polynucleotides related to the herbicide tolerance event 8291.45.36.2, expression cassette, probe, a

专利摘要:
POLYNUCLEOTIDES RELATING TO THE HERBICIDE TOLERANCE EVENT 8291.45.36.2, EXPRESSION CASSETTE, PROBE, TRANSGENIC INSERT, AS WELL AS WEED CONTROL PROCESSES, FOR IDENTIFICATION OF THE EVENT, DETERMINATION OF ZIGOSITY AND PRODUCTION OF A PLANT PRODUCTION PROTEIN IN A PLANT CELL. This invention relates to the soybean event pDAB8291. 45.36.2, which includes a new expression cassette that comprises several characteristics that confer resistance to glyphosate, aryloxyalkanoate and glufosinate herbicides. This invention also relates in part to resistant weed control processes, plant crossing and herbicide-tolerant plants. In some embodiments, the sequence of the event can be "accumulated" with other characteristics, including, for example, other herbicide tolerance gene (s) and / or insect inhibiting proteins. This invention also relates in part to detection methods, including TaqMan PCR endpoint assays, for the detection of Event pDAB8291.45.36.2 in soybeans and related plant material. Some modalities can perform high yield zygosity analysis of plant material and other modalities can be used to exclusively identify the zygosity of and the crossed soybean lines that comprise the event of the present invention. (...)
公开号:BR112013015745B1
申请号:R112013015745-3
申请日:2011-12-02
公开日:2021-01-19
发明作者:Yunxing Cui；Thom Hoffman；Ning Zhou；Stephen N. Novak；Julissa Colon；Dawn Parkhurst；Sandra Toledo；Terry Wright；Sean Russell；Bruce Held；Vaithilingam Sekar
申请人:Ms Technologies, Llc；Dow Agrosciences Llc；
IPC主号:

专利说明:

BACKGROUND OF THE INVENTION
[001] Glyphosate (N-phosphonomethylglycine), a broad-spectrum herbicide, inhibits 5-enolpyruvylchiquimate-3-phosphate synthase (EPSPS), an enzyme in the biosynthetic pathway that produces essential aromatic amino acids in plant cells. EPSPS inhibition efficiently disrupts protein synthesis and thereby kills affected plant cells. Due to the fact that glyphosate is non-selective, it kills both weeds and crop plants. Thus, it is useful with crop plants when crop plants can be modified to be resistant to glyphosate, allowing desirable plants to survive exposure to glyphosate.
[002] Recombinant DNA technology was used to isolate mutant EPSP synthases that are resistant to glyphosate. Such glyphosate resistant mutant EPSP synthases can be transformed into plants and impart glyphosate resistance to transformed plants. For the purpose of example, a glyphosate tolerance gene was isolated from the CP4 strain of Agrobacterium as described in U.S. Patent No. 5,633,435. This reference and all references cited here are hereby incorporated by reference.
[003] Other glyphosate tolerance genes were created through the introduction of mutations. These include the AroA gene isolated by Comai and described in U.S. Patent Nos. 5,094,945, 4,769,061 and 4,535,060. A single mutant was isolated, as described in U.S. Patent No. 5,310,667, by replacing an alanine residue with a glycine residue between amino acid positions 80 and 120. The double mutants have been described in U.S. Patent Nos. 6,225,114 and 5,866,775 in which, in addition to the previous mutation, a second mutation (a threonine residue by an alanine residue between positions 170 and 210) was introduced into a wild-type EPSPS gene.
[004] Another work resulted in the production of glyphosate-resistant corn by introducing a modified EPSPS gene from corn carrying mutations in residue 102 (changing threonine to isoleucine) and residue 106 (changing proline to serine) of the encoded amino acid sequence GenBank Access No. X63374. See U.S. Patent Nos. 6,566,587 and 6,040,497.
[005] Examples of events that provide glyphosate resistance in soy include the soybean strain GTS 40-3-2 (Padgette et al. 1995), the MON89788 soy event (US Patent No. 7,608,761), the US patent No. 7,608,761 refers to the MON89788 soybean event, of which each was produced by inserting the cp4 epsps gene into the soybean.
[006] The widespread adoption of the glyphosate-tolerant crop plant system and the increasing use of glyphosate have contributed to the prevalence of glyphosate resistant and difficult to control weeds in recent years. In areas where growers are face to face with glyphosate resistant weeds or a shift to more difficult to control weed species, growers can balance glyphosate weakness by tank mixing or alternating with other herbicides that will control the remaining weeds.
[007] A popular and effective tank mixing partner for controlling broad leaf leaks in many cases has been 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D, which has been used as a herbicide for more than 60 years, provides broad-spectrum post-emergence control of many annual, biennial and perennial broadleaf weeds including several important weeds in corn, soybeans and on cotton. Important weeds controlled by 2,4-D (rates of 560 - 1120 g ae / ha) in the production of row crop plants include Ambrosia artemisiifolia, Ambrosia trifida, Xanthium strumarium, Chenopodium album, Helianthus annuus, Ipo-moea sp ., Abutilon theophrasti, Conyza Canadensis and Senna obtusifolia. 2,4-D provides partial control of several important weeds including Polygonum pensylvanicum, Polygonum persicaria, Cirsium arvense, Taraxacum officinale and Amaranthus sp. including Amaranthus rudis and Amaranthus palmeri.
[008] A limitation for the additional use of 2,4-D is that its selectivity in dicotyledonous plants such as soybeans or cotton is very weak and so 2,4-D is not typically used in (and generally not close to) sensitive dicot plants. In addition, the use of 2,4-D in grass cultivation plants is somewhat limited by the nature of the damage to the cultivation plant that can occur. 2,4-D in combination with glyphosate has been used to provide a more robust burndown treatment before planting soybeans and no-till cotton; however, due to the sensitivity of these dicot species to 2,4-D, these burndown treatments have to occur at least 14-30 days before planting (Agriliance, 2005).
[009] An organism that has been extensively researched for its ability to degrade 2,4-D is Ralstonia eutropha, which contains a gene that encodes tfdA (Streber et al., 1987), an enzyme that catalyzes the first stage in mineralization pathway. (See U.S. Pat. No. 6,153,401 and GENBANK Accession No. M16730). TfdA has been reported to degrade 2,4-D (Smejkal et al., 2001). Products that result from degradation have little or no herbicidal activity compared to 2,4-D. tfdA has been used in transgenic plants to confer resistance to 2,4-D in dicotyledonous plants (for example, cotton and tobacco) normally sensitive to 2,4-D (Streber et al. (1989), Lyon et al. (1989), Lyon (1993) and US Pat. No. 5,608,147).
[0010] A number of tfdA-like genes that encode proteins capable of degrading 2,4-D have been identified from the environment and deposited in the Genbank database. Many homologues are similar to tfdA (> 85% amino acid identity). However, there are a number of polynucleotide sequences that have a significantly lower identity with tfdA (25-50%) and still have the characteristic residues associated with α-ketoglutarate dioxigenase Fe (II) dioxigenases.
[0011] An example of a gene that degrades 2,4-D with low homology (<35%) to tfdA is the aad-12 gene from Delftia acidovorans (US Patent Application 2011/0203017). The aad-12 gene encodes an S-enanciomer-specific α-ketoglutarate-dependent dioxygenase that has been used in plants to confer tolerance to certain phenoxy auxin herbicides, including, but not limited to: phenoxyacetic acid herbicides such as 2,4-D and MCPA; phonoxybutanoic acid herbicides (for example, 2,4-DB and MCPB); and pyridyloxyalanoic acid herbicides (e.g., pyridyloxyacetic acid herbicides such as triclopyr and fluroxypyr) and including acid, salt or ester forms of the active ingredient (s). (See, for example, WO 2007/053482).
[0012] Glufosinate-ammonium ("glufosinate") is a non-systemic non-systemic herbicide in the phosphinothricin class of herbicides. Used primarily for post-emergence control of a wide range of broadleaf and grass weeds, L-phosphinothricin, the active ingredient in glufosinate, controls weeds through the irreversible inhibition of glutamine synthase, an enzyme that is necessary for the detoxification of ammonia in plants. Glufosinate herbicides are sold commercially, for example, under the trade names Ignite®, BASTA® and Liberty®.
[0013] The enzyme phosphinothricin N-acetyl transferase (PAT), isolated from the soil bacterium Streptomyces viridochromogenes, catalyzes the conversion of L-phosphinothricin in its inactive form by acetylation. A plant-optimized form of the gene that expresses PAT has been used in soybeans to confer tolerance to the glufosinate herbicide. One such example of glufosinate resistant soy is event A5547-127. More recently, the use of glufosinate herbicide in combination with the glufosinate tolerance feature has been proposed as a non-selective means to efficiently control ALS and glyphosate resistant weeds.
[0014] The expression of heterologous or foreign genes in plants is influenced by where the foreign gene is inserted into the chromosome. This could be due to the chromatin structure (eg, heterochromatin) or the proximity of transcriptional regulation elements (eg, enhancers) close to the integration site (Weising et al., Ann. Rev. Genet 22: 421- 477, 1988), for example. The same gene in the same type of transgenic plant (or another organism) can exhibit a wide variation in the level of expression between different events. There may also be differences in the spatial and temporal patterns of expression. For example, differences in the relative expression of a transgene in various plant tissues may not correspond to the expected patterns of transcriptional regulatory elements present in the introduced gene construct.
[0015] Thus, large numbers of events are often created and verified with the purpose of identifying an event that expresses an introduced gene of interest at a satisfactory level for a certain purpose. For commercial purposes, it is common to produce hundreds to thousands of different events and to check those events against a single event that has the desired levels and patterns of expression of the transgene. An event that has desired levels and / or patterns of transgene expression is useful for introgression of the transgene into other fundamental genetic structures through sexual crossing using conventional crossing methods. The progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are well adapted to local growing conditions. BRIEF SUMMARY OF THE INVENTION
[0016] The present invention relates, in part, to efficient means to control weed resistance, which helps to preserve the usefulness of herbicide-tolerant technologies. In some embodiments, the present invention can provide growers with great flexibility and convenience in weed control options.
[0017] More specifically, the present invention relates in part to the soy event (Glycine max) called pDAB8291.45.36.2 ("Event pDAB8291.45.36.2") which has representative seeds deposited with the American Type Culture Collection ( ATCC) with Accession No. PTA-11335 and progeny derived from them. The present invention includes soybean plants that comprise Event pDAB8291.45.36.2 (and includes soybean plants that comprise a transgenic interest in a genomic segment comprising SEQ ID NO: 1 and SEQ ID NO: 2).
[0018] The transgenic insert present in the objective event and in the deposited seed comprises three herbicide tolerance genes: aad-12, 2mepsps and a pat gene. The aad-12 gene, derived from Delftia acidovorans, encodes the protein aryloxyalkanoate dioxigenase (AAD-12), which confers tolerance, for example, to 2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides. The 2mepsps gene, a modified EPSPS sequence isolated from corn, produces a protein that gives tolerance to glyphosate herbicides. The pat gene, from the soil bacterium Streptomyces viridochromogenes, confers tolerance to the glufosinate herbicide.
[0019] Other aspects of the invention include progeny plants, soybeans, seeds and / or parts that can be regenerated from plants and seeds and progeny comprising the soybean event pDAB8291.45.36.2, as well as food or food products made from any one. of these. The invention also includes parts of the plant from Event pDAB8291.45.36.2 that include, but are not limited to, pollen, egg, flowers, buds, roots, leaves, nuclei of vegetative cells, pollen cells and other plant cells that comprise the Event pDAB8291.45.36.2. The invention further relates to soybean plants that have tolerance to various herbicides including phenoxyacetic acid herbicides, phenoxybutanoic acid herbicides, pyridyloxyalanoic acid herbicides, glyphosate and / or glufosinate. Such soybean plants can also be accumulated with genes that confer tolerance to several other non-selective and selective herbicides, including but not limited to the dicamba, imidazolinone and HPPD herbicides. The invention also includes new genetic compositions of Event pDAB8291.45.36.2 and aspects of agronomic performance of soybean plants that comprise Event pDAB8291.45.36.2.
[0020] This invention relates in part to the cultivation of herbicide-tolerant plants and plants. This invention includes a new transformation event in soybean plants that comprises a polynucleotide, which is described here, inserted within a specific site within the genome of a soybean cell.
[0021] In some modalities, the said Event / polynucleotide can be "accumulated" with other characteristics, for example, agronomic characteristics and / or insect inhibiting proteins. However, the present invention includes plants that have the isolated event, which is described here.
[0022] In some modalities, the target herbicide tolerance event can be combined in an accumulation of crosses with an insect resistance event. In some of these modalities, the insect resistance event comprises a cry1F gene and a cry1Ac gene. Part of such events and accruals is specifically exemplified here, including soybean event 9582.812.9.1 ("Event 812") and soybean event 9582.814.19.1 ("Event 814"). Plants, plant cells and seeds, for example, which comprise any combination of the target events are included in the present invention.
[0023] Additional characteristics can be accumulated within the plant genome or within the same locus as the Event pDAB8291.45.36.2, for example, by crossing the plant, retransformation of the transgenic plant containing Event DAS-8291.45.36.2 or addition of new features through targeted integration via homologous recombination.
[0024] Other modalities include the excision of part or all of the transgenic insert and / or flanking sequences of Event DAS-8291.45.36.2. After excision, another insert and / or an additional one can be directed to the specific chromosomal site of Event DAS-8291.45.36.2. The exemplified insert can be replaced or additional insert (s) can be accumulated, in this way, with the exemplified insert of the objective soy event.
[0025] In one embodiment, the present invention encompasses a soy chromosomal target site located on chromosome 03. In some embodiments, the target site comprises a heterologous nucleic acid. In some modalities, the soy chromosomal target site is located between or within the genomic flanking sequences presented in SEQ ID NO: 1 and SEQ ID NO: 2.
[0026] In one embodiment, the present invention encompasses a method of producing a transgenic soy plant that comprises inserting a heterologous nucleic acid into a position on chromosome 03. In another embodiment, the heterologous nucleic acid is inserted into chromosome 03 near or between several polynucleotide segments exemplified as described here.
[0027] Additionally, the present invention provides assays for detecting the presence of the Objective Event in a sample (for example, soybeans). The assays can be based on the DNA sequence of the recombinant construct, inserted in the soybean genome and on the genomic sequences that flank the insertion site. Kits and conditions useful in carrying out the tests are also provided.
[0028] Thus, the present invention relates in part to the cloning and analysis of the DNA sequences of the entire exemplified insert and its border regions (in transgenic soybean lines). These strings are unique. Based on these insert and edge (and joint) sequences, event specific initiators can be and have been generated. The PCR analysis demonstrated that the Event can be identified through the analysis of the PCR amplicons generated with these sets of initiators specific to the event. Thus, these and other related procedures can be used to uniquely identify soybean strains that comprise the Event of the present invention.
[0029] The present invention also relates in part to real-time PCR or endpoint TaqMan assays for the detection of Event 8291.45.36.2. Some modalities are aimed at rehearsals that are capable of analyzing high-yield zigosity. The present invention further relates, in part, to the use of a reference gene GMFL01-25-J19 (GenBank: AK286292.1) for use in determining zygosity. These and other related procedures can be used to uniquely identify the zigosity of the Event pDAB8291.45.36.2 and cross soybean strains that comprise the event. BRIEF DESCRIPTION OF THE FIGURES
[0030] Figure 1 is a plasmid map of pDAB8291.
[0031] Figure 2 is a schematic diagram representing the locations of the initiators for the soybean event pDAB8291.45.36.2.
[0032] Figure 3 is a schematic diagram representing the locations of the primers and the deletion of the genomic DNA in the soybean event pDAB8291.45.36.2.
[0033] Figure 4 is a schematic diagram representing the locations of the initiators for the soybean event pDAB8291.45.36.2 BRIEF DESCRIPTION OF THE SEQUENCES
[0034] SEQ ID NO: 1 provides the 5 'flanking edge sequence for the objective soybean event pDAB8291.45.36.2.
[0035] SEQ ID NO: 2 provides the 3 'flanking edge sequence for the objective soybean event pDAB8291.45.36.2.
[0036] SEQ ID NO: 3 provides the 4536_WF1 initiator.
[0037] SEQ ID NO: 4 provides the 4536_WF2 initiator.
[0038] SEQ ID NO: 5 provides the 4536_WF3 initiator.
[0039] SEQ ID NO: 6 provides the 4536_WF4 initiator.
[0040] SEQ ID NO: 7 provides the 4536_WR1 initiator.
[0041] SEQ ID NO: 8 provides the 4536_WR2 initiator.
[0042] SEQ ID NO: 9 provides the 4536_WR3 initiator.
[0043] SEQ ID NO: 10 provides the 4536_WR4 initiator.
[0044] SEQ ID NO: 11 provides the initiator ED_v2_C1.
[0045] SEQ ID NO: 12 provides the PAT_11 initiator.
[0046] SEQ ID NO: 13 provides the sequence for plasmid pDAB8291.
[0047] SEQ ID NO: 14 provides the genomic flanking sequence for soybean 5 'partial and insert 5' partial.
[0048] SEQ ID NO: 15 provides partial the genomic flanking sequence of soybean to 3 'partial and insert to 3' partial.
[0049] SEQ ID NO: 16 provides a sequence of 72 pairs of bases spanning the 5 'integration junction.
[0050] SEQ ID NO: 17 provides a sequence of 142 pairs of bases spanning the 3 'integration junction.
[0051] SEQ ID NO: 18 provides the 4536_5'F primer.
[0052] SEQ ID NO: 19 provides the 4536_5'R primer.
[0053] SEQ ID NO: 20 provides the probe 4536_5'P.
[0054] SEQ ID NO: 21 provides the 4536_3'F primer.
[0055] SEQ ID NO: 22 provides the 4536_3'R primer.
[0056] SEQ ID NO: 23 provides the probe 4536_3'P.
[0057] SEQ ID NO: 24 provides the GMS116F primer.
[0058] SEQ ID NO: 25 provides the GMS116R primer.
[0059] SEQ ID NO: 26 provides the probe GMS116Probe.
[0060] SEQ ID NO: 27 provides the expected sequence of the soybean event pDAB8291.45.36.2.
[0061] SEQ ID NO: 28 provides the expected sequence of Soy Event 9582.812.9.1, including the 5 'genomic flanking sequence, the pDAB9582 T-filament insert and the 3' genomic flanking sequence.
[0062] SEQ ID NO: 29 provides the expected sequence of Soy Event 9582.814.19.1, including the 5 'genomic flanking sequence, the pDAB9582 T-filament insert and the 3' genomic flanking sequence. DETAILED DESCRIPTION OF THE INVENTION
[0063] The invention described here includes new soybean (soy) plant transformation events that comprise a cassette for the expression of several herbicide tolerance genes inserted within a specific locus within the genome of a soy cell.
[0064] The exemplified transgenic insert comprising Event pDAB8291.45.36.2 includes genetic elements for the expression of three different herbicide tolerance genes: (1) a synthetic aad-12 gene; (2) a modified EPSPS sequence with optimized corn codons that encodes a protein that contains mutations, when compared to the wild-type EPSPS polypeptide: in amino acid residues 102 (from threonine to isoleucine) and 106 (from proline to serine) and which confers resistance or tolerance to glyphosate herbicides; and (3) a pat gene that confers tolerance or resistance to glufosinate herbicides. The aad-12 gene was derived from Delphia acidovorans and encodes an aryloxyalkanoate protein enzyme dioxigenase (AAD-12) capable of deactivating herbicides that have an α-ketoglutarate group, including phenoxyalkanoate herbicides (for example, phenoxyacetic acid herbicides such as 2,4-D and MCPA; and phenoxybutanoic acid herbicides such as 2,4-DB and MCPB) and pyridyloxyalanoic acid herbicides (for example, pyridyloxy acetic herbicides such as triclopyr and fluroxypyr), including forms of acid, salt or ester of the active ingredient (s).
[0065] More specifically, the present invention relates in part to the transgenic soybean event pDAB8291.45.36.2, to the plant strains that comprise these events and to the cloning and analysis of the DNA sequences of this insert and / or the regions edge of them. The plant strains of the present invention can be detected using the sequences disclosed and suggested here.
[0066] In some embodiments, a polynucleotide segment exemplified or described here (such as SEQ ID NO: 1, SEQ ID NO: 2 and / or the insert between them, as shown in Figure 2, for example) be cut and / or subsequently redirected with additional polynucleotide sequence (s).
[0067] This invention relates in part to the crossing of plants and herbicide-tolerant plants. In some embodiments, said polynucleotide sequence may be "accumulated" with other characteristics (such as other herbicide tolerance gene (s) and / or gene (s) encoding protein inhibitors) insects or inhibitory RNA sequences, for example). However, the present invention also includes plants that have a single event, which is described here.
[0068] In some modalities, the objective herbicide tolerance event can be combined in an accumulation by crossing with an insect resistance event. In some embodiments, the Insect Resistance Event is selected from the group consisting of Event 812 and Event 814 (which are defined in more detail here below), each of which comprises a cry1F gene and a cry1Ac gene. Plants, plant cells and seeds, for example, which comprise any combination of the target events are included in the present invention.
[0069] Provisional U.S. patent application serial number 61 / 471,845, filed on April 5, 2011, relates in part to soybean strains that comprise Soy Event 9582.812.9.1 (Event 812). Seeds that comprise this Event were deposited and made available to the public without restriction (but subject to patent rights), with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, 20110. The deposit, called ATCC Deposit No PTA-11602, was made on January 20, 2011. This deposit has been made and will be maintained in accordance with and under the terms of the Budapest Treaty in relation to seed deposits for the purposes of the patent procedure.
[0070] US provisional patent applications serial numbers 61 / 511,664 (filed on July 26, 2011) and 61 / 521,798 (filed on August 10, 2011) relate in part to soybean strains that comprise the event 9582.814.19.1 (the Event 814). Seeds that comprise this Event were deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, 20110. The deposit, Patent Deposit Denomination at ATCC PTA-12006, was received by the ATCC on July 21, 2011. This deposit has been made and will be maintained in accordance with and under the terms of the Budapest Treaty in relation to seed deposits for the purposes of the patent procedure.
[0071] The present invention further includes plants, seeds and plant cells, for example, comprising SEQ ID NO: 27 (Event pDAB8291.45.36.2 (also called "Event 4536"), SEQ ID NO: 28 (Event 812) and / or SEQ ID NO: 29 (Event 814) and variants of these strings that have, for example, at least 95,%, 96%, 97%, 98% or 99% identity with It is not uncommon for some variation (such as the deletion of some segments) to occur after the integration of an insert sequence into the plant genome, which is discussed in more detail in Example 7, for example.
[0072] The present invention further provides assays for detecting the presence of the Objective Event in a sample. Aspects of the present invention include methods of planning and / or producing any diagnostic nucleic acid molecules exemplified or suggested herein, particularly those based entirely or partially on the objective flanking sequences.
[0073] In some embodiments, this invention relates to herbicide-tolerant soybean lines and their identification. The present invention relates in part to detecting the presence of the Objective Event for the purpose of determining whether the progeny of a sexual cross contains the Event of interest. In addition, a method for detecting the Event is included and is useful, for example, to comply with regulations that require pre-market approval and the labeling of foods derived from recombinant crop plants, for example. It is possible to detect the presence of the Target Event through any well-known nucleic acid detection method such as polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. Event-specific PCR assays are discussed here. (See, for example, Windels et al. (Med. Fac. Landbouww, Univ. Gent 64 / 5b: 459462, 1999) for another example). Some of these examples refer to the use of a set of primers that covers the junction between the insert's DNA and the flanker. More specifically, one primer included the sequence of the insert and a second primer included the sequence of the flanking DNA as described here.
[0074] The soybean event pDAB8291.45.36.2 and its selection and characterization in relation to stability and expression in soybean plants from generation to generation are exemplified here. Both flanking sequences for Event pDAB8291.45.36.2 have been sequenced and are described here as SEQ ID NO: 1 and SEQ ID NO: 2. Specific tests were developed for the event. This was also mapped in the soy genome (soy chromosome 03). The pDAB82- 91.45.36.2 event may suffer introgression in elite cultivars where it will confer tolerance to phenoxy auxin, glyphosate and glufosinate herbicides in hybrid and crossbred soybean lines.
The version of the gene encoding mutant 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSPS) used in preferred embodiments of the present invention has the codons of use optimized for hemicotyledons (both for dicots and monocotyledons). The wild type EPSPS gene was isolated from Zea mays and the wild type sequence was deposited under GenBank accession number X63374. See also U.S. Patent No. 6,566,587 (in particular, SEQ ID No. 3 contained therein).
[0076] In order to obtain high expression of heterologous genes in plants, it may be preferred to reengineer said genes so that they are more efficiently expressed in plant cells. A modification of the EPSPS nucleotide sequence of the wild type plant can provide such resistance when expressed in a plant cell. As described in the '587 patent, when an EPSPS polypeptide is compared with the wild type polypeptide, a modification to replace isoleucine with threonine at residue 102 and replace serine with proline at position 106 of the protein, the result is the double EPSPS polypeptide mutant (2mEPSPS) used in the objective insertion. When expressed in a plant cell, it provides tolerance to glyphosate. The present EPSPS gene, also referred to as the "2mepsps gene" or DMMG, has been optimized to improve expression in dicotyledonous plants as well as in monocotyledonous plants and in particular in soybeans. The use of codons was selected based on the use of codons preferred by hemicotyledons, that is, redesigned so that the protein is encoded by codons that have a tendency towards both the use of monocotyledons and dicotyledons. Deleterious sequences and superfluous restriction sites can be removed to increase the transcription / translation efficiency of the 2mepsps coding sequence and to facilitate the DNA manipulation steps. The present version optimized for hemicotyledons of the monocotyledon gene is further detailed in U.S.S.N. 13 / 303,502 (filed on November 23, 2011, claiming priority for December 3, 2010) entitled, "OPTIMIZED EXPRESSION OF GLIPHOSATE RESISTENCE ENCODING NUCLEIC ACID MOLECULES IN PLANT CELLS".
[0077] As previously mentioned here, the introduction and integration of a transgene into the genome of a plant involves some random events (consequently the name "event" for a certain insertion that is expressed). That is, with many transformation techniques such as transformation with Agrobacterium, the "gene gun" and WHISKERS, it cannot be predicted where in the genome a transgene will be inserted. Thus, the identification of the flanking plant's genomic DNA on both sides of the insert can be important to identify a plant that has a certain insertion event. For example, PCR primers can be designed that generate a PCR amplicon across the junction region of the insert and the host genome. This PCR amplicon can be used to identify a unique or distinct type of insertion event.
[0078] During the process of inserting an insert into the plant cell genome, it is not uncommon for some deletions or other changes to the insert and / or the flanking sequences to occur. Thus, the relevant segment of the plasmid sequence provided here could comprise some minor variations. The same is true for the flanking strings provided here. Thus, a plant comprising a polynucleotide that has some identity range with respect to the present flanking and / or insert sequences is within the scope of the present invention. The sequence identity of the present invention can be a polynucleotide sequence that has at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably at least 75% sequence identity, more preferably at least 80% identity and most preferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% sequence identity to the sequence exemplified or described here. The hybridization or hybridization conditions that are provided here can also be used to define such plants and polynucleotide sequences of the present invention. The sequence comprising the flanking sequences plus the complete insert sequence can be confirmed with reference to the deposited seed.
[0079] Since "events" are originally random events, as part of this description at least 2500 seeds of a soybean strain comprising Event pDAB8291.45.36.2 have been deposited and made available to the public without restriction (but subject to rights) patent), with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, 20110. The deposit was called ATCC Deposit No. PTA-11335. 100 packages (25 seeds per package) of Glycine max seeds ("Soybean Seed Glycine max L .: pDAB8291.45.36.2") were deposited on behalf of Dow AgroSciences LLC and MS Technologies, LLC on September 14, 2010. The deposit was tested on October 4, 2010 and on that date, the seeds were viable. This deposit has been made and will be maintained in accordance with and under the terms of the Budapest Treaty in relation to seed deposits for the purposes of the patent procedure. The deposit will be held without restriction at the ATCC depositary, which is a public depositary, for a period of 30 years or five years after the most recent application or for the effective life of the patent, whichever is longer and will be replaced if it becomes. if not viable during such period.
[0080] The deposited seeds are part of the present invention. Clearly, soybean plants can be grown from these seeds and such plants are part of the present invention. The present invention also relates to DNA sequences contained in these soybean plants that are useful for the detection of these plants and their progeny. The detection methods and kits of the present invention can be directed towards the identification of any, two or all three of these events, depending on the final test proposal.
[0081] Definitions and examples are provided here to help describe the present invention and to guide those of ordinary skill in the art to practice the invention. Unless otherwise stated, the terms must be understood in accordance with conventional usage by those of ordinary skill in the relevant art. The nomenclature for DNA bases that is presented in 37 CFR § 1.822 is used.
[0082] As used here, the term "progeny" means the offspring of any generation of a parent plant that comprises the soybean event pDAB8291.45.36.2.
[0083] A transgenic "event" is produced through the transformation of plant cells with heterologous DNA, that is, a nucleic acid construction that includes a transgene of interest, the regeneration of a plant population that results from the insertion of the transgene within the plant genome and the selection of a particular plant characterized by insertion into a particular genomic location. The term "event" refers to the original transformant and the progeny of the transformant that includes the heterologous DNA. The term "event" also refers to the progeny produced by a sexual cross between the transformant and another variety that includes genomic / transgenic DNA. Even after repeated backcrossing with a recurring parent, the DNA of the inserted transgene and the flanking genomic DNA (genomic / transgenic DNA) of the transformed parent are present in the origin of the crossing at the same chromosomal location. The term "event" also refers to the DNA of the original transformant and its progeny that comprises the inserted DNA and the flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives the Inserted DNA including the transgene of interest as the result of a sexual crossover of a parental line that includes the inserted DNA (for example, the original transformant and progeny resulting from self-crossing) and a parental line that does not contain the inserted DNA.
[0084] A "junction sequence" encompasses the point at which the DNA inserted within the genome is linked to the DNA of the native soybean genome that flanks the insertion point, the identification or detection of one or the other junction sequences in the genetic material from a plant being sufficient to diagnose the event. Included are the DNA sequences that span insertions in the soy events described here and similar lengths of flanking DNA. Specific examples of such diagnostic sequences are provided here; however, other sequences that overlap the insertion junctions or the insertion junctions and the genomic sequence, are also diagnostic and could be used in accordance with the present invention.
[0085] The present invention relates in part to the identification of the Event using such flanking, joining and insertion sequences. Related PCR primers and amplicons are included in the invention. In accordance with the present invention, PCR analysis methods using amplicons that extend along the inserted DNA and its edges can be used to detect or identify commercialized transgenic soybean varieties or strains derived from the current proprietary transgenic soybean strains.
[0086] The binary plasmid, pDAB8291 (SEQ ID NO: 13) comprises the genetic elements shown in Figure 1. The following genetic elements (T-strand edge sequences are not included) are contained within the filament region PDAB8291 tee. In Table 1, the numbering of residues of the genetic elements is provided in relation to SEQ ID NO: 13 disclosed here. Table 1: Numbering of Residues of the Genetic Elements that comprise the Binary Plasmid pDAB8291 (SEQ ID NO: 13). Genetic element Position Reference


[0087] SEQ ID NOs: 14 and 15, respectively, are the 5 'and 3' flanking sequences together with the 5 'and 3' portions of the insert sequence, as described in more detail below. below and thus include the 5 'and 3' "junction" or "transition" sequences of the insert and genomic DNA. With respect to SEQ ID NO: 14, residues 1-3088 are the 5 'genomic flanking sequence and residues 3089-3406 are residues from the 5' end of the insert. With respect to SEQ ID NO: 15, residues 1-223 are residues from the 3 'end of the insert and residues 224-1008 are the 3' genomic flanking sequence. The junction or transition sequence with respect to the 5 'end of the insert thus occurs in residues 30883089 of SEQ ID NO: 14. The junction or transition sequence with respect to the 3 'end of the insert thus occurs at residues 223224 of SEQ ID NO: 15. The polynucleotides of the present invention include those that comprise, for example, 5, 10, 20, 50, 100, 150 or 200 bases or possibly more and any increments between them, on either side of the junction sequence. Thus, a primer covering the junction sequence could comprise, for example, 5-10 bases that would hybridize to the flanking sequence and 5-10 bases that would hybridize to the insert sequence. Probes and amplicons could be similarly designed, although they could often be longer than primers.
[0088] The objective sequences (including flanking sequences) are unique. Based on these insertion and flanking sequences, specific initiators were generated for the event. The PCR analysis demonstrated that these soybean strains can be identified in different soybean genotypes by analyzing the PCR amplicons generated with these sets of primers specific to the event. Thus, these and other related procedures can be used to uniquely identify these soybean strains. The strings identified here are unique.
[0089] The detection techniques of the present invention are especially useful in association with the crossing of plants, to determine which plants of the progeny comprise a certain event, after a parent plant comprising an Event of interest is crossed with another plant strain in a effort to confer one or more additional characteristics of interest to the progeny. These methods of PCR analysis benefit soybean crossing programs as well as quality control, especially for commercialized transgenic soybean seeds. PCR detection kits for these transgenic soybean strains can now also be produced and used. This can benefit product registration and product management.
[0090] In addition, soybean flanking / genomic sequences can be used to specifically identify the geographic location of each insert. This information can be used to produce specific molecular marker systems for each event. These can be used for accelerated crossing strategies and to establish connection data.
[0091] In addition, the flanking sequence information can be used to study and characterize the transgene integration processes, the characteristics of the genomic integration site, the selection of Events, the stability of the transgenes and their flanking sequences and the gene expression (especially related to gene silencing, transgene methylation patterns, position effects and potential elements related to expression such as MARS [Matrix Binding Regions] and the like).
[0092] In light of the present description, it should be clear that the present invention includes seeds available under ATCC Deposit No. PTA-11335. The present invention also includes a herbicide-tolerant soybean plant grown from a seed deposited with the ATCC under accession number PTA-11335. The present invention further includes parts of said plant, such as leaves, tissue samples, seeds produced by said plant, pollen and the like (in which they comprise a transgenic insert flanked by SEQ ID NO: 1 and SEQ ID NO: 2).
[0093] Still further, the present invention includes descendant and / or progeny plants from plants grown from the deposited seed, preferably a herbicide-resistant soy plant in which said plant has a genome comprising a genomic DNA sequence of the type wild-type / junction of the insert DNA as described here. As used here, the term "soy" means Glycine max and includes all varieties of it and can be crossed with a soy plant.
[0094] The invention further includes breeding processes using a plant of the present invention as at least one parent. For example, the present invention includes an F1 hybrid plant that has as one or both parents any of the plants exemplified here. Still within the present invention is a seed produced by such F1 hybrids of the present invention. This invention includes a method for the production of an F1 hybrid seed by crossing an exemplified plant with a different plant (e.g., intercrossed parent) and harvesting the resulting hybrid seed. The present invention includes an exemplified plant that is a female parent or a male parent. The characteristics of the resulting plants can be enhanced through careful consideration of parental plants.
[0095] A herbicide-tolerant soybean plant of the present invention can be crossed first by sexually crossing a first parent soybean plant consisting of a soybean plant grown from the seed of any of the strains referred to here and a second parental soybean plant, thus producing a large number of first progeny plants; then selecting a first progeny plant that is resistant to an herbicide (or that has at least one of the events of the present invention); the self-crossbreeding of the first progeny plant, thus producing a large number of second progeny plants; and then by selecting the second progeny plants from a plant that is resistant to an herbicide (or that has at least one of the events of the present invention). These steps may also include backcrossing the first progeny plant or the second progeny plant with the second parent soy plant or a third parent soy plant. A soybean plantation comprising the soybean seeds of the present invention or progeny thereof can be obtained.
[0096] It should also be understood that two different transgenic plants can also be crossed to produce an offspring that contains two added exogenous genes that secrete independently. Self-crossing of appropriate progeny can produce plants that are homozygous for both added exogenous genes. Backcrossing with a parental plant and crossing with a non-transgenic plant are also considered, as is vegetative propagation. Other breeding methods commonly used for different traits and cultivation plants are known in the art. Backcross crossing has been used to transfer genes for a highly inheritable trait inherited in a simple way to a desirable homozygous cultivar or intracross crossbred, which is the recurrent parent. The source of the characteristic that will be transferred is called the parental donor. The resulting plant is expected to have the attributes of the recurring parent (eg, cultivar) and the desirable trait transferred from the donor parent. After the initial crossing, individuals who have the donor's parental phenotype are selected and repeatedly crossed (backcrossed) with the recurring parental. The resulting parent is expected to have the attributes of the recurring parent (eg, cultivar) and the desirable trait transferred from the donor parent.
[0097] The DNA molecules of the present invention can be used as molecular markers in a marker-assisted crossing (MAB) method. The DNA molecules of the present invention can be used in methods (such as AFLP markers, RFLP markers, RAPD markers, SNPs and SSRs) that identify genetically linked agronomically useful traits, as is known in the art. The herbicide resistance characteristic can be traced to the progeny of a cross with a soybean plant of the present invention (or progeny of the same and any other cultivar or variety of soybeans) using the MAB methods. DNA molecules are markers for this trait and MAB methods that are well known in the art can be used to screen for herbicide resistance trait (s) in soybean plants in which at least one soybean strain of the present invention or progeny likewise, he was a parent or an ancestor. The methods of the present invention can be used to identify any variety of soybeans that have the target event.
[0098] The methods of the present invention include a method of producing a herbicide-tolerant soy plant in which said method comprises the introgression of Event pDAB8291.45.36.2 in a soy cultivar. More specifically, the methods of the present invention can comprise the crossing of two plants of the present invention or a plant of the present invention and any other plant. Preferred methods further comprise selecting the progeny of said crossover by analyzing said progeny in relation to an Event that can be detected in accordance with the present invention. For example, the present invention can be used to track the Objective Event over crossbreeding cycles with plants that comprise other desirable characteristics, such as agronomic characteristics such as those tested here in various Examples. The plants that comprise the objective event and the desired characteristic can be detected, identified, selected and quickly used in additional crossing rounds, for example. The objective / characteristic event can also be combined by crossing and tracking according to the present invention, with one or more insect resistance characteristics and / or with additional herbicide tolerance characteristics. One modality of what was described last is a plant that comprises the objective event combined with a gene that encodes resistance to the herbicide dicamba.
[0099] Thus, the present invention can be combined with, for example, additional features encoding glyphosate resistance (eg, resistant plant or bacterial glyphosate oxidase (GOX)), glyphosate acetyl transferase (GAT), additional features for resistance to glufosinate (for example, resistance to bialaphos (bar)), characteristics that confer resistance to the herbicide that inhibits acetolactate synthase (ALS) (for example, imidazolinones [such as imazethapir], sulfonylureas, triazolopyrimidine sulfonanilide, pyrmidinilti- obenzoates and others chemicals [Csr1, SurA and others]), characteristics of resistance to bromoxynil (for example, Bxn), characteristics for resistance to the dicamba herbicide (see, for example, US 2003/0135879), characteristics for resistance to HPPD enzyme inhibitors (4-hydroxylphenyl-pyruvate-dioxigenase), characteristics for resistance to phytene desaturase inhibitors (PDS), characteristics for resistance to herb that inhibit photosystem II (for example, psbA), characteristics for resistance to herbicides that inhibit photo-system I, characteristics for resistance to herbicides that inhibit protoporphyrinogen oxidase IX (PPO) (for example, PPO-1) and characteristics for resistance to phenylurea herbicides (for example, CYP76B1). One or more of such characteristics can be combined with the present invention to provide the ability to efficiently control, delay and / or prevent weed changes and / or resistance to herbicides of various classes.
[00100] It will be considered by those skilled in the art that the aad-12 gene used in the present invention further provides resistance to compounds that are converted to auxin phenoxyacetate herbicides (e.g., 2,4-DB, MCPB, etc.). The group of butyric acid present in the herbicide 2,4-DB is converted through β-oxidation to phytotoxic 2,4-dichlorophenoxyacetic acid. Similarly, MCPB is converted by β-oxidation to phytotoxic MCPA. Butanoic acid herbicides are in themselves non-herbicides, but are converted to their respective acid by β-oxidation within susceptible plants to produce the herbicide's acetic acid form which is phytotoxic. Plants incapable of rapid β-oxidation are not harmed by butanoic acid herbicides. However, plants that are capable of rapid β-oxidation and can convert the butanoic acid herbicide to acetic form are subsequently protected by AAD-12.
[00101] Herbicide application methods are well known in the art. Such applications may include tank mixtures of more than one herbicide. Preferred herbicides for use according to the present invention are combinations of glyphosate, glufosinate and a phenoxy auxin herbicide (such as 2,4-D; 2,4-DB; MCPA; MCPB). Other preferred combinations include mixtures of glyphosate plus 2,4-D or glufosinate plus 2,4-D. These three types of herbicides can be used in advantageous combinations that would be evident to a person skilled in the art who has the benefit of the present description. One or more of the present herbicides can be applied to a field / area prior to planting it with the seeds of the present invention. Such applications can occur within 14 days, for example, of the planting of the seeds of the present invention. One or more of the present herbicides can also be applied after planting before emergence. One or more of the present herbicides can also be applied to the soil (to control weeds) or on the surface of weeds and / or on the surface of transgenic plants of the present invention. The present three herbicides can be switched or used in combination, for example, to control or prevent weeds that could be tolerant to one herbicide, but not the other. Various application times for the present three types of herbicides can be used in various ways as would be known in the art.
[00102] Additionally, the Objective Event can be accumulated with one or more additional herbicide tolerance characteristics, one or more additional entry characteristics (for example, insect resistance (for example, Event 812o or Event 814), resistance to fungi or tolerance to stress and others) or yield (for example, higher yield, improved oil profile, better quality of fibers and others), both transgenic and non-transgenic. Thus, the present invention can be used to provide a complete agronomic package of improved quality of the crop plant with the ability to flexibly and cost-effectively control any number of agronomic pests.
[00103] Methods for integrating a polynucleotide sequence within a specific chromosomal site of a plant cell through homologous recombination have been described within the art. For example, site-specific integration that is described in US Patent Application Publication No. 2009/0111188 A1, describes the use of recombinases or integrases to mediate the introduction of a donor polynucleotide sequence into a chromosomal target. . In addition, International Patent Application No. WO 2008/021207, describes zinc finger-mediated homologous recombination to integrate one or more donor polynucleotide sequences within specific genome locations. The use of recombinases such as FLP / FRT which are described in US Patent No. 6,720,475 or CRE / LOX which are described in US Patent No. 5,658,772, can be used to integrate a polynucleotide sequence within a specific chromosomal site. Finally, the use of meganucleases to target donor polynucleotides into a specific chromosomal location has been described in (Puchta et al., PNAS USA 93 (1996) pp. 5055-5060).
[00104] Other various methods for site-specific integration within plant cells are generally known and applicable (Kumar et al., Trends in Plant Sci. 6 (4) (2001) pp. 155-159). In addition, site-specific recombination systems that have been identified in various lower prokaryotic and eukaryotic organisms can be applied for use in plants. Examples of such systems include, but are not limited to; the R / RS system recombinase of the plasmid pSR1 from the yeast Zygosaccharomyces rouxii (Araki et al. (1985) J. Mol. Biol. 182: 191-203) and the Gin / gix system of the phage Mu (Maeser and Kahlmann (1991 ) Mol. Gen. Genet. 230: 170-176).
[00105] In some embodiments of the present invention, it may be desirable to integrate or accumulate new transgene (s) in the vicinity of an existing transgenic event. The transgenic event can be considered a preferred genomic locus that has been selected based on unique characteristics such as single insertion site, normal Mendelian segregation and stable expression and a superior combination of efficacy, including herbicide tolerance and agronomic performance in and over various environmental locations. The newly integrated transgenes must maintain the transgene expression characteristics of the existing transformants. In addition, the development of tests for the detection and confirmation of the newly integrated Event would be avoided since the genomic flanking sequences and the chromosomal location of the newly integrated Event are already identified. Finally, the integration of a new transgene within a specific chromosomal location that is linked to an existing transgene would accelerate the transgenes' introgression into other fundamental genetic structures through sexual crossing using conventional crossing methods.
[00106] In some embodiments of the present invention, it may be desirable to cut polynucleotide sequences from a transgenic event. For example, the excision of transgenes which is described in US Patent Application No. 13 / 011,666, describes the use of zinc finger nucleases to remove a polynucleotide sequence, which consists of a gene expression cassette, starting from an event chromosomally integrated transgenic. The polynucleotide sequence that is removed can be a selectable marker. After excision and removal of a polynucleotide sequence, the modified transgenic event can be redirected by inserting a polynucleotide sequence. The excision of a polynucleotide sequence and the subsequent redirection of the modified transgenic event provide advantages such as the reuse of a selectable marker or the ability to overcome unintended changes in the plant's transcriptome that results from the expression of specific genes.
[00107] The present invention discloses here a specific site on chromosome 03 in the soybean genome which is excellent for the insertion of heterologous nucleic acids. A 5 'flanking sequence and a 3' flanking sequence are also disclosed, which may also be useful in identifying and / or targeting the location of the insertion / targeting site on chromosome 03. Thus, the present invention provides methods for introducing heterologous nucleic acids of interest within this pre-established target site or in the vicinity of this target site. The present invention further encompasses a soybean seed and / or a soybean plant comprising any heterologous nucleotide sequence inserted at the disclosed target site or in the general vicinity of such a site. One option for performing such targeted integration is to cut and / or replace a different insert in place of the pat expression cassette exemplified here. In this general aspect, targeted homologous recombination, for example, and without limitation, can be used in accordance with the present invention.
[00108] As used here "accumulation" of genes, events or characteristics is the combination of desired characteristics within a transgenic lineage. Plant growers accumulate transgenic traits by making crosses between parents who each have a desired trait and then identifying the offspring who have both of these desired traits. Another way to accumulate genes is by transferring two or more genes into a plant's cell nucleus at the same time during transformation. Another way to accumulate genes is by re-transforming a transgenic plant with another gene of interest. For example, gene accumulation can be used to combine two or more different characteristics, including, for example, two or more different insect characteristics, insect resistance characteristic (s) and disease resistance characteristic (s), two or more herbicide resistance characteristics and / or insect resistance characteristic (s) and herbicide resistance characteristic (s). The use of a selectable marker in addition to a gene of interest can also be considered an accumulation of genes.
[00109] "Homologous recombination" refers to a reaction between any pair of nucleotide sequences that have corresponding sites that contain a similar nucleotide sequence through which the two nucleotide sequences can interact (re-combine) to form a new one recombinant DNA sequence. Similar nucleotide sequence sites are each referred to herein as a "homology sequence". Generally, the frequency of homologous combination increases as the length of the homology sequence increases. Thus, while homologous recombination can occur between two nucleotide sequences that are less than identical, the frequency of recombination (or efficiency) decreases as the divergence between the two sequences increases. The combination can be performed using a sequence of homology in each of the donor and target molecules, thus generating a "single crossover" recombination product. Alternatively, two homology sequences can be placed in each of the target and donor nucleotide sequences. The recombination between two homology sequences in the donor with two homology sequences in the target generates a "double crossover" recombination product. If the homology sequences in the donor molecule flank a sequence that will be manipulated (for example, a sequence of interest), the double crossover combination with the target molecule will result in a recombination product in which the sequence of interest replaces a DNA sequence that was originally among the homology sequences on the target molecule. The exchange of DNA sequence between the target and the donor through the Double Crossover Recombination Event is called "sequence replacement".
[00110] The Target Event enables the transgenic expression of three different herbicide tolerance proteins resulting in tolerance to herbicide combinations that would control almost all broadleaf and grass weeds. This transgenic expression cassette / insert with herbicide multitolerance characteristic can be accumulated with other herbicide tolerance characteristics (eg glyphosate resistance, glufosinate resistance, imidazolinone resistance, dicamba resistance, HPPD resistance , bromoxynil resistance and others) and insect resistance characteristics (such as Cry1F, Cry1Ab, Cry1Ac, Cry 34/45, Cry1Be, Cry1Ca, Cry1Da, Cry1Ea, Cry1Fa, vegetable insecticidal proteins ("VIPS") - including VIP3A and the like), for example. In addition, the herbicide-tolerant proteins in the transgenic expression cassette / insert of the present invention can serve as one or more selectable markers to assist in the selection of primary transformants from genetically engineered plants with a second gene or group of genes.
[00111] These combinations of characteristics give rise to new methods of controlling weed species (and the like), due to the newly acquired resistance or inherent tolerance to herbicides (eg glyphosate). Thus, new methods for weed control using Event pDAB8291.45.36.2 are within the scope of the invention.
[00112] The use of the present transgenic characteristics, accumulated or transformed individually into crop plants, provides a tool for the control of other voluntary herbicide-tolerant crop plants that do not contain genes to confer tolerance to phenoxy, pyridyloxy, glyphosate and / or glufosinate.
[00113] A preferred plant or seed of the present invention comprises in its genome the insert sequences, which are identified here, together with at least 20-500 or more contiguous flanking nucleotides on both sides of the insert, as described here . Unless otherwise stated, reference to flanking strings refers to those identified in relation to SEQ ID NO: 1 and SEQ ID NO: 2. Again, the present events include heterologous DNA inserted between the objective flanking genomic sequences immediately adjacent to the inserted DNA. It could be expected that all or part of these flanking sequences will be transferred to the progeny that receives the inserted DNA as a result of a sexual crossing of a parental lineage that includes the event.
[00114] The present invention includes tissue cultures of cells that can be regenerated from a plant of the present invention. Also included is a regenerated plant from such a tissue culture, particularly where said plant is capable of expressing all the morphological and physiological properties of an exemplified variety. The preferred plants of the present invention have all the physiological and morphological characteristics of a plant grown from the deposited seed. This invention further comprises the origin of such seed and seed which has the quality characteristics of interest.
[00115] Manipulations (such as mutation, additional transfection and additional crossing) of plants or seeds or parts of them, can lead to the creation of what can be called "essentially derived" varieties. The International Union for the Protection of New Varieties of Plants (UPOV) has provided the following guidelines for determining whether a variety has been essentially derived from a protected variety:
[00116] [A] the variety should be considered to be essentially derived from another variety ("the initial variety") when
[00117] (i) it is predominantly derived from the initial variety or from a variety that is itself predominantly derived from the initial variety, while maintaining the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety;
[00118] (ii) it can be clearly distinguished from the initial variety; and
[00119] (iii) except for the differences that result from the act of derivation, it is in accordance with the initial variety in the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety.
[00120] UPOV, Sixth Meeting with International Organizations, Geneva, October 30, 1992; document prepared by the Office of the Union.
[00121] As used here, a "lineage" is a group of plants that exhibits little or no genetic variation between individuals in relation to at least one trait. Such strains can be created through several generations of self-pollination and vegetative selection or propagation from a single parent using tissue or cell culture techniques.
[00122] As used here, the terms "cultivar" and "variety" are synonymous and refer to a strain that is used for commercial production.
[00123] "Stability" or "stable" means that in relation to the supplied component, the component is maintained from generation to generation and, preferably, at least three generations at substantially the same level, for example, preferably ± 15%, more preferably ± 10%, more preferably ± 5%. Stability can be affected by temperature, location, stress and planting time. A comparison of subsequent generations under field conditions should produce the component in a similar way.
[00124] "Commercial Utility" is defined as having good plant vigor and high fertility, so that the cultivation plant can be produced by growers using conventional cultivation equipment and oil with the described components can be extracted from the seed using equipment crushing and conventional extraction. To be commercially useful, yield, which is measured by the weight of the seed, oil content and total oil produced per acre, is within 15% of the average yield of a commercially comparable canola variety otherwise without the premium value characteristics grown in the same region.
[00125] "Agronomic elite" means that a strain has desirable agronomic characteristics such as yield, maturity, disease resistance and the like, in addition to herbicide tolerance due to the objective event (s). Agronomic characteristics, considered individually or in any combination, as shown in the Examples below, in a plant comprising an Event of the present invention, are within the scope of the present invention. Any and all agronomic characteristics and data points can be used to identify such plants, in the form of a point or at either end or both ends of a range of characteristics used to define such plants.
[00126] As one skilled in the art will recognize in the light of this description, preferred embodiments of detection kits, for example, may include probes and / or primers directed to and / or comprising "junction sequences" or "transition sequences" ( where the soybean genomic flanking sequence meets the insert sequence). For example, this includes polynucleotide probes, primers and / or amplicons designed to identify one or both of the junction sequences (where the insert meets the flanking sequence), whose coordinates are discussed elsewhere here. A common plan is to have an initiator that hybridizes to the flanking region and an initiator that hybridizes to the insert. Such initiators often each have approximately at least 15 residues in length. With this arrangement, the primers can be used to generate / amplify a detectable amplicon that indicates the presence of an Event of the present invention. These primers can be used to generate an amplicon that extends (and includes) a junction sequence as indicated above.
[00127] The "touching down" of the initiator (s) in the flanking sequence is typically not intended to hybridize beyond approximately 200 bases or so beyond the junction. Thus, typical flanking primers would be designed to comprise at least 15 residues of any of the filaments within 200 bases within the flanking sequences starting at the beginning of the insert. That is, primers that comprise a sequence of an appropriate size of (or that hybridizes to) residues within 100 through 200500 or bases as well from either or both of the junction sequences identified above are within the scope of the present invention. Insert primers can be similarly designed at any location on the insert, but residues in the insert (including the complement) within 100 to 200-500 or bases like this from the junction sequence (s) identified above, they can be used, for example, not exclusively for such an initiator design.
[00128] A person skilled in the art will also recognize that primers and probes can be designed to hybridize, under a standardized hybridization range and / or PCR conditions, with sequence segments exemplified here (or complements to them), in which the initiator or the probe is not perfectly complementary to the exemplified sequence. That is, some degree of mismatch can be tolerated. For a primer of approximately 20 nucleotides, for example, typically one or two or more nucleotides do not need to bond with the opposite strand if the wrong paired base is internal or is at the end of the primer that is opposite the amplicon. Several appropriate hybridization conditions are provided below. Synthetic nucleotide analogs, such as inosine, can also be used in the probes. Peptide nucleic acid (PNA) probes, as well as DNA and RNA probes, can also be used. What is important is that such probes and primers are diagnostic for (able to uniquely identify and distinguish) the presence of an Event of the present invention.
[00129] It should be noted that errors in PCR amplification can occur which could result in less important sequencing errors, for example. That is, unless otherwise indicated, the sequences listed here were determined by generating long amplicons from the genomic DNAs of soybeans and then cloning and sequencing. It is not uncommon to find slight differences and less important discrepancies in the sequences generated and determined in this way, due to the many rounds of amplification that are necessary to generate enough amplicon for sequencing from the genomic DNAs. A person skilled in the art should recognize and be cautioned that any adjustments necessary due to these types of sequencing errors or common discrepancies are within the scope of the present invention.
[00130] It should also be noted that it is not uncommon for part of the genomic sequence to be deleted, for example, when a sequence is inserted during the creation of an event. Thus, some differences may also appear between the flanking sequences and objective genomic sequences listed in GENBANK, for example.
[00131] The components of the "insert" are illustrated in the Figures and are discussed in greater detail below in the Examples. The DNA polynucleotide sequences of these components or fragments thereof can be used as DNA primers or probes in the methods of the present invention.
[00132] In some embodiments of the invention, compositions and methods are provided for detecting the presence of the transgenic / genomic insertion region, in plants and seeds and the like, of a soybean plant. DNA sequences are provided that comprise the transgene sequence / junction sequence of the target genomic insertion region provided here, segments that comprise a junction sequence identified herein and complements to any of such exemplified sequences and any segments thereof. The junction sequence of the insertion region encompasses the junction between the heterologous DNA inserted into the genome and the DNA of the soybean cell flanking the insertion site. Such sequences can be diagnostic for a certain event.
[00133] Based on the insert and edge sequences, event specific initiators can be generated. PCR analysis demonstrated that the soybean strains of the present invention can be identified in different soybean genotypes by analyzing the PCR amplicons generated with these sets of event-specific primers. These and other related procedures can be used to uniquely identify these soybean strains. Thus, the PCR amplicons derived from such pairs of primers are unique and can be used to identify these soybean strains.
[00134] In some embodiments, DNA sequences that comprise a contiguous fragment of the new transgene / genomic insertion region are an aspect of this invention. Included are DNA sequences that comprise a sufficient length of polynucleotides from the transgene insert sequence and a sufficient length of polynucleotides from the soybean genomic sequence from one or more of the aforementioned soybean plants and / or sequences that are useful as sequences initiators for the production of a diagnostic amplicon product for one or more of these soybean plants.
[00135] Related modalities refer to DNA sequences that comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more contiguous nucleotides from a portion of the transgene of a DNA sequence identified here or complements thereof and a similar length of soybean flanking DNA sequence (such as SEQ ID NO: 1 and SEQ ID NO: 2 and segments thereof) starting from these sequences or complements to them. Such sequences are useful as DNA primers in DNA amplification methods. The amplicons produced using these primers are diagnostic for any of the soy events referred to here. Therefore, the invention further includes the amplicons produced by such DNA primers and homologous primers.
[00136] This invention also includes methods of detecting the presence of DNA in a sample, which corresponds to the soy event referred to here. Such methods may include: (a) contacting the sample comprising DNA with a set of primers that, when used in a nucleic acid amplification reaction with DNA from at least one of these soy events, produces an amplicon that is diagnostic for the said event (s); (b) carrying out a nucleic acid amplification reaction, thereby producing amplicon; and (c) detecting the amplicon.
[00137] Additional detection methods of the present invention include a method of detecting the presence of DNA in a sample, which corresponds to said event, wherein said method comprises: (a) contact of the sample comprising DNA with a probe that hybridizes under stringent hybridization conditions with the DNA of at least one of said soy events and that does not hybridize under stringent hybridization conditions with a control soy plant (DNA that is not of the event of interest) ); (b) submitting the sample and probe to stringent hybridization conditions; and (c) detecting probe hybridization with DNA.
[00138] Still in additional modalities, the present invention includes methods of production of a soy plant that comprises the Event pDAB8291.45.36.2, in which said method comprises the steps of: (a) sexual crossing of a first lineage of parental soybean (which comprises an expression cassette of the present invention, which gives said herbicide resistance characteristic to plants of said lineage) and a second parental soybean strain (which does not have this herbicide tolerance characteristic) thus producing a large number of progeny plants; and (b) selection of a progeny plant through the use of molecular markers. Such methods may optionally comprise the additional step of backcrossing the progeny plant with the second parent soybean strain to produce a real crossbreed soybean plant comprising said herbicide tolerance characteristic.
[00139] In accordance with another aspect of the invention, methods are provided for determining the zygness of the progeny of a cross with said Event. Said methods may comprise contacting a sample, which comprises soybean DNA, with a set of primers of the present invention. Said initiators, when used in a nucleic acid amplification reaction with the genomic DNA of at least one of said soy events, produce a first amplicon that is diagnostic for at least one of said soy events. Such methods further comprise carrying out a nucleic acid amplification reaction, thereby producing the first amplicon; the detection of the first amplicon; and contact of the sample comprising the soybean DNA with said set of primers (said set of primers, when used in a nucleic acid amplification reaction with the genomic DNA of soybean plants, produces a second amplicon which comprises the Genomic DNA of the native soybean homologous to the genomic region of the soybean, and the carrying out of a nucleic acid amplification reaction, thus producing the second amplicon.The methods also include the detection of the second amplicon and the comparison of the first and second amplicons in one sample, in which the presence of both amplicons indicates that the sample is heterozygous in relation to the insertion of the transgene.
[00140] DNA detection kits can be developed using the compositions disclosed here and methods well known in the DNA detection technique. The kits are useful for identifying the DNA of the Soybean Objective Event in a sample and can be applied to methods for crossing soybean plants that contain this DNA. The kits contain DNA sequences homologous or complementary to the amplicons, for example, disclosed here or the DNA sequences homologous or complementary to the DNA contained in the genetic elements of the transgene of the target events. These DNA sequences can be used in DNA amplification reactions or as probes in a DNA hybridization method. The kits can also contain the reagents and materials needed to perform the detection method.
[00141] A "probe" is an isolated nucleic acid molecule to which a conventional detectable marker or a reporter molecule (such as a radioactive isotope, a ligand, a chemiluminescent agent or an enzyme) is attached. Such probe is complementary to a strand of a target nucleic acid, in the case of the present invention, to a strand of genomic DNA from one of said soybean events, either from a soybean plant or from a sample that includes the DNA of the event. The probes according to the present invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of such a target DNA sequence. An "isolated" polynucleotide implies that the polynucleotide is in an unnatural state - operationally linked to a heterologous promoter, for example. A "purified" protein similarly implies that the protein is in an unnatural state.
[00142] "Primers" are isolated / synthesized nucleic acids that are annealed to a complementary target DNA strand by hybridizing the nucleic acids to form a hybrid between the primer and the target DNA strand, then extended along the DNA strand targeted by a polymerase, for example, a DNA polymerizes. The primer pairs of the present invention refer to their use for amplifying a target nucleic acid sequence, for example, through polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.
[00143] Probes and primers generally have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 2 14, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 4 14, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 polynucleotides or more in length. Such probes and primers specifically hybridize to a target sequence under high stringency hybridization conditions. Preferably, the probes and primers according to the present invention have complete sequence similarity to the target sequence, although probes that differ from the target sequence and that retain the ability to hybridize to the target sequences can be designed using methods conventional.
[00144] Methods for the preparation and use of probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The PCR primer pairs can be derived from a known sequence, for example, using computer programs designed for this purpose.
[00145] Primers and probes based on the flanking DNA and insert sequences disclosed here can be used to confirm (and, if necessary, correct) the sequences disclosed by conventional methods, for example, through recloning and sequencing of such sequences.
The nucleic acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence. Any conventional hybridization or nucleic acid amplification method can be used to identify the presence of DNA from a transgenic event in a sample. The nucleic acid molecules or fragments thereof are able to specifically hybridize to other nucleic acid molecules under certain circumstances. As used here, it is said that two nucleic acid molecules are capable of hybridizing specifically to another if the two molecules are capable of forming an antiparallel double-stranded nucleic acid structure. One nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if they exhibit complete complementarity. As used here, molecules are said to exhibit "complete complementarity" when each nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be "minimally complementary" if they can hybridize to each other with sufficient stability to allow them to remain ringed with each other under at least conventional "low stringency" conditions. Similarly, molecules are said to be "complementary" if they can hybridize to each other with sufficient stability to remain ringed with each other under conventional "high stringency" conditions. Conventional stringency conditions are described by Sambrook et al., 1989. Deviations from complete complementarity are therefore permissible, as long as such deviations do not completely hamper the ability of molecules to form a double-stranded structure. For a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under particular concentrations of solvent and salt employed.
[00147] As used herein, a substantially homologous sequence is a nucleic acid sequence that will hybridize specifically to the complement of the nucleic acid sequence to which it is being compared under conditions of high stringency. The term "stringent conditions" is functionally defined in relation to the hybridization of a nucleic acid probe to a target nucleic acid (ie, to a particular nucleic acid sequence of interest) through the specific hybridization procedure discussed in Sam- brook et al., 1989, 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52 and 9.56-9.58. Consequently, the nucleotide sequences of the invention can be used for their ability to selectively form duplex molecules with complementary strands of DNA fragments.
[00148] Depending on the intended application, variable hybridization conditions can be used to achieve varying degrees of selectivity of the probe towards the target sequence. For applications that require high selectivity, relatively stringent conditions can typically be used to form the hybrids, for example, in relation to endpoint TaqMan and real-time PCR applications, 1.5 mM to approximately 4.0 mM can be selected MgCl2 at a temperature of approximately 60 ° C to approximately 75 ° C and waiting times can be varied, as described here, to increase stringency. For other hybridization techniques, relatively low salt and / or high temperature conditions may typically be employed, such as those provided by approximately 0.02 M to approximately 0.15 M NaCl at temperatures of approximately 50 ° C up to approximately 70 ° C. Strict conditions, for example, could involve washing the hybridization filter at least twice with high stringency wash buffer (0.2X SSC, 0.1% SDS, 65 ° C). The appropriate stringency conditions that promote DNA hybridization, for example, 6.0X sodium chloride / sodium citrate (SSC) at approximately 45 ° C, followed by a 2.0X SSC wash at 50 ° C are known for experts in the art. For example, the salt concentration in the washing step can be selected from a low stringency of approximately 2.0X SSC at 50 ° C to a high stringency of approximately 0.2X SSC at 50 ° C. In addition, the temperature in the washing step can be increased from low stringency conditions at room temperature, approximately 22 ° C, to high stringency conditions at approximately 65 ° C. Both the temperature and the salt can be varied or the temperature or the salt concentration can be kept constant while the other variable is changed. Such selective conditions tolerate little, if any, wrong pairing between the probe and the target mold or filtration. The detection of DNA sequences by hybridization is well known to those skilled in the art and the teachings of U.S. Patent Nos. 4,965,188 and 5,176,995 are examples of hybridization analysis methods.
[00149] In a particularly preferred embodiment, a nucleic acid of the present invention will specifically hybridize to one or more of the primers (or amplicons or other sequences) exemplified or suggested herein, including complements and fragments thereof, under conditions of high stringency. In one aspect of the present invention, a marker nucleic acid molecule of the present invention has the nucleic acid sequence that is presented here in one of the exemplified sequences or complements and / or fragments thereof.
[00150] In another aspect of the present invention, a marker nucleic acid molecule of the present invention shares between 80% and 100% or 90% and 100% sequence identity with such nucleic acid sequences. In a further aspect of the present invention, a marker nucleic acid molecule of the present invention shares between 95% and 100% sequence identity with such a sequence. Such sequences can be used as markers in plant breeding methods to identify the progeny of genetic crosses. Hybridization of the probe to the target DNA molecule can be detected by any of a number of methods known to those skilled in the art, these may include, but are not limited to, fluorescent labels, radioactive labels, antibody-based labels and chemiluminescent markings.
[00151] Regarding the amplification of a target nucleic acid sequence (for example, by PCR) using a pair of particular amplification primers, "stringent conditions" are conditions that allow the pair of primers to hybridize only to the target nucleic acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably produce a single amplification product, amplicon.
[00152] The term "specific for (a target sequence)" indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.
[00153] As used herein, "amplified DNA" or "amplicon" refers to the nucleic acid amplification product of a target nucleic acid sequence that forms part of a nucleic acid template. For example, to determine whether the soybean plant resulting from a sexual cross contains the genomic DNA from the transgenic event from the soybean plant of the present invention, the DNA extracted from a sample of soybean tissue can be subjected to the amplification method nucleic acid using a primer pair that includes a primer derived from the flanking sequence in the plant genome adjacent to the inserted heterologous DNA insertion site and a second primer derived from the inserted heterologous DNA to produce an amplicon that is diagnostic for the presence of the Event DNA. The amplicon is of a length and has a sequence that is also diagnostic in relation to the event. The amplicon can vary in length from the combined length of the primer pairs plus a nucleotide base pair and / or the combined length of the primer pairs plus approximately 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 , 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135 , 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 , 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 18 2, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 38 2, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500, 750, 1000, 1250, 1500, 1750, 2000 or more nucleotide base pairs (plus or minus any of the increments listed above). Alternatively, a pair of primers can be derived from the flanking sequence on both sides of the inserted DNA in order to produce an amplicon that includes the nucleotide sequence of the entire insert. A member of a pair of primers derived from the plant's genome sequence may be located at a distance from the inserted DNA sequence. This distance can vary from one nucleotide base to approximately twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers that can be formed in the DNA thermal amplification reaction.
[00154] Nucleic acid amplification can be performed using any of the various nucleic acid amplification methods known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, inter alia, in U.S. Patent No. 4,683,195 and in U.S. Patent No. 4,683,202. PCR amplification methods were developed to amplify up to 22 kb of genomic DNA. These methods as well as other methods known in the art of DNA amplification can be used in the practice of the present invention. The DNA insert sequence of the heterologous transgene or the genomic flanking sequence of a soybean objective Event can be verified (and corrected, if necessary) by amplifying such sequences from the Event using primers derived from the sequences provided here followed by the sequencing of Standardized DNA from the PCR amplicon or cloned DNA.
[00155] The amplicon produced by these methods can be detected by a large number of techniques. Agarose gel electrophoresis and ethidium bromide staining is a common, well-known method of detecting DNA amplicons. Another such method is Genetic Bit Analysis in which a DNA oligonucleotide is drawn that overlaps both the adjacent flanking DNA genomic sequence and the inserted DNA sequence. The oligonucleotide is immobilized in wells of a microwell plate. After PCR of the region of interest (using a primer in the inserted sequence and one in the adjacent flanking genomic sequence), a single-stranded PCR product can be hybridized to the immobilized oligonucleotide and serves as a template for a base extension reaction isolated using a DNA polymerase and specific labeled ddNTPs for the next expected base. The reading can be fluorescent or ELISA-based. A signal indicates the presence of the flanking insert / sequence due to the amplification, hybridization and extension of successful isolated bases.
[00156] Another method is the Pirosequencing technique that is described by Winge (Innov. Pharma. Tech. 00: 18-24, 2000). In this method, an oligonucleotide is drawn that overlaps the adjacent genetic DNA and the DNA junction of the insert. The oligonucleotide is hybridized with the single-stranded PCR product from the region of interest (one primer in the inserted sequence and one in the flanking genomic sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apinase, 5 'adenosine phosphosulfan and luciferin. DNTPs are added individually and the incorporation results in a light signal that is measured. A light signal indicates the presence of the transgene insert / flanking sequence due to the amplification, hybridization and extension of several successful bases.
[00157] Fluorescence polarization is another method that can be used to detect an amplicon of the present invention. Following this method, an oligonucleotide is drawn that overlaps the genomic flanking sequence and the junction of the inserted DNA. The oligonucleotide is hybridized with the single-stranded PCR product from the region of interest (a primer in the inserted DNA and one in the genomic DNA flanking sequence) and incubated in the presence of a DNA polymerase and a fluorescently labeled ddNTP. The extension of a single base results in the incorporation of ddNTP. Incorporation can be measured in the form of a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgene insert / flanking sequence due to the successful amplification, hybridization and extension of a single base.
[00158] TAQMAN (PE Applied Biosystems, Foster City, Calif.) Is a method of detecting and quantifying the presence of a DNA sequence. Briefly, a FRET oligonucleotide probe is designed that overlaps the genomic flanking sequence and the DNA junction of the insert. The FRET probe and PCR primers (one primer in the insert's DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. During specific amplification, Taq DNA polymerase cleans and releases the fluorescent cluster away from the extinction cluster on the FRET probe. A fluorescent signal indicates the presence of the flanking / insert sequence of the transgene due to successful amplification and hybridization.
[00159] Molecular "Beacons" have been described for use in sequence detection. Briefly, a FRET oligo-nucleotide probe is designed that overlaps the flanking genomic sequence and the DNA junction of the insert. The FRET probe's unique structure results in a secondary structure that keeps the fluorescent and extinction groups in close proximity. The FRET probe and PCR primers (one primer in the DNA sequence insert and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, hybridization of the FRET probe to the target sequence results in the removal of the secondary structure of the probe and in the spatial separation of the fluorescent and extinction groups. A fluorescent signal appears. A fluorescent signal indicates the presence of the flanking / insert genomic sequence of the transgene due to successful amplification and hybridization.
[00160] Having disclosed a location in the soybean genome that is excellent for an insertion, the present invention further includes a soybean seed and / or a soybean plant comprising at least one coding sequence other than aad12 / pat / 2mepsps within or around a general neighborhood of this genomic location. One option is to replace a different insert in place of the one shown here. Under these general aspects, targeted homologous recombination, for example, can be used in accordance with the present invention. This type of technology is the objective, for example, of WO 03/080809 A2 and the corresponding published U.S. patent application (U.S. 2003/0232410). Thus, the present invention includes plants and plant cells that comprise a heterologous insert (in place of or with several copies of the exemplified insert), flanked by all or a part that can be recognized from the flanking sequences identified here as SEQ ID NO: 1 and SEQ ID NO: 2. An additional copy (or additional copies) of the example insert or any of its components could also be targeted for insertion in this / these ways.
[00161] All patents, patent applications, provisional patent applications and publications referred to or cited here are incorporated by reference in their entirety to the extent that they are not inconsistent with the explicit teachings of this specification.
[00162] The following examples are included to illustrate procedures for practicing the invention and to demonstrate certain preferred embodiments of the invention. These examples are not to be construed as limiting. It should be considered by those skilled in the art that the techniques disclosed in the examples below represent specific approaches used to illustrate the preferred modes for their practice. However, those skilled in the art should, in the light of the present description, consider that many changes can be made in these specific embodiments while the same or similar results are still obtained without departing from the spirit and scope of the invention. Unless otherwise stated, all percentages are by weight and all proportions of solvent mixtures are by volume unless otherwise stated.
[00163] The following abbreviations are used unless otherwise indicated. bp base pair ° C degrees Celcius DNA deoxyribonucleic acid DIG digoxigenin EDTA ethylenediaminetetraacetic acid kb kilobase μg microgram μL microliter mL milliliter M molar OLP overlap probe PCR polymerase chain reaction PTU plant transcription unit sodium Sod dodecyl sulfate transcription unit SOP standard operating procedure SSC a buffer solution containing a mixture of sodium chloride and sodium citrate, pH 7.0 TBE a buffer solution containing a mixture of Tris base, boric acid and EDTA, pH 8.3 volts EXAMPLES Example 1: Transformation and Selection of 2mEPSPS and AAD-12 of Soy Event 8291.45.36.2
[00164] The transgenic soybean (Glycine max) containing the soybean event 8291.45.36.2 was generated through Agrobacterium-mediated transformation of soybean cotyledon explants. The disarmed Agrobacterium strain EHA101 (Hood et al., 2006) carrying the binary vector pDAB8291 (Figure 1) containing a selectable marker, pat and the genes of interest, aad-12 and 2mepsps v2, within the DNA region of filament T, was used to initiate the transformation.
[00165] Agrobacterium-mediated transformation was performed using a modified procedure by Zeng et al. (2004). Briefly, soybean seeds (cv Maverick) were germinated in a basal medium and in the cotyledon nodes were isolated and infected with Agrobacterium. The sprout initiation, sprout elongation and rooting means were supplemented with cefotaxime, timentin and vancomycin to remove Agrobacterium. Glufosinate selection was used to inhibit the growth of untransformed shoots. The selected shoots were transferred to the rooting medium for root development and then transferred to the soil mixture to acclimatize the young plants.
[00166] The terminal leaflets of selected young plants had their leaves brushed with glufosinate to select alleged transformants. The selected young plants were transferred to the greenhouse, allowed to acclimatize and then the leaves were brushed with glufosinate to reconfirm tolerance and considered to be supposed transformants. Samples of the selected plants were taken and molecular analyzes were performed to confirm the selectable marker gene and / or the gene of interest. The T0 plants were allowed to self-fertilize in the greenhouse to give rise to the T1 seed.
[00167] This event, Soy Event 8291.45.36.2, was generated from an independent transformed isolate. T1 plants were backcrossed and suffered introgression in elite varieties over subsequent generations. The Event was selected based on its unique characteristics such as single insertion site, normal Mendelian segregation and stable expression and a superior combination of efficacy, including herbicide tolerance and agronomic performance. The following examples contain the data that were used to characterize the Soy Event 8291.45.36.2. Example 2: Characterization of Protein AAD-12, 2mEPSPS and PAT in Soy Event 8291.45.36.2
[00168] The biochemical properties of the AAD-12 protein, 2mEPSPS and recombinant PAT derived from the transgenic soybean event pDAB8291.45.36.2 were characterized. The quantitative enzyme-linked immunosorbent assay (ELISA) was used to characterize the biochemical properties of the protein and confirm the expression of the AAD-12, PAT and 2mEPSPS protein. Example 2.1: Expression of AAD-12 Protein in Plant Tissues
[00169] AAD-12 protein levels were determined in the soybean event pDAB8291.45.36.2. The AAD-12 protein that can be extracted soluble was measured using an immuno-absorbent assay method linked to the quantitative enzyme (ELISA) starting from the soybean leaf tissue.
[00170] Soy tissue samples were isolated from the test plants and prepared for expression analysis. The AAD-12 protein was extracted from the soy plant tissues with a phosphate-buffered saline solution containing the Tween-20 detergent (PBST) containing 0.5% Bovine Serum Albumin (BSA). The vegetable tissue was centrifuged; the aqueous supernatant was collected, diluted with an appropriate buffer and analyzed using an ELISA kit for AAD-12 in a sandwich format. The kit was used following the protocol suggested by the manufacturer.
[00171] Detection analysis was performed to investigate expression stability and inheritance capacity both vertically (between generations) and horizontally (between strains of the same generation) in the soybean event pDAB8291.45.36.2. In the T4 generation, the expression of the soybean event pDAB8291.45.36.2 was stable (not segregating) and coherent among all strains. Studies of expression levels in the field were carried out on the soybean event pDAB8291.45.36.2; the average expression among all strains was approximately 200 - 250 ng / cm2. Example 2.2: Expression of the 2mEPSPS Protein in Plant Tissues
[00172] The 2mEPSPS protein levels were determined in the soybean event pDAB8291.45.36.2. The 2mEPSPS protein that can be extracted soluble was measured using an immunosorbent assay method linked to the quantitative enzyme (ELISA) starting from the soybean leaf tissue.
[00173] Soy tissue samples were isolated from the test plants and prepared for expression analysis. The 2mEPSPS protein was extracted from the soy plant tissues with a phosphate-buffered saline solution containing the Tween-20 detergent (PBST) containing 0.5% Bovine Serum Albumin (BSA). The vegetable tissue was centrifuged; the aqueous supernatant was collected, diluted with appropriate buffer when necessary and analyzed using an ELISA kit for 2mEPSPS in a sandwich format. The kit was used following the protocol suggested by the manufacturer.
[00174] Detection analysis was performed to investigate expression stability and inheritance capacity both vertically (between generations) and horizontally (between strains of the same generation) in the soybean event pDAB8291.45.36.2. In the T4 generation, the expression of the soybean event pDAB8291.45.36.2 was stable (not segregating) and coherent among all strains. Studies of expression levels in the field were carried out on the soybean event pDAB8291.45.36.2. The average expression among all strains was approximately 5,000 - 10,000 ng / cm2. Example 2.3: Expression of PAT Protein in Plant Tissues
[00175] PAT protein levels were determined in the soybean event pDAB8291.45.36.2. The PAT protein that can be extracted soluble was measured using an immunoabsorbent assay method linked to the quantitative enzyme (ELISA) starting from the soybean leaf tissue.
[00176] Soy tissue samples were isolated from the test plants and prepared for expression analysis. PAT protein was extracted from soy plant tissues with a phosphate-buffered saline solution containing the Tween-20 detergent (PBST) containing 0.5% Bovine Serum Albumin (BSA). The plant tissue was centrifuged; the aqueous supernatant was collected, diluted with appropriate buffer when necessary and analyzed using an ELISA kit for PAT in an interleaved format. The kit was used following the protocol suggested by the manufacturer.
[00177] Detection analysis was performed to investigate expression stability and inheritance capacity both vertically (between generations) and horizontally (between strains of the same generation) in the soybean event pDAB8291.45.36.2. In the T4 generation, the expression of the soybean event pDAB8291.45.36.2 was stable (not segregating) and coherent among all strains. Studies of expression levels in the field were carried out on the soybean event pDAB8291.45.36.2. The average expression among all strains was approximately 15 - 20 ng / cm2. Example 3: Cloning and Characterization of the DNA Sequence in the Insert and Flanking Edge Regions of the Soy Event pDAB8291.45.36.2
[00178] To characterize and describe the genomic insertion site, the sequence of the edge regions of the genomic DNA flanking the soybean event pDAB8291.45.36.2 was determined. In total, 4,414 bp of the genomic sequence of the soybean event pDAB8291.45.36.2 were confirmed, comprising 3,088 bp of the 5 'flanking edge sequence (SEQ ID NO: 1), 785 bp of the flanking edge sequence to 3 '(SEQ ID NO: 2). PCR amplification based on the edge sequences of the pDAB8291.45.36.2 soy event validated that the edge regions were of soy origin and that the junction regions are unique sequences for the pDAB8291.45.36.2 event. The junction regions could be used for event specific identification of the soybean event pDAB8291.45.36.2. The insertion site of filament T was characterized by the cloning of a genomic fragment corresponding to the region of the flanking edge sequences identified in the genome of non-transgenic transgenics. The comparison of the soybean event pDAB8291.45.36.2 with the genomic sequence of the wild type revealed a deletion of 48 bp starting from the original locus. In general, the characterization of the insert and the edge sequence of the soybean event pDAB8291.45.36.2 indicated that an intact copy of filament T was present in the soybean genome. Table 2. Primers and sequences used to analyze DNA in the





Example 3.1: Confirmation of Soy Genomic Sequences
[00179] The 5 'and 3' flanking edges were aligned with chromosome 03 of the "shotgun" sequence of the entire Glycine max genome, indicating that the transgene of the soybean event pDAB8291.45.36.2 was probably inserted on chromosome 03 of the soybean genome. To confirm the insertion site of the transgene of the soybean event pDAB8291.45.36.2 in the soybean genome, a PCR was performed with different pairs of primers (Figure 2 and Table 3). The genomic DNA from the soybean event pDAB8291.45.36.2 and other transgenic strains or non-transgenic strains of soybean was used as templates. Thus, to confirm that the 5 'end edge sequences are correct, specific primers for 2mepsps, for example, ED_v2_C1 (SEQ ID NO: 11) and two primers designed according to the cloned 5' end edge sequence , named 4536_WF1 (SEQ ID NO: 3) and 4536_WF4 (SEQ ID NO: 6), were used for the amplification of the DNA segment that encompasses the 2mepsps gene to the 5 'end edge sequence. Similarly, for confirmation of, a specific primer for pat, for example, PAT_11 (SEQ ID NO: 12) and two primers designed according to the cloned 3 'end edge sequence and / or its alignment sequence on the chromosome 03 of the soybean genome, named 4536_WR1 (SEQ ID NO: 7) and 4536_WR4 (SEQ ID NO: 10), were used for the amplification of the DNA segments that span the pat gene to the 3 'end edge sequence. DNA fragments with predicted sizes were amplified only starting from the genomic DNA of the soybean event pDAB8291.45.36.2 with each pair of primers, a primer located on the flanking edge of the soybean event pDAB8291.45.36.2 and a specific primer for the transgenic, but not from the DNA samples of other transgenic soybean strains or non-transgenic control. The results indicate that the 5 'and 3' cloned edge sequences are the flanking edge sequences of the T-filament insert in the soybean event pDAB8291.45.36.2.
[00180] To further confirm the insertion of DNA into the soybean genome, an amplification by PCR covering the two soybean border sequences has been completed. Two primers, 4536_WF1 (SEQ ID NO: 3) and 4536_WF4 (SEQ ID NO: 6), were designed according to the 5 'end edge sequence and two primers, 4536-WR1 (SEQ ID NO: 7) and 4536-WR4 (SEQ ID NO: 10), were designed according to the 3 'end edge sequence. As expected, PCR amplification with the 4536_WF1 (SEQ ID NO; 3) and 4536_WR1 (SEQ ID NO: 7) primer pair amplified an approximately 3.0 kb DNA fragment from non-GM soy controls and others transgenic soybean lines, but not from the sample of the soybean event pDAB8291.45.36.2. Similarly, PCR reactions completed with the primer pair of 4536_WF4 (SEQ ID NO: 6) and 4536_WR4 (SEQ ID NO: 10) produced a DNA fragment of approximately 878 bp from the genomic DNA of the non-transgenic soybean controls and other transgenic soybean strains except the sample from the soybean event pDAB8291.45.36.2. These results demonstrated that the soybean event transgene pDAB8291.45.36.2 was inserted within the chromosome 03 site of the soybean genome. Example 3.2: Confirmation of Soy Genomic Sequences
The amplified DNA fragments of 3 kb, using the pair of primers 4536_WF1 and 4536_WR1, from the non-transgenic soybean line Maverick were cloned and sequenced. The sequence was aligned with the 5 'and 3' edge sequences identified from the soybean event pDAB8291.45.36.2. This demonstrated that the cloned DNA sequence contained the locus where the T strand of pDAB8291 was integrated within the soybean event pDAB8291.45.36.2. The alignment analysis also revealed a 48 bp deletion from the original locus (Figure 3). Analysis of the open reading frame (ORF) using Vector NTI (Version 11, Invitrogen) revealed that filament T in the soybean event pDAB8291.45.36.2 was inserted within a 249 bp ORF located in the soybean genomic region of the locus original that was cloned. Example 4: Characterization of the Soy Event pDAB8291.45.36.2 Through Southern Blot
[00182] Southern blot analysis was used to establish the integration pattern of the soybean event pDAB8291.45.36.2. These experiments generated data that demonstrated the integration and integrity of the aad-12, pat and 2mepsps v2 transgenes within the soybean genome. The soybean event pDAB8291.45.36.2 was characterized as a simple full-length integration event containing a single copy of the aad-12, pat and 2mepsps v2 PTU of the plasmid pDAB8291.
[00183] Southern data suggested that a T-filament fragment was inserted into the genome of the soybean event pDAB8291.45.36.2. A detailed Southern blot analysis was performed using a probe specific for the insert of aad-12, pat and 2mepsps v2, contained in the T filament integration region of pDAB8291 and descriptive restriction enzymes that have cleavage sites located within the plasmid and they produce hybridization fragments internal to the plasmid or fragments that comprise the junction of the plasmid with the genomic DNA of the soybean (border fragments). The molecular weights indicated starting from Southern hybridization for the combination of the restriction enzyme and the probe were unique for the Event and established their identification standards. These analyzes also showed that the plasmid fragment had been inserted into the genomic DNA of the soybean without rearrangements of the aad-12, pat and 2mepsps v2 PTU. Example 4.1: Soy Leaf Sample Collection and Genomic DNA Isolation (gDNA)
[00184] Genomic DNA was extracted from leaf tissue collected from individual soybean plants containing the soybean event pDAB8291.45.36.2. In addition, the gDNA was isolated from a conventional soybean plant, Maverick, which contains the fundamental genetic structure that is representative of the substance lineage, with the absence of the aad-12 and 2mepsps v2 genes. Individual genomic DNA was extracted from lyophilized leaf tissue following the standardized cetitrimethylammonium bromide CTAB method. After extraction, the DNA was quantified spectrofluorometrically using the Pico Green reagent (Invitrogen, Carlsbad, CA). The DNA was then visualized on an agarose gel to confirm values from the Pico Green analysis and to determine the quality of the DNA. Example 4.2: Digestion and DNA Separation
[00185] For the Southern blot molecular characterization of the soybean event pDAB8291.45.36.2, ten micrograms (10 μg) of genetic DNA were digested. The genomic DNA of soybean pDAB8291.45.36.2 and of the Maverick non-transgenic soybean strain was digested by adding approximately five units of selected restriction enzyme per μg of DNA and the corresponding reaction buffer in each DNA sample. Each sample was incubated at approximately 37 ° C overnight. The restriction enzymes BstZ17I, HinDIII, NcoI, NsiI and PacI were used individually for digestions (New England Biolabs, Ipswich, MA). In addition, a positive hybridization control sample was prepared by combining the plasmideal DNA, pDAB8291 with the genomic DNA of the non-transgenic soybean variety, Maverick. The cocktail of plasmideal DNA / genetic DNA was digested using the same procedures and the restriction enzyme as the test samples. After the digestions were incubated overnight, NaCl was added to a final concentration of 0.1 M and the digested DNA samples were precipitated with isopropanol. The precipitated DNA pellet was resuspended in 20 μL of 1X loading buffer (0.01% bromophenol blue, 10.0 mM EDTA, 5.0% glycerol, 1.0 mM Tris pH 7.5) . The DNA samples and molecular size markers were then electrophoresed using 0.85% agarose gels with 0.4X TAE buffer (Fisher Scientific, Pittsburgh, PA) at 35 volts for approximately 18-22 hours to achieve the separation of fragments. The gels were stained with ethidium bromide (Invitrogen, Carlsbad, CA) and the DNA was visualized under ultraviolet (UV) light. Example 4.3: Southern Transfer and Membrane Treatment
[00186] Southern blot analysis was performed essentially as described by, Memelink, J .; Swords, K .; Harry J .; Hoge, C .; (1994) Southern, Northern and Western Blot Analysis. Plant Mol. Biol. F1 Manual: 1-23. Briefly, after electrophoretic separation and visualization of DNA fragments, the gels were depurinated with 0.25 M HCl for approximately 20 minutes and then exposed to a denaturing solution (0.4 M NaOH, 1.5 M NaCl) for approximately 30 minutes followed by a neutralization solution (1.5 M NaCl, 0.5 M Tris pH 7.5) for at least 30 minutes. Southern transfer was performed overnight on nylon membranes using a 10x SSC wick system. After the transfer, the DNA was linked to the membrane by cross-linking through UV followed by rapid washing of the membrane with a 2x SSC solution. This process produced Southern blot membranes ready for hybridization. Example 4.4: DNA Probe Labeling and Hybridization
[00187] DNA fragments attached to the nylon membrane were detected using a labeled probe. The probes were generated through a PCR-based incorporation of a digoxigenin-tagged nucleotide (DIG), [DIG-11] -dUTP, within the amplified DNA fragment starting from plasmid pDAB8291 using specific primers for the gene elements. The production of DNA probes through PCR synthesis was performed using a PCR DIG Probe Synthesis Kit (Roche Diagnostics, Indianapolis, IN) following the procedures recommended by the manufacturer.
[00188] The labeled probes were analyzed by agarose gel electrophoresis to determine their quality and quantity. A desired amount of labeled probe was then used for hybridization with the target DNA on the nylon membranes for the detection of specific fragments using the procedures essentially as described for DIG Easy Hyb Solution (Roche Diagnostics, Indianapolis, IN). Briefly, the blots on the nylon membrane containing the fixed DNA were briefly washed with 2x SSC and prehybridized with 20-25 mL of DIG Easy Hyb solution preheated in the hybridization bottles at approximately 45-55 ° C for approximately 2 hours in a hybridization oven. The pre-hybridization solution was then decanted and replaced with approximately 15 mL of the pre-heated DIG Easy Hyb solution containing a desired amount of specific probes denatured by boiling in a water bath for approximately five minutes. The hybridization step was then conducted at approximately 45-55 ° C overnight in the hybridization oven.
[00189] At the end of probe hybridization, DIG Easy Hyb solutions containing the probes were decanted into clean tubes and stored at approximately -20 ° C. These probes could be reused twice according to the procedure recommended by the manufacturer. The blots on the membrane were washed briefly and washed twice in clean plastic containers with low stringency washing buffer (2x SSC, 0.1% SDS) for approximately five minutes at room temperature, followed by washing twice with buffer high stringency wash cycle (0.1x SSC, 0.1% SDS) for 15 minutes each at approximately 65 ° C. The blots on the membrane were washed briefly with 1x maleic acid buffer from the DIG Wash and Block Buffer Set (Roche Diagnostics, Indianapolis, IN) for approximately 5 minutes. This was followed by blocking in a 1x blocking buffer for 2 hours and an incubation with anti-DIG-AP (alkaline phosphatase) antibody (Roche Diagnostics, Indianapolis, IN) in 1x blocking buffer also for a minimum of 30 minutes. After 2-3 washes with 1x wash buffer, specific DNA probes remain attached to the blots on the membrane and the DNA patterns marked with DIG were visualized using the CDP-Star Chemiluminescent Nucleic Acid Detection System (Roche Diagnostics, Indiana-polis , IN) following the manufacturer's recommendation. The blots were exposed to the chemiluminescent film for one or more time points to detect fragments of hybridization and to visualize the molecular size patterns. The films were developed with an All-Pro 100 Plus film developer (Konica Minolta, Osaka, Japan) and the images were scanned. The number and sizes of the detected bands were documented for each probe (Table 5). DIG-labeled DNA Molecular Weight Marker II (DIG MWM II) and DIG-labeled DNA Molecular Weight Marker VII (DIG MWM VII), visible after DIG detection as described, were used to determine the size of the hybridization fragment on Southern blots. Table 5. Length of the probes used in the Southern analysis of the soybean event pDAB8291.45.36.2
Example 4.5: Southern Blot results
[00190] The sizes of fragments expected and observed with a digestion product and a particular probe, based on the restriction enzyme sites known from aad-12 and 2mepsps PTU, are given in Table 6. The sizes of fragments expected are based on plasmideal map of pDAB8291 and the observed fragment sizes are approximate results from these analyzes and are based on the indicated sizes of the DIG-labeled DNA Molecular Weight Marker II and Mark VII fragments.
[00191] Two types of fragments have been identified from these hybridization digestion products: internal fragments in which known enzyme sites flank the probe region and are completely contained within the insertion region of the aad-12 and 2mepsps PTU PTU and edge fragments where a known enzyme site is located at one end of the probe region and a second site is expected in the soybean genome. The sizes of the edge fragments vary by Event because, in most cases, the integration sites of the DNA fragment are unique for each event. The edge fragments provide a means for locating a restriction enzyme site relative to the integrated DNA and for assessing the number of DNA insertions. Southern blot analyzes completed over several generations of soybean counting Event pDAB8291.45.36.2 produced data that suggest that an intact aad-12 and 2mepsps intact low-copy plasmid pDAB8291 was inserted into the soybean genome of the event soybean pDAB8291.45.36.2. Table 6. Hybridization Fragments Predicted and Observed in Southern Blot Analysis



[00192] The restriction enzymes NcoI and HinD III bind and cleave unique restriction sites on plasmid pDAB8291. Subsequently, these enzymes were selected to characterize the insertion of the aad-12 gene in the soybean event pDAB8291.45.36.2. It was predicted that edge fragments greater than 4,078 bp or greater than 3,690 bp would hybridize with the probe after digestion with HinD III and NcoI respectively (Table 6). Isolated aad-12 hybridization bands of approximately 4,800 bp and approximately 5,900 bp were observed when HinDIII and NcoI were used, respectively. Hybridization of the probe with bands of this size suggests the presence of a single insertion site for the aad-12 gene in the soybean genome of the soybean event pDAB8291.45.36.2. The restriction enzymes BstZ17I, NsiI and PacI were selected to release a fragment containing the aad-12 plant transcription unit (PTU; promoter / gene / terminator) (Table 6). The predicted fragments of approximately 5,000, approximately 5,000 and approximately 6,800 bp were observed with the probe after digestions with BstZ17I, NsiI and PacI, respectively. The results obtained with enzyme digestion of the pDAB8291.45.36.2 sample followed by probe hybridization indicated that an intact aad-12 PTU from plasmid pDAB8291 was inserted into the soybean genome of the soybean event pDAB8291.45.36.2. In addition, the molecular weight sizes of the hybridization bands produced for the HinDIII, NcoI, NsiI and BstZ17I restriction fragments indicate that the aad-12 PTU PTU also contained the pat pat PTU.
The restriction enzymes BstZ17I, NcoI and NsiI bind and cleave restriction sites on plasmid pDAB8291. Subsequently, these enzymes were selected to characterize the insertion of the 2mepsps gene in the soybean event pDAB8291.45.36.2. It was predicted that edge fragments greater than 4,870 bp, greater than 3,772 or greater than 5,207 bp hybridized with the probe after digestions with BstZ17I, NcoI and NsiI, respectively (Table 6). Hybridization bands of 2mepsps isolated of approximately 11,000 bp, approximately 4,000 bp and approximately 6,000 bp were observed when BstZ17I, NcoI and NsiI were used, respectively. Hybridization of the probe with bands of this size suggests the presence of a single insertion site for the 2mepsps gene in the soybean genome of the soybean event pDAB8291.45.36.2. The restriction enzyme PacI was selected to release a fragment containing the 2mepsps plant transcription unit (PTU; promoter / gene / terminator) (Table 6). The predicted fragments of approximately 6,800 bp were observed with the probe after digestions with PacI. The results obtained with digestion with the enzyme from the pDAB8291.45.36.2 sample followed by probe hybridization indicated that an intact 2mepsps PTU of the plasmid pDAB8291 was inserted into the soybean genome of the soybean event pDAB8291.45.36.2. Example 4.6: Absence of Structural Sequences
[00194] A Southern blot analysis was also performed to verify the absence of the spectinomycin resistance gene, the Ori Rep element and the trfA replication initiation protein (trf A element) in the soybean event pDAB8291.45.36.2. No specific hybridization to resistance to spectinomycin, the Ori Rep element or the trf A element is expected when positive (pDAB8291 plus Maverick) and negative (Maverick) controls are included for Southern analysis. After digestion with Hind III digestion and hybridization with the specific specR probe, a size band of approximately 9,300 bp could be expected to be observed in the positive control sample (pDAB8291 plus maverick), but absent in the negative control samples and in the soybean event pDAB8291.45.36.2. Similarly, a band of expected size of approximately 9,200 bp was detected in the positive control sample (pDAB8291 plus maverick), but absent in the samples of the negative control and soybean event pDAB8291.45.36.2 after digestion with Pac I and hybridization with the mixture of specific probe for OriRep and specific probe for trfA. These data indicate the absence of the spectinomycin resistance gene, the Ori Rep element and the trfA replication initiation protein in the soybean event pDAB8291.45.36.2. Example 5: Agronomic, Yield and Herbicide Tolerance Assessment
[00195] The agronomic characteristics and herbicide tolerance of the soybean event pDAB8291.45.36.2 were studied in yield tests in several geographic locations during a single growing season. No significant unintended agronomic differences were observed between the soybean event pDAB8291.45.36.2 and the Maverick control plants. The results of the study demonstrate that the soybean event pDAB8291.45.36.2 was agronomically equivalent to the Maverick control plants. In addition, the soybean event pDAB8291.45.36.2 provided tolerance to robust herbicide when sprayed with a glyphosate and 2,4-D tank mixture.
[00196] The following agronomic characteristics were measured and recorded for all test records at each location.
[00197] 1.) Emergence: Calculated by dividing the support count by the number of seeds planted in a one-meter section multiplying by 100.
[00198] 2.) Stamina of the Seedling in V1: Stamina is a general estimate of the health of the batch. The results are rated on a scale of 0-100% with 0% representing a lot with all the dead plants and 100% representing lots that looked very healthy.
[00199] 3.) Generally classified visual damage to the cultivation plant, chlorosis and necrosis on 1 day, 7 days and 14 days after the chemical application of V3. Observations were made regarding any signs of epinastia that were typical of damage caused by 2,4-D. Epinastia is shown as the curvature or inclination of leaves and stems. All damage to crop plants used a scale from 0 to 100%, where 0% indicates no damage and 100% indicates total plant death.
[00200] 4.) Flowering date: This measure records the date when 50% of the plants in the lot begin to bloom. The number of days from planting to when 50% of the plants in each lot were blooming was recorded.
[00201] 5.) Sustaining count in R2 or R1: It is a visual estimate of the average vigor of the plants in each lot, determined by counting the number of plants in a representative section of one meter of a row per lot and taking note growth stage R2 or R1.
[00202] 6.) Generally classified visual damage to the cultivation plant, chlorosis and necrosis on 1 day, 7 days and 14 days after the chemical application of R2. Observations were made for any signs of epinastia that were typical of damage caused by 2,4-D. Epinastia is shown as the curvature or inclination of leaves and stems. All damage to crop plants used a scale from 0 to 100%, where 0% indicates no damage and 100% indicates total plant death.
[00203] 7.) Incidence of diseases in the growth stage R6: It is a visual estimate of disease incidence used to record the severity of disease in the batch. Classified on a scale of 0-100%. Where 0% indicates no disease is present and 100% indicates that all plants in the lot have become ill.
[00204] 8.) Damage caused by insects in the growth stage R6: It is a visual estimate of damage caused by insects used to record the severity of damage caused by insects in the lot. The percentage of plant tissue in the batch damaged by insects was recorded using a scale of 0-100%. Where 0% indicates no insect damage is present and 100% indicates that all plants in the lot had insect damage.
[00205] 9.) Height of the plant at senescence: The average height of the plants in each lot was recorded. Plants were measured from the surface of the soil to the tip after the leaves had fallen off. The measurements were recorded in centimeters. If the lot was felled, a representative group of plants was raised to obtain a measure.
[00206] 10.) Days until maturity. Data recorded when 95% of the pods in a batch reached physiological maturity and the plants had a dry color. The number of days until the end since planting was calculated.
[00207] 11.) Felling: A visual estimate of the felling severity at the time of harvesting was recorded. Recorded on a scale of 0 to 100% with 0% indicating no tipping and 100% indicating that all plants in a lot were dropped on the ground.
[00208] 12.) Shattering: A visual estimate of the shredding of pods was recorded at the time of harvest. Recorded as an estimate of the percentage of pods smashed per batch. Scale 0-100% with 0% indicating no shattering and 100% indicating that all pods have shattered.
[00209] 13.) Yield: Recorded the weight of the grain harvested from each batch. The entire 2-row lot was harvested and the weight and moisture of the seed were recorded. Calculations were made for bu / acre by adjusting the humidity by 13%.
[00210] 14.) 100 gram seeds: For each batch 100 seeds were counted and the weight was recorded in grams.
[00211] The herbicide tolerance of the soybean event pDAB8291.45.36.2 was evaluated after the application of a mixture in 2,4-D tank and glyphosate at 2,185 g ae / ha mixed with 2% by weight by weight of sulfate of ammonium (AMS). The herbicides were sprayed in the form of a sequential V3 / R2 herbicide treatment. This herbicide treatment was completed by spraying the soybean plants in the V3 growth stage followed by a second sequential application in the R2 growth stage. The V3 growth stage is characterized when the unifoliate and the first three trifoliate leaves are fully developed. The growth stage R2 is characterized by a single flower open in one of the two uppermost nodules in the main stem with a fully developed leaf.
[00212] These tests were prepared using a complete randomized block design with four replicates for each treatment. Each lot was 2 rows wide and the rows were spaced 76.2 cm (30 inches) from each other. The lots were planted up to a total length of 381 cm (12.5 feet) with a passage of 76.2 to 91.44 cm (2.5 to 3.0 feet) between the lots. Maverick control plants were expected to die from herbicide applications so that they were grown in a separate lot; away from the ranks of transgenic soy plants.
[00213] The results of the soybean event pDAB8291.45.36.2 sprayed with the mixture in a 2,4-D tank and glyphosate when compared with those of the soybean event pDAB8291.45.36.2 not sprayed are summarized. Table 7 presents the averages of an analysis that compares the sprayed soybean event pDAB8291.45.36.2 with a mixture in a 2,4-D tank and glyphosate with the non-sprayed soybean event pDAB8291.45.36.2. The herbicide application did not damage the soybean event pDAB8291.45.36.2, these plants had performance equivalent to that of the soybean event pDAB8291.45.36.2 not sprayed in relation to the reported agronomic characteristics listed in Table 7. With the exception of some initial transient damage 1 and 7 daa (days after application) in the V3 development stage and 1, 7 and 14 daa in the R2 development stage, the soybean event pDAB8291.45.36.2 exhibited robust tolerance to the 2-tank mix , 4-D and glyphosate. In contrast, none of the Maverick plants survived after being sprayed with the herbicides. Table 7. Comparison of sprayed soybean event pDAB8291.45.36.2


[00214] The agronomic equivalence of the soybean event pDAB8291.45.36.2 and the control strain, Maverick, was evaluated. These tests were assembled using a block design with two replicates. Each lot was 2 rows apart and spaced 30 inches apart. The plots were planted to a total length of 2.5 feet with a passage of 2.5 to 3.0 feet between the plots.
[00215] Table 8 presents the averages from the analysis comparing the agronomic equivalence of the soybean Event pDAB- 8291.45.36.2 with the control strain, Maverick. The agronomic data are indicative that the soybean event pDAB8291.45.36.2 has a performance equivalent to that of the Maverick plants and does not result in significant agronomically significant differences. Table 8. Comparison of the soybean event pDAB8291.45.36.2 with the Maverick control lines in yield tests.
Example 6: Event-specific Taqman trial
[00216] Two TAQMAN assays specific to the Event were developed to detect the presence of the soybean event pDAB8291.45.36.2 and to determine the status of plant zygosity in the crossbreeding populations. The soybean event pDAB- 8291.45.36.2 contains the T filament of the binary vector pDAB8291 (Figure 1). For the specific detection of the soybean event pDAB8291.45.36.2, specific Taqman primers and probes were designed according to the DNA sequences located at the 5 'insert-to-plant junction (SEQ ID NO: 14) or 3 '(SEQ ID NO: 15) (Figure 4). An Event-specific assay for the pDAB- 8291.45.36.2 soy event was designed to specifically detect a 72 bp DNA fragment (SEQ ID NO: 16) that encompasses the 5 'integration junction using two primers and a probe Target-specific MGB synthesized by Applied Biosystems (ABI) containing the FAM reporter as its 5 'end. Another Event specific trial for the pDAB8291.45.36.2 soy event was planned to target specifically a 142 bp DNA fragment (SEQ ID NO: 17) that encompasses the 3 'integration junction using two specific primers and a target specific MGB probe synthesized by ABI containing the FAM reporter at its 5 'end. The specificity of this Taqman detection method for the soybean event pDAB8291.45.36.2 was tested against 11 soybean events of molecular accumulation other than EPDM and aad-12 and a variety of non-transgenic control soybean (Maverick) in the form of duplex with the soy-specific endogenous reference gene, GMFL01-25-J19 (Glycine max cDNA, GenBank: AK286292.1). Example 6.1: Isolation of gDNA
[00217] GDNA samples from 11 different soy events and non-GM soy varieties were tested in this study. Genomic DNA was extracted using the modified Qiagen MagAttract Planta DNA kit (Qiagen, Valencia, CA). Fresh soybean leaf discs, 8 per sample, were used for gDNA extraction. The gDNA was quantified using the Pico Green method according to the manufacturer's instructions (Molecular Probes, Eugene, OR). The samples were diluted with DNase-free water resulting in a concentration of 10 ng / μL for the purpose of this study. Example 6.2: Taqman Assay and Results
[00218] Taqman specific primers and probes were designed for a specific Taqman assay for the soybean event pDAB8291.45.36.2. These reagents can be used with the conditions listed below to detect the transgene within the soybean event pDAB8291.45.36.2. Table 9 lists the primer and probe sequences that were developed specifically for the detection of the soybean event pDAB8291.45.36.2. Table 9. Initiators and Probes for Taqman PCR.


[00219] The multiplex PCR conditions for amplification are as follows: 1X Roche PCR Buffer, 0.4 μM Initiator for event specific forward, 0.4 μM Inverse Initiator specific for event, 0.4 μM Initiator GMS116 F, 0.4 μM of Initiator GMS116 R, 0.2 μM of Probe specific to the event, 0.2 μM of Probe GMS116, 0.1% of PVP, 20 ng of gDNA in a total reaction of 10 μL . The cocktail was amplified using the following conditions: i) 95 ° C for 10 min., Ii) 95 ° C for 10 s, iii) 60 ° C for 30 s, iv) 72 ° C for 1 sv) repeat the steps ii-iv for 35 cycles, v) 40 ° C at rest. Real-time PCR was performed on the Roche LightCycler 480. Data analysis was based on the measurement of the crossing point (Cp value) determined by the LightCycler 480 software, which is the number of PCR cycles when the rate of change in fluorescence reaches its maximum.
[00220] The Taqman detection method for the soybean event pDAB8291.45.36.2 was tested against 11 different soybean events with 2mEPSPS and aad-12 molecular accumulation and duplex non-transgenic soybean variety with the gene of specific endogenous soybean reference, GMFL01-25-J19 (GenBank: AK286292.1). The assays specifically detected the DAB8291.45.36.2 soy event and did not produce or amplify any false positive results from the controls (ie, the 11 soy events with 2mEPSPS and aad-12 different molecular accumulation and non-soy varieties). transgenic). Event specific primers and probes can be used to detect the soybean event pDAB8291.45.36.2 and these conditions and reagents are applicable for zygosity assays. Example 7: Expected Soy Event Sequence PDAB8291.45.36.2
[00221] SEQ ID NO: 27 provides the expected sequence of the soybean event pDAB8291.45.36.2. This sequence contains the 5 'genomic flanking sequence, the expected T-filament insert from pDAB8291 and 3' genomic flanking sequences. In relation to SEQ ID NO: 27, residues 1-781 are the 3 'genomic flanking sequence, residues 782 - 11,103 are the T filament insert of pDAB8291 and residues 11.104 - 14,191 are the flanking sequence to 5 '. The junction or transition sequence in relation to the 3 'end of the insert occurs as well in residues 781-782 of SEQ ID NO: 27. The junction or transition sequence in relation to the 5 'end of the insert thus occurs in residues 11.103 -11.104 of SEQ ID NO: 27.
[00222] It should be noted that SEQ ID NO: 27 is the expected representation of the soybean event pDAB8291.45.36.2 and was assembled from an alignment of SEQ ID NO: 1, SEQ ID NO: 2 and filament T of pDAB8291. The actual sequence of the T-filament insert of the soybean event pDAB8291.45.36.2 may slightly deviate from SEQ ID NO: 27. During the transformation process of introducing a T-filament insert into the plant cell genome, it is not uncommon for some deletions or other changes to the insert to occur. In addition, errors in PCR amplification can occur that could result in less significant sequencing errors. For example, the flanking sequences listed here were determined by producing amplicons from the genomic DNAs of soybeans and then cloning and sequencing the amplicons. It is not uncommon to find slight differences and less significant discrepancies in the sequences generated and determined in this way, given the many rounds of amplification that are necessary to generate sufficient amplicon for the sequencing of genomic DNAs. A person skilled in the art should recognize and be advised that any adjustments necessary due to these types of common scheduling errors or discrepancies are within the scope of the present invention. Thus, the relevant segment of the plasmid sequence provided here could comprise some minor variations. Thus, a plant comprising a polynucleotide that has some identity range with the target insert sequence is within the scope of the present invention. The sequence identity of SEQ ID NO: 27 can be a polynucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with an exemplified or described sequence here. The sequence of the flanking sequences plus the sequence of the insert can be confirmed with reference to the deposited seed. Thus, some differences between SEQ ID NO: 27 and the actual T-filament insert from the soybean event pDAB8291.45.36.2 can be identified.

权利要求:
Claims (11)
[0001]
1. Polynucleotide, characterized by the fact that it is selected from the group consisting of SEQ ID NO: 13, 14-17, and 27.
[0002]
2. Polynucleotide, according to claim 1, characterized by the fact that it comprises SEQ ID NO: 27.
[0003]
3. Expression cassette used to generate SEQ ID NO: 27 in the genome of a plant, characterized by the fact that it comprises nucleotides 782 to 11,103 of SEQ ID NO: 27 which comprises: a. a first plant transcription unit that expresses a glyphosate herbicide tolerance gene; B. a second plant transcription unit that expresses a phenoxyacetic acid herbicide tolerance gene, a phenoxybutanoic acid herbicide tolerance gene and / or a pyridyloxyalanoic acid tolerance gene; and c. a third plant transcription unit that expresses a gene for tolerance to the herbicide glufosinate.
[0004]
4. Process for the identification of SEQ ID NO: 27 in a sample, characterized by the fact that it comprises the detection of a junction sequence of SEQ ID NO: 27, which is present in the seed deposited under the ATCC Access number PTA -11335, with a probe comprising SEQ ID NO: 20 or 23 and at least one primer in which the primers comprise SEQ ID NO: 18, 19, 21 and 22 that specifically bind to or amplify said junction sequence, said junction sequence comprising residues 3088-3089 of SEQ ID NO: 14 or residues 223-224 of SEQ ID NO: 15.
[0005]
5. Process according to claim 4, characterized by the fact that it further comprises the amplification of a DNA fragment of a nucleic acid present in said sample using a polymerase chain reaction with at least two initiators, in which one first primer comprising SEQ ID NO: 19 or 21 specifically binds to an insert sequence within SEQ ID NO: 13 or the complement thereof and a second primer comprising SEQ ID NO: 18 or 22 specifically binds to a sequence within a flanking sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
[0006]
6. Process for determining the zygosity of the event of a soybean plant comprising the soybean event SEQ ID NO: 27, which is present in the seed deposited under ATCC Accession number PTA-11335, the said event comprising a construction of transgene, said transgene construction being flanked by a 5 'flanking soybean genomic DNA and a 3' flanking soybean DNA, characterized by the fact that it comprises: 7. obtaining a DNA sample from the DNA genomics of the aforementioned soybean plant; 8. production of a sample in contact through the contact of said DNA sample with a. a first event initiator comprising SEQ ID NO: 19 or 21 and a second event initiator comprising SEQ ID NO: 18 or 22, wherein said first event initiator specifically binds to said transgene construct, said second initiator of the event specifically binds to said 5 'soybean genomic flanking DNA or to 3' soybean genomic flanking DNA and in which said first event initiator and said second event initiator produce an event amplicon when subjected to the conditions from TaqMan PCR b. a direct reference primer comprising SEQ ID NO: 24 and a reverse reference primer comprising SEQ ID NO: 25 which produce a reference amplicon starting from an endogenous soy reference gene when subjected to TaqMan PCR c conditions. a fluorescent event probe comprising SEQ ID NO: 20 or 23 that hybridizes to said event d amplicon. a fluorescent reference probe comprising SEQ ID NO: 26 that hybridizes to said reference amplicon; and. submission of said sample in contact with endpoint TaqMan PCR conditions based on fluorescence; f. quantifying said fluorescent event probe that hybridized to said event amplicon; quantifying said fluorescent reference probe that hybridized to said reference amplicon; comparing the quantities of the hybridized fluorescent event probe with the hybridized fluorescent reference probe; and determining the zygosity of SEQ ID NO: 27 by comparing the fluorescence ratios of the hybridized fluorescent event probe and the hybridized fluorescent reference probe.
[0007]
7. Process according to claim 6, characterized by the fact that it is used to cross the introgression of the event in another soybean strain.
[0008]
8. Probe, characterized by the fact that a sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 15 and their complementary ones.
[0009]
9. Production process of a transgenic soybean plant, said plant comprising a genome, said process characterized by the fact that it comprises the insertion of a transgenic insert in a DNA segment of said genome to generate SEQ ID NO: 27, present in seeds deposited under the ATCC accession number PTA-11335, in the said genome, in which the insert comprises nucleotides 782 to 11,103 of SEQ ID NO: 27 and in which the said insertion stage comprises the crossing of the introgression of a transgenic event comprising SEQ ID NO: 27, and wherein said insertion step further comprises the crossing of said plant with a plant containing the event comprising SEQ ID NO: 27.
[0010]
10. Process of producing a protein in a plant cell, characterized by the fact that it comprises inserting a transgenic insert into a segment of DNA in the plant's genome to generate SEQ ID NO: 27, present in seeds deposited under the number ATCC access code PTA-11335, in said genome.
[0011]
Process according to claim 9 or 10, characterized in that the plant further comprises an insect resistance polynucleotide segment that SEQ ID NO: 28 or SEQ ID NO: 29.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US10973229B2|2021-04-13|Stacked herbicide tolerance event 8264.44.06.1, related transgenic soybean lines, and detection thereof

---

BR112013015745B1|2021-01-19|polynucleotides related to the herbicide tolerance event 8291.45.36.2, expression cassette, probe, as well as processes for event identification, zygosity determination, production of a transgenic soy plant and production of a protein in a plant cell

---

US9732353B2|2017-08-15|Stacked herbicide tolerance event 8264.42.32.1, related transgenic soybean lines, and detection thereof

---

ES2714432T3|2019-05-28|Soybean event 9582.814.19.1 insect resistant and herbicide tolerant

---

US8785728B2|2014-07-22|AAD-12 event 1606 and related transgenic soybean lines

---

BR102013001773A2|2015-09-08|herbicide tolerant cotton event pdab4468.19.10.3

---

AU2013280641B2|2019-04-04|Insect resistant and herbicide tolerant soybean event pDAB9582.816.15.1

---

WO2013112525A2|2013-08-01|HERBICIDE TOLERANT COTTON EVENT pDAB4468.18.07.1

---

WO2012033808A2|2012-03-15|Methods for determining the presence or zygosity of aad-12 soybean event 1606

同族专利:

---

公开号 | 公开日

---

US20130338006A1|2013-12-19|

---

BR112013015745A2|2017-09-26|

---

US9540656B2|2017-01-10|

---

WO2012075429A1|2012-06-07|

---

AR084162A1|2013-04-24|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US20030237110A9|1998-05-12|2003-12-25|Incyte Pharmaceuticals, Inc.|Polynucleotides and polypeptides derived from corn seedling|

---

US20040031072A1|1999-05-06|2004-02-12|La Rosa Thomas J.|Soy nucleic acid molecules and other molecules associated with transcription plants and uses thereof for plant improvement|

---

US20070083945A1|2000-03-10|2007-04-12|Byrum Joseph R|Nucleic acid molecules and other molecules associated with plants|

---

US7834146B2|2000-05-08|2010-11-16|Monsanto Technology Llc|Recombinant polypeptides associated with plants|

---

US20030031757A1|2001-08-03|2003-02-13|Kraft Food Holdings, Inc.|Stable and bioavailable iron fortified beverages|

---

JP2004027091A|2002-06-27|2004-01-29|Nisshin Oillio Ltd|Compressed soybean oil and method for producing the same|

---

UA87808C2|2002-07-29|2009-08-25|Монсанто Текнолоджи, Ллс|Cereal crops pv-zmir13 and compositions and methods for detection thereof|

---

US7045684B1|2002-08-19|2006-05-16|Mertec, Llc|Glyphosate-resistant plants|

---

CA2516221C|2003-02-18|2014-05-13|Monsanto Technology Llc|Glyphosate resistant class i 5-enolpyruvylshikimate-3-phosphate synthase |

---

PL1668141T3|2003-09-29|2013-04-30|Monsanto Technology Llc|Methods for enhancing drought tolerance in plants and methods thereof|

---

FR2865738B1|2004-02-03|2008-06-20|Agronomique Inst Nat Rech|USE OF GENES ENCODING POTASSIVE CHANNELS TO MODIFY A PHENOTYPE RELATING TO A SIZE OF AT LEAST ONE PLANT STORAGE ORGAN|

---

EP2862934B1|2004-03-26|2019-05-29|Dow AgroSciences LLC|Cry1F and Cry1AC transgenic cotton lines and event-specific identification thereof|

---

BRPI0509460B8|2004-04-30|2017-06-20|Dow Agrosciences Llc|herbicide resistance genes|

---

US7750207B2|2004-09-01|2010-07-06|Monsanto Technology Llc|Zea mays ribulose bisphosphate carboxylase activase promoter|

---

CA2588243C|2004-09-29|2013-06-11|Pioneer Hi-Bred International, Inc.|Corn event das-59122-7 and methods for detection thereof|

---

EP1807519B1|2004-10-29|2012-02-01|Bayer BioScience N.V.|Stress tolerant cotton plants|

---

AP2693A|2005-05-27|2013-07-16|Monsanto Technology Llc|Soybean event MON89788 and methods for detection thereof|

---

NZ608188A|2005-10-28|2014-10-31|Dow Agrosciences Llc|Novel herbicide resistance genes|

---

US7951995B2|2006-06-28|2011-05-31|Pioneer Hi-Bred International, Inc.|Soybean event 3560.4.3.5 and compositions and methods for the identification and detection thereof|

---

CN100558902C|2007-01-24|2009-11-11|中国农业科学院油料作物研究所|Transgene rape exogenous origin gene integrator incident Rf1 exogenous insertion vector flanking sequence and application thereof|

---

CA2686835C|2007-05-09|2020-04-21|Dow Agrosciences Llc|Novel herbicide resistance genes|

---

US8809059B2|2007-09-21|2014-08-19|Basf Plant Science Gmbh|Plants with increased yield|

---

EP2195438B1|2007-10-05|2013-01-23|Dow AgroSciences LLC|Methods for transferring molecular substances into plant cells|

---

WO2009110924A1|2008-03-03|2009-09-11|Ms Technologies Llc|Antibodies immunoreactive with mutant 5-enolpyruvlshikimate-3-phosphate synthase|

---

WO2009152359A2|2008-06-11|2009-12-17|Dow Agrosciences Llc|Constructs for expressing herbicide tolerance genes, related plants, and related trait combinations|

---

ES2563485T3|2008-07-01|2016-03-15|Monsanto Technology Llc|Recombinant DNA constructs and procedures to modulate the expression of a target gene|

---

US8344209B2|2008-07-14|2013-01-01|Syngenta Participations Ag|Plant regulatory sequences|

---

WO2010015627A2|2008-08-05|2010-02-11|Dsm Ip Assets B.V.|Adipoyl-7-adca producing strains|

---

US20120156718A1|2008-12-17|2012-06-21|Basf Plant Science Gmbh|Production of Ketocarotenoids in Plants|

---

WO2010085705A2|2009-01-22|2010-07-29|Syngenta Participations Ag|Mutant hydroxyphenylpyruvate dioxygenase polypeptides and methods of use|

---

US9062324B2|2009-11-23|2015-06-23|M.S. Technologies Llc|Herbicide tolerant soybean plants and methods for identifying same|

---

WO2011066382A1|2009-11-24|2011-06-03|Dow Agrosciences Llc|Control of aad dicot volunteers in monocot crops|

---

WO2011066360A1|2009-11-24|2011-06-03|Dow Agrosciences Llc|Detection of aad-12 soybean event 416|

---

BR112012012511A2|2009-11-24|2015-09-15|Dow Agrosciences Llc|event 416 of the aad-12 gene related to transgenic soybean strains and their event-specific identification|

---

RU2608650C2|2010-12-03|2017-01-23|ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи|Stacked herbicide tolerance event 8264.44.06.1 related transgenic soybean lines and detection thereof|

---

UY34199A|2011-07-13|2013-02-28|Dow Agrosciences Llc|? 8264.42.32.1 EVENT OF TOLERANCE STACKED ON HERBICIDES, TRANSGENIC SOY LINES RELATED TO AND THEIR DETECTION ?.|EP2646559A2|2010-12-03|2013-10-09|MS Technologies, LLC|Optimized expression of glyphosate resistance encoding nucleic acid molecules in plant cells|

---

BR102012019436B1|2011-07-26|2021-02-02|Dow Agrosciences Llc|soybean event detection method pdab9582.814.19.1|

---

CN103826445A|2011-07-26|2014-05-28|陶氏益农公司|Insect resistant and herbicide tolerant soybean event 9582.814.19.1|

---

WO2013075817A1|2011-11-21|2013-05-30|Bayer Intellectual Property Gmbh|Fungicide n-[methyl]-carboxamide derivatives|

---

CN105906567B|2011-11-30|2019-01-22|拜耳知识产权有限责任公司|Antifungal N- bicyclic alkyl and N- tricyclic alkylcarboxamide derivative|

---

TWI557120B|2011-12-29|2016-11-11|拜耳知識產權公司|Fungicidal 3-[methyl]-2-substituted-1,2,4-oxadiazol-5-one derivatives|

---

TWI558701B|2011-12-29|2016-11-21|拜耳知識產權公司|Fungicidal 3-[methyl]-2-sub stituted-1,2,4-oxadiazol-5-one derivatives|

---

WO2013110594A1|2012-01-25|2013-08-01|Bayer Intellectual Property Gmbh|Active compound combinations containing fluopyram and biological control agent|

---

UA122197C2|2012-06-25|2020-10-12|ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі|Insect resistant and herbicide tolerant soybean event pdab9582.816.15.1|

---

MX369284B|2012-09-14|2019-11-04|Bayer Cropscience Lp|Hppd variants and methods of use.|

---

UA114647C2|2012-10-19|2017-07-10|Байєр Кропсайнс Аг|Method for enhancing tolerance to abiotic stress in plants using carboxamide or thiocarboxamide derivatives|

---

UA114822C2|2012-10-19|2017-08-10|Байєр Кропсайнс Аг|Active compound combinations comprising carboxamide derivatives|

---

EP2908640B1|2012-10-19|2019-10-02|Bayer Cropscience AG|Method of plant growth promotion using carboxamide derivatives|

---

EP2908641B1|2012-10-19|2018-01-10|Bayer Cropscience AG|Method for treating plants against fungi resistant to fungicides using carboxamide or thiocarboxamide derivatives|

---

WO2014090765A1|2012-12-12|2014-06-19|Bayer Cropscience Ag|Use of 1-[2-fluoro-4-methyl-5-phenyl]-5-amino-3-trifluoromethyl)-1 h-1,2,4 tfia zole for controlling nematodes in nematode-resistant crops|

---

DK2964767T3|2013-03-07|2020-03-23|BASF Agricultural Solutions Seed US LLC|TOXICATIONS AND PROCEDURES FOR USE THEREOF|

---

CA2909725A1|2013-04-19|2014-10-23|Bayer Cropscience Aktiengesellschaft|Method for improved utilization of the production potential of transgenic plants|

---

WO2014177514A1|2013-04-30|2014-11-06|Bayer Cropscience Ag|Nematicidal n-substituted phenethylcarboxamides|

---

TW201507722A|2013-04-30|2015-03-01|Bayer Cropscience Ag|N-carboxamides as nematicides and endoparasiticides|

---

CA2942171A1|2014-03-11|2015-09-17|Bayer Cropscience Lp|Hppd variants and methods of use|

---

CN104611308B|2015-02-13|2019-01-11|北京大北农科技集团股份有限公司|Herbicide resistance protein, its encoding gene and purposes|

---

EP3097782A1|2015-05-29|2016-11-30|Bayer CropScience Aktiengesellschaft|Methods for controlling phytopathogenic nematodes by combination of fluopyram and biological control agents|

---

CN108884470A|2015-09-11|2018-11-23|拜耳作物科学股份公司|HPPD variant and application method|

---

CN105567682B|2016-01-12|2019-01-29|吉林省农业科学院|Transgenic soybean event B4J8049 external source Insert Fragment flanking sequence and its application|

---

MX2019005835A|2016-11-23|2019-10-30|BASF Agricultural Solutions Seed US LLC|Axmi669 and axmi991 toxin genes and methods for their use.|

---

EP3559241A1|2016-12-22|2019-10-30|Basf Agricultural Solutions Seed Us Llc|Use of cry14 for the control of nematode pests|

---

CA3046232A1|2016-12-22|2018-06-28|BASF Agricultural Solutions Seed US LLC|Elite event ee-gm4 and methods and kits for identifying such event in biological samples|

---

JP2020513753A|2016-12-22|2020-05-21|ビーエーエスエフ アグリカルチュラル ソリューションズ シード ユーエス エルエルシー|Elite event EE-GM5 and methods and kits for identifying such events in biological samples|

---

AR110756A1|2017-01-18|2019-05-02|Bayer Cropscience Lp|USE OF BP005 FOR PLANT PATHOGEN CONTROL|

---

EP3571303A1|2017-01-18|2019-11-27|Basf Agricultural Solutions Seed Us Llc|Bp005 toxin gene and methods for its use|

---

US20200040313A1|2017-03-07|2020-02-06|BASF Agricultural Solutions Seed LLC|Hppd variants and methods of use|

---

BR112019021938A2|2017-04-21|2020-05-05|Bayer Cropscience Lp|crop safety improvement method|

---

WO2019083810A1|2017-10-24|2019-05-02|Basf Se|Improvement of herbicide tolerance to 4-hydroxyphenylpyruvate dioxygenaseinhibitors by down-regulation of hppd expression in soybean|

---

US20210032651A1|2017-10-24|2021-02-04|Basf Se|Improvement of herbicide tolerance to hppd inhibitors by down-regulation of putative 4-hydroxyphenylpyruvate reductases in soybean|

---

WO2019233863A1|2018-06-04|2019-12-12|Bayer Aktiengesellschaft|Herbicidally active bicyclic benzoylpyrazoles|

---

WO2021013721A1|2019-07-22|2021-01-28|Bayer Aktiengesellschaft|5-amino substituted pyrazoles and triazoles as pest control agents|

---

TW202118754A|2019-07-23|2021-05-16|德商拜耳廠股份有限公司|Novel heteroaryl-triazole compounds as pesticides|

---

WO2021013719A1|2019-07-23|2021-01-28|Bayer Aktiengesellschaft|Novel heteroaryl-triazole compounds as pesticides|

---

WO2021022069A1|2019-08-01|2021-02-04|Bayer Cropscience Lp|Method of improving cold stress tolerance and crop safety|

---

EP3701796A1|2019-08-08|2020-09-02|Bayer AG|Active compound combinations|

---

WO2021058659A1|2019-09-26|2021-04-01|Bayer Aktiengesellschaft|Rnai-mediated pest control|

---

WO2021064075A1|2019-10-02|2021-04-08|Bayer Aktiengesellschaft|Active compound combinations comprising fatty acids|

---

WO2021069569A1|2019-10-09|2021-04-15|Bayer Aktiengesellschaft|Novel heteroaryl-triazole compounds as pesticides|

---

TW202128664A|2019-10-09|2021-08-01|德商拜耳廠股份有限公司|Novel heteroaryl-triazole compounds as pesticides|

---

WO2021089673A1|2019-11-07|2021-05-14|Bayer Aktiengesellschaft|Substituted sulfonyl amides for controlling animal pests|

---

WO2021097162A1|2019-11-13|2021-05-20|Bayer Cropscience Lp|Beneficial combinations with paenibacillus|

---

WO2021099271A1|2019-11-18|2021-05-27|Bayer Aktiengesellschaft|Active compound combinations comprising fatty acids|

---

TW202134226A|2019-11-18|2021-09-16|德商拜耳廠股份有限公司|Novel heteroaryl-triazole compounds as pesticides|

---

TW202136248A|2019-11-25|2021-10-01|德商拜耳廠股份有限公司|Novel heteroaryl-triazole compounds as pesticides|

---

US20210238579A1|2020-01-31|2021-08-05|Pairwise Plants Services, Inc.|Suppression of shade avoidance response in plants|

---

WO2021165195A1|2020-02-18|2021-08-26|Bayer Aktiengesellschaft|Heteroaryl-triazole compounds as pesticides|

---

EP3708565A1|2020-03-04|2020-09-16|Bayer AG|Pyrimidinyloxyphenylamidines and the use thereof as fungicides|

---

WO2021209490A1|2020-04-16|2021-10-21|Bayer Aktiengesellschaft|Cyclaminephenylaminoquinolines as fungicides|

---

WO2021211926A1|2020-04-16|2021-10-21|Pairwise Plants Services, Inc.|Methods for controlling meristem size for crop improvement|

---

WO2021213978A1|2020-04-21|2021-10-28|Bayer Aktiengesellschaft|2-aryl-substituted condensed heterocyclic derivatives as pest control agents|

---

WO2021224323A1|2020-05-06|2021-11-11|Bayer Aktiengesellschaft|Novel heteroaryl-triazole compounds as pesticides|

---

WO2021224220A1|2020-05-06|2021-11-11|Bayer Aktiengesellschaft|Pyridine amides as fungicidal compounds|

---

WO2021228734A1|2020-05-12|2021-11-18|Bayer Aktiengesellschaft|Triazine and pyrimidine amides as fungicidal compounds|

---

WO2021233861A1|2020-05-19|2021-11-25|Bayer Aktiengesellschaft|Azabicyclicamides as fungicidal compounds|

---

WO2021247477A1|2020-06-02|2021-12-09|Pairwise Plants Services, Inc.|Methods for controlling meristem size for crop improvement|

---

WO2021245087A1|2020-06-04|2021-12-09|Bayer Aktiengesellschaft|Heterocyclyl pyrimidines and triazines as novel fungicides|

---

WO2021249995A1|2020-06-10|2021-12-16|Bayer Aktiengesellschaft|Azabicyclyl-substituted heterocycles as fungicides|

---

US20210395767A1|2020-06-17|2021-12-23|Pairwise Plants Services, Inc.|Methods for controlling meristem size for crop improvement|

---

WO2021255118A1|2020-06-18|2021-12-23|Bayer Aktiengesellschaft|Composition for use in agriculture|

---

WO2021255071A1|2020-06-18|2021-12-23|Bayer Aktiengesellschaft|3--5,6-dihydro-4h-1,2,4-oxadiazine derivatives as fungicides for crop protection|

---

WO2021255091A1|2020-06-19|2021-12-23|Bayer Aktiengesellschaft|1,3,4-oxadiazoles and their derivatives as fungicides|

---

WO2021255169A1|2020-06-19|2021-12-23|Bayer Aktiengesellschaft|1,3,4-oxadiazole pyrimidines as fungicides|

---

WO2021255089A1|2020-06-19|2021-12-23|Bayer Aktiengesellschaft|1,3,4-oxadiazole pyrimidines and 1,3,4-oxadiazole pyridines as fungicides|

---

WO2021255170A1|2020-06-19|2021-12-23|Bayer Aktiengesellschaft|1,3,4-oxadiazole pyrimidines as fungicides|

---

EP3929189A4|2020-06-25|2021-12-29|Bayer Animal Health Gmbh|Novel heteroaryl-substituted pyrazine derivatives as pesticides|

---

WO2022002818A1|2020-07-02|2022-01-06|Bayer Aktiengesellschaft|Heterocyclene derivatives as pest control agents|

---

WO2022033991A1|2020-08-13|2022-02-17|Bayer Aktiengesellschaft|5-amino substituted triazoles as pest control agents|

---

EP3915971A4|2020-12-16|2021-12-01|Bayer Ag|Phenyl-sn-phenylamidines and the use thereof as fungicides|

法律状态:
2017-10-03| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|

---

2017-10-24| B08G| Application fees: restoration [chapter 8.7 patent gazette]|

---

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

---

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

---

2020-02-27| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

---

2020-07-28| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

---

2020-11-03| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2021-01-19| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 02/12/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

US41970910P| true| 2010-12-03|2010-12-03|

---

US61/419,709|2010-12-03|

---

US201161471845P| true| 2011-04-05|2011-04-05|

---

US61/471,845|2011-04-05|

---

US201161511664P| true| 2011-07-26|2011-07-26|

---

US61/511,664|2011-07-26|

---

US201161521798P| true| 2011-08-10|2011-08-10|

---

US61/521,798|2011-08-10|

---

PCT/US2011/063133|WO2012075429A1|2010-12-03|2011-12-02|Stacked herbicide tolerance event 8291.45.36.2, related transgenic soybean lines, and detection thereof|

---