double-null of an A1D2 genotype, with no triple-null homozygote identified. The wild type SBEIIa gene in genome B was shown to be heterozygous, that is, both mutant and wild type alleles and were presented for genome B.
The 41 seeds that had distorted but not germinated starch granules were analyzed for their SBEIIa genotype. Many of these were observed to be triple-null, that is, showing an absence of any amplification product for the SBEIIa genes and, therefore, having six null alleles for SBEIIa. This confirmed that triple-null seeds could be generated, but these seeds had defects that affected germination. The germs from some of these seeds were removed and cultivated using tissue culture media under conditions to promote germination of the germs. Some germs have successfully germinated, resulting in green seedlings. However, when these seedlings were transferred to the soil, they grew insufficiently and did not produce fertile wheat plants.
From these data, it was concluded that a homozygous mutant triple-null seed based on the exclusion mutations generated by HIB, and seedlings derived from these seeds and having six null alleles of SBEIIa and entirely without SBEIIa, was recoverable from these crosses, but it was affected in germination and growth, indicating an essential role for some SBEIIa in these processes. In contrast, double-null mutants for SBEIIa that were heterozygous for the third null allele and therefore having five null alleles of SBEIIa were recovered, grew normally and were fertile, albeit with reduced fertility.
Analysis of S28 strain protein expression. The SBEIIa expression protein in the developing endosperm obtained from a complete ear of an S28 plant was analyzed by Western blotting using an SBEIIa specific antibody. All 15 endosperm on the ear showed a pattern without the isoforms of the AE and D genome of SBEIIa (double-null AD) with only one band of SBEIIa present, expressed from genome B. Out of the 15 endosperms, 7 had the level of expression of SBEIIa of genome B considerably smaller than others and that of the control strain, NB1. Based on the intensity of the band, the expression of SBEIIa in each endosperm was quantified.
The remaining starch granules from the endosperm were purified using 90% Percoll. Following resuspension in 200 μl of water, the granules were examined microscopically. It was observed that all endosperm having a SBEIIa expression level that was less than about 36% of the wild type had starch granules with distorted morphology typical of a phenotype with a high amylose content. A range of SBEIIa protein expression levels was observed in the developing grains from an S24 plant's ear, up to less than 5% of the wild type. Endosperms with lower SBEIIa levels also showed altered starch granule morphology; the phenotypes were, therefore, completely correlated in this experiment. The levels of SBEIIb expression in all of these endosperm were also analyzed using a specific SBEIIb antibody. The results clearly showed that there was a concomitant reduction in the expression of SBEIIb corresponding to the reduction in the expression of SBEIIa.
Discussion. Analysis of seed from plants with the A1B2 mutant genotype (summarized in Table 18) having four SBEIIa mutant alleles indicated that the amylose content was only slightly elevated for that genotype, yielding an amylose level of less than 40%. In comparison, data from S14, S22, S24 and S28 seeds demonstrated that the addition of the SBEIIa fifth mutant allele raised the amylose level to about 67%. Consequently, the increase in the number of four null SBEIIa alleles to a minimum of five mutant SBEIIa alleles was critical to increasing the amylose level to more than 50% (w / w), in fact more than 60% (w / w ). This conclusion was in line with the predictions made from the data in Example 2. The observed relationship of the allelic composition to the amylose content indicated that the total number of SBEIIa mutant alleles in the plant was important in determining the amylose content (Table 18 ). It was also concluded that the number of SBEIIb mutant alleles also played a role, although less important than the number of SBEIIa mutant alleles.
It was also concluded that homozygous triple-null mutant seeds and seedlings having six null SBEIIa alleles and entirely without SBEIIa could be generated from the simple null mutants containing exclusions generated by HIB, but these were affected in germination and growth, indicating a role essential for some SBEIIa in these processes. In contrast, the double-null mutants for SBEIIa that were heterozygous for the third null allele and therefore having five null alleles of SBEIIa were recovered, grew normally and were fertile.
EXAMPLE 8: OTHER ATTEMPTS TO PRODUCE TRIPLE-NULL MUTANES COMPLETELY WITHOUT SBEIIA OR SBEIIB The observed inability to generate a triple-null mutant completely without SBEIIa in the Example above may have been dependent on the particular mutant plants used as mothers in the cross. To test, a second set of crosses using additional parental mutants, also obtained from HIB mutagenesis, was performed, summarized in Table 19. The F2 seeds from the 38 crosses were harvested and the DNA extracted. At least 96 DNAs, each of 25 crosses, 12 of which are from the crosses targeted in the production of an A1B2D2 genotype (triple-null mutant), but using different parental lineages from those described in Example 7, were examined by PCR to determine the tendency for segregation. No viable triple-nulls were identified for any of these crossings. The recovery of double nulls also varies depending on the cross, but in most cases the expected genotypes were obtained. The F2 seeds from six of the A1B2D2 crosses were also examined microscopically to identify seeds having a phenotype with a high amylose content. Such seeds were identified at a moderate frequency.
Sorting seeds from cross A2B2D2, 08 / mm-l. Among the crosses listed in Table 19, 12 were crosses between a main with an A2 genotype and a main with a B2D2 genotype, that is, both parents were wild type for all three SBEIIb genes, with the objective of generating triple mutants. - SBEIIa nulls having the A2B2D2 genotype. DNA preparations of approximately 672 F2 seeds obtained by crossing 08 / mm-l were examined by PCR. The proportions of segregation were distorted from the expected Mendelian proportions, with double-nulls identified significantly smaller than expected (Table 20). Nevertheless, all possible combinations of double-zero mutations were identified in viable seed. No triple-nulls of the A2B2D2 genotype were identified among the 672 seeds, even though by Mendelian segregation about 10 would have been expected.
In parallel, the F2 seeds from the 08 / mm-1 cross were examined by microscopy to identify any seeds with a distorted / high amylose starch granule (HA) phenotype. Of the 576 F2 seeds that were examined, seeds with the HA phenotype were not identified. This seed population must have included a low frequency of seeds having 5 mutant alleles of SBEIIa, being a homozygous mutant in two genomes and a heterozygous / wild type mutant in the third genome for SBEIIa. The lack of seeds observed with an HA phenotype at the A2B2D2 crossing indicated that 5 mutant alleles of SBEIIa, in the absence of any mutant alleles of SBEIIb, did not appear to be sufficient to provide a phenotype with a high content of amylose (> 50% amylose) . That is, a reduction in SBEIIb levels in relation to the wild type further to the greatly reduced SBEIIa level in the context of 5 mutant alleles of SBEIIa and one wild type allele of SBEIIa, or an equivalent level of SBEIIa activity in an endosperm having partial loss of function mutations in one or more SBEIIa genes, it was necessary to provide more than 50% amylose.
Screening of F2 seeds from eleven additional crosses between single SBEIIa mutant parents (wild type by SBEIIb) and double SBEIIa mutant parents in the other two genomes also did not identify any viable triple-null mutant seeds of the A2B2D2 genotype.
Intersections involving Type 3 mutations. Intersections involving Type 3 mutations were performed with the objective of finding homozygous mutants having two, four or six mutant alleles of SBEIIb combined with four mutant alleles of SBEIIa, and determining the phenotype of the resulting plants and their grain. Table 21 summarizes the results of screening crosses involving Type 3 mutations. Triple-nulls were identified from crossings A3B3D3 and A3B2D2, both of which showed wild-type starch granule morphology.
EXAMPLE 9: ANOTHER SCREENING FOR HIGH AMYLOSIS MUTANTS In other attempts to produce SBEIIa triple-mutants from identified single mutants, an altered strategy was adopted. This strategy added the stage of some initial backcrosses of the simple mutants after their identification, in order to remove unbound and unrelated mutations from the M2 plants having the simple SBEIIa mutations. This was included to reduce the effect of the mutated history, due to the high level of mutagenic treatment used, which would have produced additional mutations in the plants regardless of the desired SBEIIa mutations which could have detrimental effects when the mutations were combined. These initial backcrosses were carried out by crossing the M2 mutants with plants from the winter wheat Apache cultivar or the spring wheat Chara cultivar.
Initially, 13 crosses were performed to combine the mutations in all three genomes, and molecular analysis was done on the DNA of 21,400 F2 seeds in half, with the second half of each seed retained to preserve the strain. A preliminary screening to detect the mutations used dominant SSR markers that were genome specific for SBEIIa or SBEIIb. From that, 21 seeds were identified as putative triple-null mutants and 793 seeds as putative double mutants (Table 22) due to the absence of genome-specific amplification products.
Assays based on TaqMan Q-PCR of wheat seed genotypes. The first round of screening using dominant markers as described above could not distinguish between seeds that were heterozygous or homozygous wild type for any SBEIIa gene. A PCR assay based on TaqMan was therefore developed to distinguish heterozygotes and homozygotes for the gened and SBEIIa in the third genome, and to confirm the genotypes of the initial screening. Because TaqMan analysis was performed on half of the seeds and because the wheat endosperm is triploid (3n) for each genome, two types of profiles were possible for heterozygous endosperm for the wild type SBEIIa allele in the third genome, or 2n, where the wild type allele was provided by the main maternal, or 1n, where the wild type allele was provided by the main paternal through pollen. The Q-PCR-based assays TaqMan used the Applied Biosystems 7900HT Fast Real-Time PCR System (ABI, Foster City, CA) to detect the copy number of the SBEIIa gene in the third genome of putative double mutant wheat seeds. The assays used genomic DNA extracted from half of the seeds by magnetic sphere methods (Nucleomag, Cat Ref No. 744 400.24). DNA was loaded into 384 well plates and duplex Q-PCR reactions were performed in duplicate for each plate. PCR reactions were designed to amplify a 65 bp fragment from exon 21 of the SBEIIa genes using the SBE2a primers QPCRABDF4 (forward primer): 5'-ACGATGCACTCTTTGGTGGAT-3 '(SEQ ID NO: 31) and SBE2a QPCRABDR4 (reverse primer) ): 5'-ACTTACGGTTGTGAAGTAGTCGACAT (SEQ ID NO: 32). The probe used to deliver the fluorescene signal during the Q-PCR reactions was SBE2a QPCRABDS4 (probe TaqMan® MGB, FAM) 5'-CAGCAGGCTTGATCAT-3 '(SEQ ID NO: 33). A sequence of an endogenous gene, GamyB, was used as an internal control to normalize the signal value of each sample, using the GamyB1F primers (direct primer): 5'-GATCCG AATAGCTGGCTCAAGTAT-3 '(SEQ ID NO: 34) and GamyB2R (reverse primer): 5 '-GGAGACTGC AGGTAGGGATC AAC-3' (SEQ ID NO: 35). The reaction conditions were as follows: "hot start" (95 ° C, 10 min) followed by 40 cycles of denaturation (95 ° C, 15 sec), annealing (58 ° C, 60 sec). The reaction products were analyzed using Relative Quantification management software integrated with the 7900HT Fast Real-Time PCR system.
Using this TaqMan assay, all of the 21 putative triple-null mutants were confirmed to be double-null, not triple-null. The incorrect identification in the initial screening was considered due to false negatives, perhaps caused by the low quality of the standard DNA. When 14 of the seeds were examined for starch granule morphology by light microscopy, all 14 were observed to have a wild type granule phenotype, which was consistent with the seeds being double-null mutants, not triple-null mutants. The assays also identified few putative double mutant seeds that were 2n heterozygous in the third genome, from M76, M77, M82, M83 and M86 crosses. However, those results need to be confirmed since it was difficult to distinguish the heterozygous 2n genotype from the homozygous 3n genotype, even in the presence of the SBEIIa double-zero background. This will be confirmed in the next generation of progeny. The assays also showed that any double-null mutants of SBEIIa that were heterozygous mutants of SBEIIa / wild type in the third genome were obtained from the crosses M79, M81, M74, M75, M78 and M80. The M84 and M85 crosses yielded the highest number of double-null homozygous mutants of SBEIIa clearly identified that were good candidates for being 1n heterozygous (SBEIIa / wild type mutant) in the third genome. Some 2n heterozygotes have also been identified, but need to be confirmed.
At these crosses, the numbers of SBEIIa single and double-null mutants were less than the expected frequency of Mendelian segregation. This segregation distortion has also been studied. Where the expected frequency of simple homozygous mutants must have been 25%, in some crosses the frequency was much lower, ranging from 1% to 25%. The number of double homozygous mutants in the progeny of crosses to produce triple-null mutants, theoretically, should be about 6% (1/4 * 1/4) per combination (6% AB, 6% AD, and 6% of AB). The actual number of double mutants identified was much lower and ranged from 0 to 5.2%. This suggested that some combinations of mutations were harmful to the plant, for example, for seed development, leading to a lesser recovery of combinations of mutations than expected. Two crossings, M74 and M75, show the lowest frequencies compared to the expected. It was noted that the parents used at those crosses had not been backcrossed with Apache or Chara before the crosses were performed, suggesting that additional, unrelated mutations in the parents resulting from the mutagenic treatment may have played a role in distorting segregation proportions. Even for M76 and M86 crosses that yielded a greater number of single mutants, the frequency of double-null mutants was low, particularly for some combinations. For example, for M76 crossing, the frequencies of single nulls in genome D and genome A were 23% and 17%, respectively, while the frequency of double-null mutants in both genomes A and D was only 0.8%. This suggested that some combinations of SBEIIa mutations were less favorable to the plant than others, and consequently counter-selected. The proportion of mutants containing five SBEIIa mutant alleles (double-nulls that were heterozygous mutants in the third genome) was also very low. The expected frequency would be 9% (1/4 * 1/4 * 1/2 * 3) while the highest percentage observed was 1.1% for the M84 and M85 crosses.
The correlation between frequencies of single and double homozygous mutants at M74 and M75 crosses was very good for SBEIIa mutations in genomes A and D (0.789 and 0.558, respectively) while much lower (0.386) for genome B. One possible explanation would be that one of the parents (19,832 (D1) / 20,257 (A2) [08 / bl2]) used at the M74 and M75 crosses was a heterozygote in the different first place instead of a double homozygous mutant.
Under these conditions, the probability of obtaining a triple-null mutant (6 null mutant alleles of SBEIIa) was very low and much less than the expected frequency of 1/64. However, the self-fertilization of the double mutants that were also heterozygous in the third genome, in particular of the M84 and M85 crosses, should confirm whether the triple-null mutants are recoverable from these parental mutants. The progeny of the self-pollinated plants will be analyzed to identify any triple mutant seeds.
EXAMPLE 10: SCREENING FOR MUTANT WHEAT SEEDS BY NIR A fast, non-destructive, high-yield method was developed to examine simple seeds for a phenotype that was associated with high amylose content. The PCR-based screening methods described in Examples 4 to 6, while successful in detecting mutants in a population of 15,000 seeds, required the preparation of DNA from each half of the seed after cutting each seed manually, so it was time consuming and time consuming. boring. It was determined that Near Infrared Spectroscopy (NIRS) could be used to distinguish between phenotypes with a high amylose content and normal amylose. Near Infrared Spectroscopy (NIRS) is a non-destructive technology that has been used to determine some properties of wheat seed (McClure, 2003). The analysis of NIRS from simple wheat seed for a waxy starch phenotype (low amylose) has been developed in durum wheat by Delwiche et al. (2006). Dowell et al (2009) developed an automatic classification system for single seed NIR to separate durum wheat and waxy, partially waxy and normal wheat. To the knowledge, this method has not been used previously to distinguish seeds with a high amylose content in hexaploid wheat.
Development and validation of a reduced biochemical reference method to measure the apparent amylose content in the ground seed material. In order to calibrate the NIRS measurements according to the apparent amylose content in individual seeds, a mathematical model had to be established to correlate the NIRS spectrum data and a biochemical method measuring the apparent amylose content in the same sample, in that simple seeds case. Standard iodometric methods, for example, the method described in Example 1, routinely use an amount of seed that is combined before solubilization of the starch, providing bulky (combined) starch that is normally defatted before the colorimetric measurement of the amylose content based on on the iodine bond. To be suitable for use for NIRS calibration purposes, this method has been modified, simplified and reduced to allow measurement of the apparent amylose content in single seeds, thus allowing variation in the amylose content between seeds. The term "apparent amylose content" is used in this context because the modified method did not purify the starch from the milled grain, the lipids interacting with the amylose in the starch were not removed, and the results were expressed as a percentage of fresh seed weight instead of as a percentage of the starch isolated from the seed. For these reasons, the values obtained for "apparent amylose content" were much lower than the values obtained using the standard method as described in Example 1.
As a first step, this method was developed by assessing the linearity between the colorimetric response and the amylose content using ground wheat grain without starch purification. The material with a high amylose content used for this was wheat grain transformed with the hp5'-SBEIIa construct and having reduced SBEIIa (WM, Line 85.2c, see Example 2) and wheat with the normal level of amylose which was a wheat wild type (WMC) grown at the same time and under the same conditions. The milled WM grain contained about 80% amylose as determined by the standard method of Example 1, while the milled WMC grain had an amylose content of about 25%. Samples with different proportions from WM to WMC were prepared from ground seed material, but not purified again. Approximately 17 mg of samples were used for the assay. The mixtures of WM and WMC were weighed precisely in 1.5 ml microcentrifuge tubes. To solubilize the starches in the samples, 1 ml of DMSO was added per 17 mg of sample and then the mixtures heated in a 95 ° C water bath for 90 min with an occasional vortex. A 10 μl aliquot of each mixture was added in 1.98 ml of water and treated with 10 μl of 0.3% I2 + 3% KI in 0.01N NaOH solution. The absorbance of each mixture was measured at 605 nm and absorbance values were converted to percent amylose using a standard curve. The standard curve was made using corn amylopectin (Sigma catalog no. A7780) and potato amylose (Sigma, A0512) in proportions from 0% to 100% amylose and treated in the same way as ground wheat samples.
The results showed a linear relationship between the level of WM incorporation and the apparent amylose content, showing that the simplified iodometric method could be used to calibrate NIRS and that starch purification was not necessary for this purpose.
Test of the biochemical reference method to measure the apparent amylose content in half of the seeds. The seeds of the (control) strains WM and WMC obtained from field trial experiments conducted in Arizona and Washington were used for this test. In total, 47 seeds in half with germs removed were individually placed in 1.5 ml microcentrifuge tubes and weighed just before the addition of 0.6 ml of DMSO for each. The tubes were incubated in a 95 ° C water bath for 20 min after which the samples were crushed in the tubes using a glass rod. The volume of each mixture was adjusted to precisely 1 ml of DMSO per 17 mg of sample, after which the tubes were incubated at 95 ° C in a water bath for an additional 70 min with an occasional vortex. Apparent amylose was measured by taking 10 μl aliquots of each mixture and treating them with 10 μl of 0.3% I2 + 3% KI in 0.01N NaOH solution and diluted in 2 ml with H20, as before . The absorbance of each sample was measured at 605 nm and absorbance values were converted to the percentage of “apparent amylose” using a standard curve as described above.
Using this method, the apparent amylose content of WM seeds ranged from 20% to 41% (average 27%) while the apparent amylose content of WMC seeds ranged from 7.5% to 17% (average 11.4%). The reasons why these values were much less than the amylose content as determined by the method of Example 1 are described above. This simplified method, therefore, allowed seeds with a high amylose content to be distinguished from those with a wild-type amylose content.
NIRS calibration. Single-seeded NIRS scans on WM and WMC seeds were obtained using a Multifunction Analyzer (MPA) NIRS spectrometer (Bruker Optics, F-77420 Champs Sur Marnes, France). Each seed was placed at the bottom of a glass tube with aluminum foil and scanned twice. The spectra were recorded using a Bruker MPA Multifunction Analyzer spectrometer (Bruker Optics) fitted with a fiber probe. The spectra were recorded using 32 reference scans and 16 sample scans ranging from 4000 to 12,500 cm-1 at a resolution of 16 cm-1 resulting in 1100 data points. The fiber optic probe used was the IN 261 probe for solids.
To determine the correlation between apparent amylose levels and NIR readings, 226 individual WM or WMC seeds with an apparent amylose content ranging from 6 to 44% were analyzed. First, duplicated NIRS spectra were acquired for each seed, after which the apparent amylose content was biochemically measured for each seed according to a method described above. The spectral outliers (6 samples) were identified as spectra that were compared abnormally to the spectra of the total data set and eliminated, and the remaining spectra analyzed with Min-max pretreatment of normalization. Partial Least Square software with full cross-validation (one out) was used to create the model. The spectral window used for the development of the model was 9827-7150 cm-1 and 6271-4481 cm-1. The number of PLS factors used to develop the calibration was 14. The accuracy of the calibration model was expressed by the standard error of cross validation (SECV) and the coefficient of determination (R2). The effectiveness of a calibration was shown by the RPD, which is the ratio of the standard error of forecast (RMSECV) to the standard deviation of the reference data of the set.
A positive correlation (R2 = 0.702) was obtained between the data and biochemicals and the spectral data of NIR (Figure 15). It was concluded that the model was robust enough to distinguish wheat seeds with a high amylose content from wheat seeds with normal amylose, but it is not yet accurate enough to accurately measure the amylose content in any seed. The method was therefore able to examine a very large population of seeds to enrich the grains with phenotype with high amylose content. This has been validated as follows.
Validation of NIRS. In order to validate the NIR method in distinguishing the grain with high amylose content and control grain, another 60 WM seeds and 34 WMC seeds were scanned twice by NIR and the apparent expected amylose content was calculated. When the apparent amylose values thus determined were plotted to obtain the distribution profile for the WM and WMC populations, it was seen that the two groups were mainly separated with a slight overlap (Figure 16). According to these results, seeds having an apparent predicted amylose phenotype determined by NIRS equal to or above 30% could be considered as good candidates for being a seed with a high amylose content.
NIRS screening of F2 seeds from wheat crossings. NIRS screening was performed to detect mutant seeds that have a high amylose content. The screening used 2,700 F2 seeds from two different crosses: M80 and M85 which were, respectively: 21,142 (B2) / Type I-20257 (A1) [08 / h-111] // Type 1-19,832 (D1) / CHARA and 5.706 (D2) / 21.668 (B2) // 20.257 (A1) / CHARA). The screening was therefore aimed at identifying the seeds with an A1B2D1 or A1B2D2 genotype, respectively. Two spectra of NIRS were recorded per seed as described above.
Seeds that yielded an apparent predicted value of amylose above 34% in at least one of the two duplicate screens first were selected for another analysis. Out of the 2,700 seeds, 27 seeds were selected and were then evaluated by light microscopy to determine the starch granule morphology. Each seed was carefully scraped to preserve the germ, yet obtain enough endosperm material to be examined. Four seeds out of 27 were observed to have distorted starch granule morphology. These four seeds started to have the highest apparent amylose content expected from NIR screening and were the only ones where both apparent predicted amylose values were above 30%. The other 23 seeds showed normal granule morphology (wild type).
Molecular data in seeds selected by NIRS screening. PCR analysis was performed on the four seeds to determine the SBEIIa genotype of each. The initial assays used dominant PCR markers that show the presence or absence of each SBEIIa gene in the three genomes. Three of the seeds were shown to be double-null mutants while the fourth was a putative triple-null mutant. However, when tested again with a co-dominant PCR marker (see below), all four seeds were shown to be double-null mutants for SBEIIa (that is, without SBEIIa in both genomes) and heterozygous for a mutant SBEIIa gene in the third genome. Therefore, these seeds contained 5 mutant alleles of SBEIIa and at least two mutant alleles of SBEIIb.
When the germ of each seed was put in the conditions to germinate, none of them germinated successfully, perhaps because it was damaged or the combination of mutations was very harmful.
In order to try to identify more candidates, another NIRS screening was performed on more progeny F2 seeds from crosses M80 and M85, with less stringent selection of candidate seeds. The selection criterion for the second exam was that one of the predicted values of apparent amylose had to be above 30% and the second at least 23%. A new set of 22 seeds was selected to evaluate the starch granule by light microscopy. Out of those 22 candidates, 1 seed, BD85; 9F08 (P279-F08-834), showed a distorted starch granule phenotype. This mutant was also analyzed by PCR and showed to be a double-null mutant of SBEIIa in genomes A and B and heterozygous for the mutant gene of SBEIIa in the D genome. It was successfully germinated for multiplication.
EXAMPLE 11: DETECTION OF STARCH BRANCHING ENZYME ALLELS WITH CHANGED STARCH BINDING AFFINITY Populations of mutagenized wheat grains produced by treatment with sodium azide of chemical mutagens or EMS were examined to identify mutants that had mutations of points in the SBEIIa genes and therefore potentially reduced, but not abolished, SBEIIa-A, -B or -D activity, or SBEIIb-A, -B or -D activity (partial mutants) relative to wild type wheat. Screening for mutants was based on measuring the amount of SBEIIa or SBEIIb proteins by using Western blotting with antibodies specific to SBEIIa or SBEIIb (see Example 2), or by affinity-based techniques, as follows. This screening was also expected to detect mutants with point mutations that were totally lacking the SBEIIa-A, -B, or -D activity as well as the mutants with partial activity.
Native gel electrophoresis of grain protein extracts including starch branching enzymes through a polyacrylamide matrix containing glycogen, amylopectin, amylose or β-limit dextrin (affinity gel electrophoresis) provides a method for identifying SBEIIa alleles or SBEIIb encoding SBEIIa or SBEIIb with altered starch binding capacity. Since the starch branching enzyme active site contains a starch binding site, SBEII polypeptides with altered binding efficacy are likely to have changes in catalytic rate and / or affinity. In particular, polypeptides with reduced binding efficacy were expected to have reduced SBEII activity.
The following methods were used, based on Morell et al., (1997); and Kosar-Hashemi et al, (2006) with some modifications.
Protein preparation. The soluble proteins were extracted by homogenizing the endosperm isolated from the developing seeds (about 15 days post-anthesis) in 50 mM phosphate buffer, pH 7.5 containing 5 mM EDTA, 5 mM DTT, 0.4% protease inhibitor cocktail and 20% glycerol. After centrifugation at 14,000 g for 10 min, the supernatant was used for gel electrophoresis. The protein concentration in the extracts was estimated using a Coomassie Plus Protein Assay Reagent.
Affinity Electrophoresis. In a two-dimensional (2D) affinity electrophoresis technique to separate the SBEII protein isoforms, the aliquots (40 or 100 μg) of the protein extracts were loaded onto the gel of the first dimension, a non-denatured polyacrylamide gel mold in a 16 cm Hoefer SE600 vertical plate gel unit. The resolving component of the second dimension gel was a 6% non-denaturing gel (14 x 16 cm or 16 x 16 cm, 1.5 mm thick) containing 10% glycerol with an appropriate amount of target polysaccharide (amylopectin , β-limit dextrin or glycogen) immobilized within the gel structure. A stacking gel (polysaccharide free) was poured into 1 cm from the top of the glass plate formation using a comb to form cavities. The gels run overnight at 4 ° C at constant voltage (100V for glycogen and β-limit dextrin and 135V for gels containing amylopectin).
Alternatively, a dimensional system was used to separate the SBEIIa proteins in which the protein extracts (20 μg) were loaded onto a non-denaturing polyacrylamide gel. The resolving component of the gel was a 6% non-denaturing gel containing 10% glycerol with 0.15% β-limit dextrin immobilized within the gel structure, while the stacking gel was free of polysaccharide. The gels were run at 4 ° C with a constant current of 20mA per gel and a maximum voltage of 200V.
SBEIIb proteins can also be separated into a Bis-Tris of 4 to 12% gradient gel (Invitrogen). The gel is run at 4 ° C with a constant current of 20mA per gel and a maximum voltage of 200V.
Immunological Detection. For immunological detection of SBEII proteins that follow electrophoresis, the proteins were transferred from the gels to nitrocellulose membranes using a TE 70 PWR semi-dry transfer unit (Amersham Biosciences). The transfer buffer contained 39 mM glycine, 48 mM Tris, 0.0375% SDS and 20% methanol. The transfer was carried out for 1 to 1.5 h with a constant current of 0.8 mA / cm2. The membrane was blocked with 5% skimmed milk prior to Western blotting using specific rabbit polyclonal wheat primary antibody SBEIIa.
The migration patterns of the SBEII isoforms encoded by the homoalleles of genomes A, B and D of wheat showed differences between different wheat varieties when analyzed by a gel electrophoresis method of dimensional affinity. In some varieties, the clear separation of homeoforms A, B and D was possible, allowing simple classification of polymorphisms in mutagenized populations of those varieties. For example, affinity gel electrophoresis of protein extracts from the endosperm of the wild type wheat varieties Sunstate and NB1 showed a clear separation of the SBEIIa- A, - B and -D isoforms. The enzyme branching alleles with reduced affinity for starch migrated a greater distance through the polysaccharide-containing polyacrylamide gel than the respective native homoalleles. Alleles containing lines with reduced expression or an absence of expression of a particular homoallele were identified by the presence / absence of a band in the homozygous state and through densitometry to measure the intensity of the band in heterozygous lines. To validate this method, the mutant plants of SBEIIa and SBEIIb that were identified by genotypic analysis (Example 6) were confirmed to be without SBEIIa or SBEIIb specific proteins by affinity gel electrophoresis, consistent with their genotypes. These experiments validated this method of protein analysis for detecting mutants with a reduction in the amount or activity of an SBEII isoform.
The screening of a population of 2100 mutagenized wheat strains of the Sunstate variety, treated with sodium azide as described in Zwar and Chandler (1995), using β-limit dextrin affinity gel electrophoresis led to the identification of 18 mutants that had altered mobility in the affinity gels of one of the SBEIIa proteins (affinity mutants) or null mutants for one of the SBEIIa genes based on a lack of protein detectable by that gene. The constant dissociation (Kd) of starch enzyme interactions for each of the SBEIIa isoforms in one of the affinity mutants was calculated by measuring the change in enzyme mobility as a function of the β-limit dextrin concentration in an affinity gel 1 -D as described in Kosar-Hashemi et al., 2006. This affinity mutant had SBEIIa proteins with the following Kd values: 0.53g / L, 0.52g / L and 1.69g / L for the isoforms of
SBEIIa-A, SBEIIa-B and SBEIIa-D respectively (Figure 13). The higher Kd value observed for isoform D compared to that of isoforms A and B indicated a lower, reduced affinity of this isoform for starch binding, indicating that this strain was a mutant affinity mutant for the SBEIIa-D gene. It is expected that the isoform of the genome D (SBEIIa-D) of this strain has a lower enzymatic activity, but not total loss of activity, compared to the other two isoforms. This expectation is confirmed by the SBEII activity assays in the presence of null SBEIIa-A and SBEIIa-B alleles.
The single SBEIIa mutants identified from the Sunstate mutagenized sodium azide population were then crossed with the previously identified double-null HIB mutants to isolate the triple mutants that do not have SBEIIa activity from the two genomes with total or partial loss of activity in the third genome. . Four crosses to isolate A1B2D2, two crosses each to isolate A2B2D2 and A2B2D1 and one cross to isolate the A1B2D1 genotypes were performed. Examination of the starch granule morphology of F2 seeds from one of the A1B2D2 crosses by seeds identified by microscopy with gravely distorted starch granules similar to those found in wheat with a high amylose content (at least 70% amylose). The amylose genotype and phenotype of these seeds are confirmed by the analysis of the SBEIIa alleles in the seeds and progeny and by the extraction and analysis of starch from the progeny grain. Eight crosses were also performed between single affinity mutants to produce double SBEIIa affinity mutants. This included the crosses generated in order to isolate the A2B2, A2D2 and B2D2 affinity double mutants. F2 progenies are analyzed by the methods described above to identify the homozygous double affinity mutants.
EXAMPLE 12: PROPERTIES OF STARCH GRANULES AND WHEAT GRAIN STARCH WITH HIGH AMYLOSE CONTENT. Changes in starch granule morphology and birefringence. The properties of starch and starch granules were examined in the transgenic high amylose wheat described in Example 2. Scanning electron microscopy was used to identify macroscopic changes in the size and structure of the starch granule. Compared to the untransformed control, the starch granules of the endosperm that have reduced SBEIIa expression exhibited significant morphological changes. They were highly irregular in shape and many of the A granules (> 10 μm in diameter) appeared to be sickle-shaped. In contrast, both A and B starch granules (<10 μm in diameter) of the endosperm having reduced SBEIIb expression and unchanged SBEIIa expression were smooth, spherical or ellipsoid in shape and imperceptible in the wild-type wheat starch granules .
When viewed microscopically in polarized light, wild-type starch granules typically show a strong birefringence pattern. However, birefringence has been greatly reduced for granules containing wheat with a high amylose content. Less than 10% of the starch granules of the strains having reduced expression of SBEIIa and 70% to 80% of amylose content was birefringent when viewed in polarized light. For strains that essentially do not have SBEIIb expression, but with wild type SBEIIa expression, no change in birefringence was observed compared to untransformed controls. In the strains suppressed by SBEIIb and wild type, approximately 94% of the starch granules exhibited complete birefringence. The data are given in Table 23. Loss of birefringence, therefore, correlated closely with high amylose content.
Amylose content of transgenic wheat grain. The amylose content of transgenic wheat grain was evaluated by two independent methods, that is, an iodometric method and a size exclusion chromatography (SEC) method. The iodometric determination of amylose content was based on the measurement of the color change induced when the iodine alloys in the linear regions of α-1,4 glycan, with reference to a standard curve generated using known concentrations of purified potato amylose and amylopectin, as described in Example 1. The size exclusion chromatography method was based on the separation, by column chromatography, of amylose and amylopectin that was not debranched, followed by the measurement of the starch concentration in the eluted fractions of the column. The three grain genotypes were analyzed. Firstly, plants transformed with the hp-SBEIIa construct and having very low levels of SBEIIa expression; secondly, plants containing the hp-SBEIIb construct and with no detectable expression of SBEIIb, but wild type for SBEIIa; and thirdly, the untransformed wild-type control (NB1). The grain of plants without expression of SBEIIb (008) had an amylose content of 27.3% determined by the iodometric method and 32% by the SEC method. This was not significantly different from the amylose content of the NB1 untransformed control strain (31.8% iodometric, 25.5% SEC).
However, in the grain that has reduced expression of SBEIIa (line 087), the amylose content was significantly high (88.5% iodometric, 74.4% SEC). The difference in these two figures for lineage 087 was considered to be in the presence of some “material intermediary” that binds to iodine as well as amylose and was measured in the iodometric test as amylose, but was separated in column chromatography with major amylopectin.
Starch chain length distribution by FACE. The chain length distribution of isoamylase de-branched starch was determined by fluorophore-assisted carbohydrate electrophoresis (FACE). This technique provides a high resolution analysis of the distribution of chain lengths in the range of DP 1 to 50. From the molar difference graph in which the normalized distribution of the untransformed control chain length was subtracted from the normalized distribution of the transgenic strains , it was observed that there was a marked decrease in the proportion of chain lengths of DP 6 to 12 and a corresponding increase in chain lengths greater than DP 12 in the starch of the grain that has reduced SBEIIa expression. No statistically significant changes in the distribution of the starch chain length of the hp-SBEIIb strains were observed when compared to the wild type.
Molecular weight of amylopectin and amylose. The molecular weight distribution of starch was determined by size exclusion HPLC (SE-HPLC). The HPLC system comprised of a GBC pump (LC 1150, GBC Instruments, Vic, Australia) equipped with Auto Sampler (GBC, LCI 610) and Evaporative Light Scattering Detector (ELSD) (ALLTech, Deerfield, USA). The Ultrahydrogel® 1000 column, Ultrahydrogel® 250 column and protection column (7.8 mm x 300 mm, Waters, Japan) were used and maintained at 35 ° C during the HPLC operation. The ammonium acetate buffer (0.05 M; pH 5.2) was used as the mobile phase at a flow rate of 0.8 mL min-1.
The molecular weight of amylopectin in SBEIIa reduced grain starch appeared to be much less than that of amylopectin in NB1 (wild type, non-transgenic) starches and the reduced grain of SBEIIb (peak position of 7166 kDa versus 45523, 43646 kDa) . In contrast, the molecular weight of amylose from the reduced grain of SBEIIb was not significantly different compared to that of the wild type grain of the unprocessed variety NB1. The data are in Table 24. The total starch content in the endosperm of wheat with reduced expression of SBEIIa.
Analysis of total starch content in the grain as a percentage of grain weight revealed a slight reduction in the starch content of the endosperm of the hp-SBEIIa strain (43.4%) compared to 52% in the control and 50.3% in the strain of hp-SBEIIb (Table 23). This indicated that there was some reduction in the total synthesis of starch when the expression of SBEIIa was reduced by the inhibitory construct.
Starch Bloating Strength (SSP). The swelling strength of the gelatinized starch starch was determined following the small-scale test by Konik-Rose et al, (2001) which measured the absorption of water during the gelatinization of the starch. The estimated value of SSP was significantly lower for the starch of the reduced SBEIIa strain with a figure of 3.51 compared to the control starch (9.31) and reduced grain of SBEIIb (10.74) (Table 23).
Starch Bonding Properties. The starch glue viscosity parameters were determined using a Rapid Viscosity Analyzer (RVA) essentially as described in Regina et al, (2004). The temperature profile for the RVA comprised the following stages: keep at 60 ° C for 2 min, heat at 95 ° C for 6 min, keep at 95 ° C for 4 min, cool to 50 ° C for 4 min, and hold at 50 ° C for 4 min. The results (Table 25) showed that peak and final viscosities were significantly lower in SBEIIa reduced grain starch compared to control wheat starch.
Starch gelatinization properties. The starch gelatinization properties were studied using differential scanning calorimetry (DSC) as described in Regina et al., (2004). DSC was performed on a Perkin Elmer Pyris 1 differential scanning calorimeter. Starch and water were premixed in a 1: 2 ratio and approximately 50 mg plated in a DSC pan that was sealed and left to equilibrate overnight. A heating rate of 10 ° C per minute was used to heat the test and reference samples from 30 to 130 ° C. The data were analyzed using the software available with the instrument. The results (Table 26) clearly showed a delayed end of the gelatinization temperature (72.6 ° C) for the reduced SBEIIa starch compared to the control (66.6 ° C). The peak gelatinization temperature was also higher in the reduced SBEIIa starch (63.51 ° C) compared to the control starch (61.16 ° C).
EXAMPLE 13: ANALYSIS OF WHEAT FLOUR WITH HIGH AMYLOSE CONTENT DURING PROCESSING. Pressure processing studies in collaboration with CSIRO Alimento and Nutritional Sciences, Werribee. The structural characterization of wheat starches with high amylose content compared to native starch was performed using Low Angle X-Ray Scattering (SAXS). The study was designed to include a) characterization of raw wheat flour and b) real-time analysis of the gelatinization process while cooking in pressure samples of flour or starch at temperatures greater than 100 ° C and c) changing structures in the cooling by a period from 0 to 10 days, and retrogradation. The study used samples of wheat flour of varying levels of amylose ranging from about 25% (wild type) to about 75%, increasing at intervals of about 10%.
Three sets of flour samples were included in the experiments. Firstly, with pure strains without clustering of wheat with a high amylose content of the reduced SBEIIa strains, an AC45.1 mid-level amylose wheat strain that was transformed with the hp-combo construct having about 50% amylose ( Example 2) and control wheat (NB1). Second, with wheat material grouped from the transformed strains as described in Example 2, grouping samples at 10% intervals increasing the amylose content. Third, comparison of flour from different species including wheat (high amylose content, wild type, and wheat without SSIIa), barley (wild type, high amylose content by reduced SBEIIa and SBEIIb, and high content of reduced amylose by SSIIa ), and high amylose corn. The results of the analysis of resistant starch in wheat material grouped with a range of amylose content revealed a linear increase in resistant starch of an amylose content of> 40%.
EXAMPLE 14: PRODUCTION OF BREADS AND OTHER FOOD PRODUCTS One of the most effective ways to distribute a grain such as wheat with a high amylose content in the diet is through bread. To show that wheat with a high amylose content could be quickly incorporated into breads and to examine the factors that allowed the quality of bread to be retained, samples of flour were produced, analyzed and used in baking. The following methods were employed.
Methods. The wheat grains were conditioned to 16.5% moisture content overnight and ground with a scale from the Buhler laboratory in BRI Ltd, Australia, or using a Quadromat Junior mill followed by sieving, to achieve a final particle size of 150 μm. The protein and moisture content of the samples were determined by infrared reflection (NIR) according to Method AACC 39-11 (1999), or by the Dumas method and air-oven according to Method AACC 44-15 A ( AACC5 1999).
Mixture of Micro Z-arm. The ideal water absorption values of the wheat flour were determined with the Micro Z-arm mixer, using 4 g of test flour per mixture (Gras et al, (2001); Bekes et al, (2002). constant with speeds of rotation for the fast and slow blades of 96 and 64 rpm, respectively, was used during all the mixtures. The mixture was carried out in triplicate, each one for 20 minutes. Before adding water to the flour, the baseline was automatically recorded (30 sec) by mixing only solid components. Water was added in one step using an automatic water pump. The following parameters were determined from the individual mixing experiments taking the averages: WA% - Absorption of Water was determined at the mass consistency of the Brabender 500 Unit (BU); Mass Development Time (DDT): time to peak resistance (sec).
Mixograms. To determine the ideal parameters for mixing the dough with the modified wheat flour, samples with variable water absorption corresponding to the water absorption determined by the Micro Z-arm mixer, were mixed in a 10 g CSIRO Mixograph prototype keeping the mass constant total mass. For each of the flour samples, the following parameters were recorded: MT - mixing time (sec); PR - peak resistance of the Mixographer (Arbitrary Units, AU); BWPR - peak resistance bandwidth (Arbitrary Units, AU); RBD - breaking of resistance (%); BWBD - break in bandwidth (%); TMBW - time for minimum bandwidth (s); and MBW - maximum bandwidth (Arbitrary Units, A.U.).
Microextension test. The mass extension parameters were measured as follows: the masses were mixed to the peak of mass development in a 10 g Mixograph prototype. The extension tests at 1 cm / s were performed on a TA.XT2i texture analyzer with a modified geometry Kieffer mass and gluten extension platform (Mann et al, 2003). The mass samples for extension test (~ 1.0 g / test) were molded with a Kieffer molder and rested at 30 ° C and 90% RH for 45 min before the extension test. The R_Max and Ext Rmax were determined from the data with the help of the Exceed Expert software (Smewing, The measurement of dough and gluten extensibility using the SMS / Kieffer rig and the TA.TX2 texture analyzer handbook, SMS Ltd: Surrey, UK, 1995; Mann, (2002).
An illustrative recipe based on 14 g of flour as 100% was as follows: flour 100%, salt 2%, dry yeast 1.5%, vegetable oil 2%, and renewer (ascorbic acid 100 ppm, fungal amylose 15 ppm, xylanase 40 ppm, soy flour 0.3%, obtained from Goodman Fielder Pty Ltd, Australia) 1.5%. The level of water addition was based on the micro Z-arm water absorption values that were adjusted for the complete formula. Flour (14 g) and the other ingredients were mixed for the peak time of dough development in a 35g Mixograph. The molding and panorama were carried out in two-stage verification steps at 40 ° C at 85% RH. Bakery was carried out in a Rotel oven for 15 min at 190 ° C. The volume of the bread (determined by the canola seed displacement method) and weight measurements were taken after cooling on a shelf for 2 hours. The loss of liquid water was measured by weighting the loaves over time.
Flour or wholegrain flour can be mixed with flour or wholegrain flour from unmodified wheat or other cereals such as barley to provide the desired dough and baking or nutritional qualities. For example, Chara or Glenlea cv flour has a high dough resistance while Janz cv has a medium dough resistance. In particular, the levels of glutenin subunits of high and low molecular weight in the flour are positively correlated with the resistance of the dough, and still influenced by the nature of the alleles present.
Flour from transgenic wheat strains that have reduced SBEIIa was used at 100%, 60% and 30% addition levels, for example, all flours came from various wheat strains or 60% or 30% were added to the Control flour Bakery (B. extra). The percentages are of the total flour in the bread formulation. Four lines of transgenic wheat were used as follows: 072 (reduced SBEIIa), 212 (a cross-derived wheat line, reduced SBEIIa x triple-null SBEI wheat), H7 (a cross-derived wheat line, reduced SBEIIa x wheat of SSIIa triple-null) and 008 (reduced SBEIIb) were tested together with an untransformed control wheat (NB1). All wheat was ground in a Brabender Quadramat Junior mill. All mixtures had water absorptions determined in the 4 g Z-arm mixer and ideal mixing times determined in the 10 g mixograph as described above. These conditions were used in the preparation of the 10 g test breads.
Mixing Properties. In addition to the control lines (Bakery Control, NB1 and 008), all other wheat lines yielded highly high water absorption values (Figure 17 (a)). Strains 212 and 072 yielded increasing absorption values with increasing levels of addition, including up to a maximum of 95% water absorption in 100% addition of flour 212. The increased incorporation levels of flour from these lines also led to a decrease in the ideal mixing times of the Mixographer (Figure 17 (b)). As with water absorption data, uncontrolled strains showed a strong reduction in specific bread volume (bread volume / bread weight) with increasing levels of addition. The effect was particularly strong for the 212 strain.
These studies show that breads with commercial potential, including acceptable crumb structure, texture and appearance could be obtained using wheat flour with a high amylose content mixed with control flour samples. In addition, high amylose wheat is used in combination with preferred genetic background characteristics (for example, preferred high and low molecular weight glutenins), the addition of enhancers such as gluten, ascorbate or emulsifiers, or the use of different styles in the making of bread (for example, making sponge or dough bread, sour dough, mixed grains, or wholegrain flour) to provide a range of products with particular utility and nutritional efficacy to improve gut and metabolic health.
Other food products: yellow alkaline noodles (YAN) (100% flour, 32% water, 1% Na2C03) were prepared in a Hobart mixer using the standard BRI Research Pasta Manufacturing Method (AFL 029). The pasta sheet was formed on the stainless steel rolls of an Otake machine. After resting (30 min), the pasta sheet was reduced and cut into strips. The dimensions of the pasta were 1.5 x 1.5 mm.
Instant noodles (100% flour, 32% water, 1% NaCl and 0.2% Na2C03) were prepared in a Hobart mixer using the BRI Research standard pasta manufacturing method (AFL 028). The pasta sheet was formed on the stainless steel rolls of an Otake pasta machine. After resting (5 min), the pasta sheet was reduced and cut into strips. The dimensions of the pasta were 1.0 x 1.5 x 25 mm. The strips of pasta were cooked for 3.5 minutes and then fried in oil at 150 ° C for 45 sec.
Sponge Bread and Pasta (S&D). BRI Research's dough and sponge baking involves a two-step process. In the first stage, the sponge was made by mixing part of the total flour with water, yeast and yeast foods. The sponge was allowed to ferment for 4 h. In the second stage, the sponge was incorporated with the rest of the flour, water and other ingredients to make the dough. The sponge stage of the process was made with 200 g of flour and was given 4 h for fermentation. The dough was prepared by mixing the remaining 100 g of flour and other ingredients with the fermented sponge.
Pasta-Spaghetti. The method used for mass production was as described in Sissons et at, (2007). The test sample flours of high amylose wheat (reduced SBEIIa) and control wheat (NB1) were mixed with Manildra semolina in various percentages (test sample: 0, 20, 40, 60, 80, 100%) to obtain flour mixes for dough preparation on a smaller scale. The samples were corrected to 30% humidity. The desired amount of water was added to the samples and mixed quickly before being transferred to a 50 g flour bowl for a further 2 min mixture. The resulting mass, which resembled crumbs the size of a coffee bean, was transferred to a stainless steel chamber and rested at a pressure of 7000 kPa for 9 min at 50 ° C. The dough was then extruded at a constant rate and cut to lengths of approximately 48 cm. Two batches of dough were made for each sample. The dough was dried using a Thermoline Humidity and Temperature Cabinet (TEC 2604) (Thermoline Scientific Equipment, Smithfield, Australia). The drying cycle consisted of a maintenance temperature of 25 ° C followed by an increase to 65 ° C for 45 min then a period of about 13h at 50 ° C followed by cooling to 25 ° C. The humidity was controlled during the cycle. The dry paste was cut into 7 cm strips for subsequent testing.
EXAMPLE 15: IN VITRO MEASUREMENTS OF GLYCEMIC INDEX (GI) AND RESISTANT STARCH (AIR) FROM FOOD SAMPLES
The Glycemic Index (GI) of food samples including bread made as described here was measured in vitro as follows: the food samples were homogenized with a home food processor. A sample amount representing approximately 50 mg of carbohydrate was weighed in a 120 ml plastic sample container and 100 μl of carbonate buffer without α-amylose. Approximately 15 to 20 seconds after the addition of carbonate buffer, 5 ml of Pepsin solution (65 mg of pepsin (Sigma) dissolved in 65 ml of 0.02M HCl, pH 2.0, made up to the day of use) were added, and the mixture incubated at 37 ° C for 30 minutes in an alternative water bath at 70 rpm. Following the incubation, the sample was neutralized with 5 ml of NaOH (0.02M) and 25 ml of 0.2M acetate buffer, pH 6 added. 5 ml of the enzyme mixture containing 2 mg / ml of pancreatin (α-amylase, Sigma) and 28U / ml of amyloglycosidase from Aspergillus niger (AMG, Sigma) dissolved in Na acetate buffer (sodium acetate buffer, 0, 2 M, pH 6.0, containing 0.20 M calcium chloride and 0.49 mM magnesium chloride) were then added, and the mixture incubated for 2 to 5 minutes. 1 ml of the solution was transferred to each vial in a 1.5 ml tube and centrifuged at 3000 rpm for 10 minutes. The supernatant was transferred to a new tube and stored in a freezer. The remainder of each sample was covered with aluminum foil and the containers incubated at 37 ° C for 5 hours in a water bath. An additional 1 ml of the solution was then collected from each flask, centrifuged and the supernatant transferred as previously performed. It was also stored in a freezer until absorbances could be read.
All samples were thawed at room temperature and centrifuged at 3000 rpm for 10 minutes. The samples were diluted as needed (1 in 10 dilutions normally sufficient), 10 μl of supernatant transferred from each sample to 96 well microtiter plates in duplicate or triplicate. A standard curve for each microtiter plate was prepared using glucose (0 mg, 0.0625 mg, 0.125 mg, 0.25 mg, 0.5 mg and 1.0 mg). 200 μl of Glucose Trinder reagent (Microgenetics Diagnostics Pty Ltd, Lidcombe, NSW) was added to each well and the plates incubated at room temperature for approximately 20 minutes. The absorbance of each sample was measured at 505 nm using a plate reader and the amount of glucose calculated with reference to the standard curve.
The level of Resistant Starch (AR) in food samples including bread made as described here was measured in vitro as follows. This method describes the preparation of the sample and in vitro digestion of starch in foods, as normally eaten. The method has two sections: first, the starch in the food has been hydrolyzed under simulated physiological conditions; second, the by-products were removed by washing and the residual starch determined after homogenization and drying of the sample. The starch quantified at the end of the digestion treatment represented the content of resistant starch in the food.
On day 1, the food samples were processed in a manner simulating consumption, for example, by homogenization with a domestic food processor for consistency as would be achieved by chewing. After homogenization, an amount of food representing up to 500 mg of carbohydrate was weighed in a 125 mL Erlenmeyer flask. A carbonate buffer was prepared by dissolving 121 mg of NaHCO3 and 157 mg of KC1 in approximately 90 mL of purified water, adding 159 μL of 1 M CaCl2.6H2O solution and 41 μL of 0.49 M MgCl2.6H2O, adjusting the pH to 7 to 7.1 with 0.32 M HCl, and adjusting the volume to 100 mL. This buffer was stored at 4 ° C for up to five days. A solution of artificial saliva containing 250 units of α-amylase (Sigma A-3176 Type VI-B of the pig pancreas) per ml of the carbonate buffer was prepared. An amount of the artificial saliva solution, approximately equal to the weight of the food, was added to the flask. About 15 to 20 sec after adding saliva, 5 ml of pepsin solution in HCl (1 mg / ml of pepsin (Sigma) in 0.02 M HCl, pH 2.0, made on the day of use) were added in each bottle. The mixture of amylase and then pepsin mimicked human chewing of the food before swallowing it. The mixture was incubated at 37 ° C for 30 min with shaking at 85 rpm. The mixture was then neutralized with 5 ml of 0.02M NaOH. 25 ml of acetate buffer (0.2 M, pH 6) and 5 ml of pancreatin enzyme mixture containing 2 mg / ml pancreatin (Sigma, pig pancreas at 4 x USP activity) and 28U were added per bottle amyloglycosidase (AMG, Sigma) from Aspergillus niger in acetate buffer, pH6. Each flask was closed with aluminum foil and incubated at 37 ° C for 16 hours in an alternative water bath set at 85 rpm.
On day 2, the contents of each flask were transferred quantitatively to a 50 ml polypropylene tube and centrifuged at 2000 x g for 10 min at room temperature. The supernatants were discarded and each pellet was washed three times with 20 mL of water, with a gentle vortex in the tube with each wash to break the pellet, followed by centrifugation. 50 μL of the last water wash was tested with a Glucose Trinder reagent for the absence of free glucose. Each pellet was then resuspended in approximately 6 mL of purified water and homogenized three times for 10 seconds using an Ultra Turrax TP18 / 10 with an S25N-8G dispersion tool. The contents are quantitatively transferred to a 25 ml volumetric flask and made to give volume. The contents were mixed vigorously and returned to the polypropylene tube. A 5 mL sample of each suspension was transferred to a 25 mL culture tube and the shell was immediately frozen in liquid nitrogen and lyophilized.
On day 3, the total starch in each sample was measured using reagents provided in the Megazyme Starch Total Starch Procedure kit. Starch standards (Regular Corn Starch, Sigma S-5 5296) and a reagent blank assay were prepared. The samples, controls and reagent blanks were wetted with 0.4 mL of 80% ethanol to aid dispersion, followed by the vortex. Immediately, 2 mL of DMSO was added and the solutions mixed by vortexing. The tubes were placed in a boiling water bath for 5 min, and 3 mL of thermostable α-amylase (100 U / ml) in MOPS buffer (pH 7, containing 5 mM CaCl2 and 0.02% sodium azide ) added immediately. The solutions were incubated in the boiling water bath for another 12 min, with a vortex mixture at 3 min intervals. The tubes were then placed in a 50 ° C water bath and 4 ml of sodium acetate buffer (200 mM, pH 4.5, containing 0.02% sodium azide) and 0.1 ml of amyloglycosidase a 300 U / ml added. The mixtures were incubated at 50 ° C for 30 min with gentle mixing at 10 min intervals. The volumes were made up to 25 mL in a volumetric flask and mixed cavity. The aliquots were centrifuged and 2000 x g for 10 min. The amount of glucose in 50 μL of supernatant was determined with 1.0 mL of Glucose Trinder reagent and measuring absorbance at 505 nm after incubating the tubes at room temperature in the dark for a minimum of 18 min and a maximum of 45 min .
The flour breads from four transgenic wheat lines, ie 072 (reduced SBEIIa), 212 (a crossbred wheat line, reduced SBEII x triple-null wheat from SBEI), H7 (a wheat line derived from the cross) crossbreeding, reduced SBEIIa x SSIIa triple-null wheat) and 008 (reduced SBEIIb) were tested with unprocessed control wheat (NB1) by AR and IG after levels of 100%, 60% and 30% flour incorporation, the remainder 40% or 70% of flour being of the wild type grain. High incorporation of flour 212, 072 and H7 resulted in significant increases in RA (Figure 18 (a) and reductions in the predicted GI (Figure 18 (b)). The magnitude of the changes was greater when using the Line 212 flour. For example , bread made with 100% addition of this flour with a high amylose content has an AR content of about 10% which represented an increase of 150% above that to 30% of the inclusion level of a 9-fold increase compared to controls of NB 1. Increasing the extent of incorporation of flour from strains 008 had no effect on the AR and GI of the resulting breads and the results were comparable to those of the control bread.
EXAMPLE 16: PROCESSING OF WHEAT WITH HIGH AMYLOSE CONTENT AND RESULTING AIR LEVELS A small-scale study was conducted to determine the content of resistant starch (RA) in the grain processed from wheat with high amylose content that has been crushed or is crushed. in flakes. The technique involved conditioning the grains at a humidity level of 25% for one hour, followed by cooking the grains. Following cooking, the beans were crushed using a small-scale roll. The flakes were then roasted in an oven at 120 ° C for 35 min. Two roll widths and three cooking times were used in approximately 200 g of wheat samples with high amylose content having reduced SBEIIa (HAW, line 85.2c) and wild type, control wheat (cv. Hartog). The tested roll widths were 0.05 mm and 0.15 mm. The cooking times tested were 60 ', 45'and 35'.
This study showed a clear and substantial increase in the amount of RA in wheat with a high content of processed amylose compared to the control. (Table 27, Figure 18). Also, there appeared to be some effect of processing conditions at the RA level. For example with the grain with a high amylose content, the long cooking times led to a slight reduction in the AR level, probably due to the high gelatinization of the starch during cooking (Table 27). The wider opening of the roll generated a higher level of AR except for the longest cooking time. This may have been due to the high shear damage of the starch granules when the grains were rolled into narrower openings, slightly reducing the levels of AR. The narrower openings of the roller led to higher levels of RA in Hartog control, albeit at much lower total RA levels. In contrast to the results with high amylose content, high cooking times led to higher levels of RA possibly due to high starch gelatinization in very long cooking times contributing to the retrogradation of more starch during processing and subsequent cooling.
Consolidated data in RA of several products. The RA data obtained from various products such as pasta, sponge bread and pasta and spaghetti, prepared as described in Example 10, are shown in Table 28. Not all levels of incorporation have been tested by all products, but levels of incorporation 20%, 40% and 60% were used in most of the analyzed products. The results showed a linear relationship between the AR content and the level of incorporation of flour with a high amylose content.
EXAMPLE 17: ISOLATION OF PLANTS THAT HAVE SPECIAL MUTATIONS IN SBEIIA A population of mutated plant strains was developed after EMS mutagenesis of the seeds of the Arche or Apache wheat cultivars, using standard EMS treatment conditions. About 5,000 Apache and 900 Arche individual M1 plants were grown from the self-fertilized mutagenized seed and seeds from each plant and subsequent generations of progeny maintained as potentially mutant strains, each derived from an individual M1 plant. The strains were examined for mutations in the three homologous SBEIIa genes by sequencing Solexa of the next generation (Illumina). To do this, 7 groups of DNA were prepared, each by grouping the DNA of about 130 M1 families from the Arche population and 96 from the Apache population. PCR was performed on the DNAs grouped by 3 or 4 regions per homologous gene, targeting the exonic regions including gene fitting sites. Genome specific primers are defined in Table 29.
The 10 amplicons (amplification products) from the same groups of DNA were fused after normalization of the PCR products, and sequencing was done with one flow cell per group of DNA. Sequence data were analyzed to select from all of the polymorphisms the most likely due to mutations instead of sequencing errors, based on the frequencies of the observed polymorphisms. 64 putative mutants from the Arche population and 48 from the Apache population were observed from the first sequence analysis covering the exonic regions and fitting locations. SNP assays were designed for each polymorphism based on kaspar technology, and genotyping was performed on 130 families in each group that was positive for the particular polymorphism. In this way, the individual mutant lineage containing each mutant gene was identified and the SBEIIa mutant sequences confirmed.
By this method, 31 mutant strains from the Apache population and 9 from the Arche population were identified each having an SBEIIa mutation, and M2 kernels from each retained. From each mutant strain, depending on availability, about 10 M2 seeds were cut in half, half without the germ was used for DNA extraction and analysis, the other half with the germ was saved by sowing. A total of 5 mutants were confirmed in half of the seeds of the Arche population and 28 of the Apache population: The corresponding seeds were sown to produce progeny plants to confirm that the mutations were inherited in the Mendelian model by repeating the analysis on the leaf material. M2 plant, providing much better DNA quality. These analyzes confirmed 19 mutants, 4 from the Arche population and 15 from the Apache population and allowed their position depending on their DNA and the deduced protein sequences encoded by the mutants.
The mutants obtained included those that had SBEIIa genes mutated with stop codons in the protein coding regions of the SBEIIa genes in the B or D genomes, causing premature termination of the translation of the SBEIIa proteins, and strains with mutations of docking sites in the SBEIIa-B or -D genes. Such mutations were expected to be null mutations. Point mutations in the SBEIIa-A, SBEIIa-B and SBEIIa-D genes such as acid substitution mutations have also been obtained and their impact on the structure of predicted encoded proteins using Blosum 62 and Pam 250 matrices.
The plants of the 8 most promising mutant strains were crossed with SBEIIa double-null mutants of the appropriate genotype including A1D2, A2D2, A1B2, B2D2 genotypes in order to produce the triple mutant plants and seeds in the F2 generation. Fertile plants producing seed with at least 50% amylose in the starch content are selected. The mutant plants were also crossed with durum wheat (Soldur cultivar) to introduce the mutations into the tetraploid wheat. Table 1. Starch branching enzyme genes characterized from cereals.

Table 2. Subclassification of amino acid
Table 3. Conserved Amino Acid Substitutions
Table 4. Genome specific primers for
Table 5. Nucleotide sequences of primers
Table 6. Primers designed to amplify parts of the SBEIIa gene specifically from the wheat A genome - detected polymorphisms and fragment sizes
Table 7. Primers designed to amplify parts of the SBEIIa gene specifically from the extra wheat B / D genome

Table 8. Primers designed to amplify parts of the SBEIIa gene specifically
Table 9. Genome specific primers for wheat SBEIIa genes
Table 10. Nucleotide sequences of
5 Table 11. Expression of SBEII versus content of
Table 12. List of microsatellite markers tested on mutants

Table 13. Mutants identified from the HIB population and microsatellite mapping data

Table 14. SBEII double-null mutants identified
Table 15. Crosses performed between single and double-null mutants

Table 16. Amylose content in the starch of the progeny grain of the crosses between double-null and single-null mutants Strains Genotype% amylose
Table 17. Fertility observations in 5 F2 progeny plants
Table 18 AlD2 (hetB2) AlB2 (hetD2) All) 2 (hetB2)

Table 19. Other crosses between single and double-null mutants

Table 20. Frequency of genotypes observed for normal seed germination of an A2B2D2 cross. Numbers in parentheses indicate the expected frequency based on Mendelian segregation

Table 21. Other crosses between single and double-null mutants
Table 22. Putative double and triple-null mutants in SBEIIa genes in an initial screening using dominant markers.
Table 23. Characterization of starch starch in grain 5 of transgenic wheat lines
Table 24. Molecular weight distribution of starch fractions from transgenic wheat lines
Table 25. RVA parameters of hp5'-SBEIIa transgenic wheat starch
Table 26.

Table 27. AR content in milled and flaked grain products
Table 28. Resistant starch content in food products at a variable level of wheat incorporation with
Table 29. Genome specific primers

权利要求:
Claims (15)
[0001]
1. METHOD TO PRODUCE WHEAT FLOUR,, characterized by grinding wheat grain (TRITICUM AESTIVUM) comprising a germ, an endosperm, starch and a reduced amount or activity of protein II (SBEII) of total starch branching enzyme, in that the germ comprises a loss of function mutation in each of the 5 to 12 alleles of the endogenous SBEII genes selected from the group consisting of SBEIIA-A, SBEIIA-B, SBEIIA-D, SBEIIB-A, SBEIIB-B and SBEIIB- D, such that the amount or activity of the total SBEII protein in the grain is between 2% and 30% of the amount or activity of the total SBEII protein in the wild type wheat grain, said 5 to 12 alleles including 4, 5 or 6 SBEIIA alleles each comprising a loss of function mutation, where when the number of SBEIIA alleles comprising a loss of function mutation is only 4, then the number of SBEIIB alleles comprising a loss of function mutation is 6, in that the 4, 5 or 6 alleles of SBEIIA that comprise a mutation loss function comprise at least one point mutation in a SBEIIA gene or a 1-30 base pair deletion in a SBEIIA gene, in which at least one of the loss of function mutations is an introduced mutation, and in which the grain comprises an amylose content of at least 50% (w / w) as a proportion of the total starch in the grain.
[0002]
2. METHOD, according to claim 1, characterized in that the flour is whole wheat flour.
[0003]
METHOD, according to either of claims 1 or 2, characterized in that: (i) the grain germ is homozygous for alleles in each of 2 or 3 SBEIIA genes, each of the homozygous alleles comprising a loss mutation of function, (ii) the grain comprises an allele which comprises a partial loss of function mutation that expresses an SBEIIA or SBEIIB enzyme which in quantity and / or activity corresponds to between 2% and 60%, or between 10% and 50%, of the amount or activity of the corresponding wild type allele, (iii) the number of null alleles of the SBEIIA genes in the germ is 2 or 4, (iv) the SBEIIA 4, 5 or 6 alleles, each comprising a mutation of loss of function, each one is null, (v) the grain does not have null alleles of the SBEIIB genes, or the number of null alleles of the SBEIIB genes in the grain is 2, 4 or 6, (vi) the grain comprises two SBEIIB alleles each comprising a partial loss of function mutation, (vii) the grain comprises null S gene alleles BEIIA in genome A, genome B, genome D, genomes A and B, genomes A and D, or genomes B and D, (viii) the grain comprises null alleles of the SBEIIB gene in genome A, genome B, genome D, genomes A and B, genomes A and D, genomes B and D, or all three of genomes A, B and D, (ix) the grain comprises only one or only two SBEIIB proteins that have starch branching enzyme activity when produced in the developing endosperm, or just one or just two SBEIIB proteins that are detectable by Western blot analysis (x) the grain comprises a null mutation which is an exclusion mutation in the A, B or D genome that excludes at least part of a SBEIIA gene and at least a part of the SBEIIB gene in the genome, (xi) the grain comprises a null mutation which is a deletion mutation in genome A, B or D that deletes the entire SBEIIA gene and / or all SBEIIB gene in that genome, (xii) the grain comprises a null mutation in an SBEIIA gene that is an amino acid substitution mutation, ( xiii) the germ comprises a null mutation in one SBEIIA gene, or null mutations in more than one SBEIIA gene, in which each mutation is independently selected from the group consisting of an exclusion mutation, an insertion mutation, a mutation of local splice, a premature translation termination mutation and a framing mutation, (xiv) the grain has only one SBEIIA protein as determined by Western blot analysis, in which said protein is encoded by one of the SBEIIA-A genes , SBEIIA-B, and SBEIIA-D e have reduced starch branching enzyme activity when produced in the development of wheat endosperm when compared to an SBEIIA protein encoded by the corresponding wild type gene, or (xv) a combination of more than one of (i) to (xiv).
[0004]
4. METHOD according to any one of claims 1 to 3, characterized in that the grain comprises an amount or activity of the total SBEII protein between 2% and 15%, or between 3% and 10%, or between 2% and 20% or between 2% and 25% of the amount or activity of the total SBEII protein in the wild type wheat grain.
[0005]
5. METHOD according to any one of claims 1 to 4, characterized in that the grain comprises an amount or activity of the SBEIIA protein less than 2%, or between 2% and 15%, or between 3% and 10%, or between 2% and 20% or between 2% and 25% of the quantity or activity of the SBEIIA protein in the wild type wheat grain.
[0006]
Method according to any one of claims 1 to 5, characterized in that the grain comprises an amylose content of at least 60% (w / w) or at least 67% (w / w) as a proportion of the total starch in the grain.
[0007]
METHOD, according to any one of claims 1 to 6, characterized in that the grain is free of any exogenous nucleic acid encoding an RNA that reduces the expression of a SBEIIA gene.
[0008]
METHOD according to any one of claims 1 to 7, characterized in that the grain has starch that comprises one or more of the properties selected from the group consisting of: (i) comprising at least 2% resistant starch, (ii) comprising a reduced glycemic index (GI), (iii) comprise a reduced amount of amylopectin, (iv) comprise distorted starch granules, (v) reduced starch granule birefringence, (vi) reduced swelling volume, (vii) length distribution modified chain and / or branching frequency, (viii) increased peak gelatinization temperature, (ix) reduced viscosity, (x) high molecular weight of amylopectin, (xi) reduced percentage of starch crystallinity, and (xii) reduced percentage of crystalline starch type A or type B, in relation to starch or granules of wild type wheat starch.
[0009]
9. WHEAT FLOUR, produced using the method as defined in any one of claims 1 to 8, the flour characterized by comprising a reduced amount or activity of the total SBEII protein, wherein the DNA of the whole flour or flour comprises a mutation of loss of function in each of the 5 to 12 alleles of the endogenous SBEII genes selected from the group consisting of SBEIIA-A, SBEIIA-B, SBEIIA-D, SBEIIB-A, SBEIIB-B and SBEIIB-D, such that the amount or activity of the total SBEII protein in the grain is between 2% and 30% the amount or activity of the total SBEII protein in the wild type wheat grain, said 5 to 12 alleles including 4, 5 or 6 alleles of SBEIIA each comprising a loss mutation of function, where when the number of SBEIIA alleles comprising a loss of function mutation is only 4, then the number of SBEIIB alleles comprising a loss of function mutation is 6, where the 4, 5 or 6 alleles of SBEIIA that comprise a mutation by loss of fu nition comprise at least one point mutation in a SBEIIA gene or a 1-30 base pair deletion in a SBEIIA gene, in which at least one of the loss of function mutations is an introduced mutation, in which the flour starch has an amylose content of at least 50% (w / w), and in which the flour or flour has been refined by fractionation, bleaching and / or heat treatment to stabilize the wheat flour.
[0010]
10. WHEAT FLOUR, according to claim 9, characterized in that the flour is whole flour.
[0011]
11. FLOUR according to either of claims 9 or 10, wherein the flour starch is characterized by one or more of: (i) comprising at least 60% (w / w), or at least 67% (w / p) amylose, (ii) comprise at least 2% resistant starch, (iii) comprise a reduced glycemic index (GI), (iv) comprise a reduced amount of amylopectin, (v) comprise distorted starch granules, ( vi) reduced starch granule birefringence, (vii) reduced swelling volume, (viii) modified chain length distribution and / or branching frequency, (ix) gelatinization, (x) (xi) increased peak viscosity , high molecular weight of amylopectin temperature, of (xii) a reduction in the percentage of crystallinity, and / or (xiii) a reduction in the percentage of crystalline starch type A or B, in relation to granules of wild-type wheat starch or starch .
[0012]
12. FOOD INGREDIENT, characterized in that it comprises wheat flour, as defined in any one of claims 9 to 11.
[0013]
13. FOOD PRODUCT, characterized in that it comprises a food ingredient in an amount of at least 10% on a dry weight basis, wherein the ingredient is flour, as defined in any one of claims 9 to 11, or the food ingredient as defined in claim 12.
[0014]
14. FOOD PRODUCTION METHOD, characterized in that it comprises the step of adding the food ingredient of claim 12 to another food ingredient, thereby producing the food.
[0015]
15. STARCH PRODUCTION METHOD, characterized in that it comprises the step of extracting starch from the flour, as defined in any one of claims 9 to 11, thereby producing the starch.

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法律状态:
2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A23L 7/152 (2016.01), A23L 7/10 (2016.01), C08B 30 |

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-05-15| B15K| Others concerning applications: alteration of classification|Ipc: C12N 15/52 (2006.01), C12N 9/10 (2006.01), C12N 15 |

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2019-06-04| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-12-03| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-07-21| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-12-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-02| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 04/11/2011, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-05-25| B09W| Decision of grant: rectification|Free format text: RETIFICACAO DA PUBLICACAO DEVIDO A INCORRECOES NO TITULO DO RELATORIO DE EXAME TECNICO DO PARECER DE DEFERIMENTO. |

优先权:

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申请号 | 申请日 | 专利标题

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US41028810P| true| 2010-11-04|2010-11-04|

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US61/410,288|2010-11-04|

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PCT/AU2011/001426|WO2012058730A1|2010-11-04|2011-11-04|High amylose wheat|

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