methods of increasing seed production, biomass, growth rate, vigor, tolerance to nitrogen deficiency

专利摘要:
PRODUCTION INCREASE METHOD, BIOMASS, GROWTH RATE, STRENGTH, OIL CONTENT, FIBER PRODUCTION, FIBER QUALITY, TOLERANCE TO ABIOTIC STRESS AND / OR EFFICIENCY IN THE USE OF NITROGEN OF A PLANT, NON-POLYUCLEOTIC, POLYNUCLEUTOID, ISOLATED POLYPEPTIDE, PLANT AND PLANT CELL, in which isolated polynucleotides encoding a polypeptide at least 80% homologous to the amino acid sequence selected from the group consisting of SEQ ID NO: 799, 488-798, 800-813, 4852-5453 , 5460, 5461, 5484, 5486-5550, 5553, and 5558-8091; and isolated polynucleotides comprising nucleic acid sequences at least 80% identical to SEQ ID NO: 460, 1-459, 461-487, 814-1598, 1600-1603, 1605-1626, 1632-1642, 16454850 or 4851; nucleic acid constructions are also provided comprising these, isolated encoded polynucleotides, transgenic cells and transgenic plants comprising these and methods of use for increasing yield, biomass, growth rate, vigor, oil content, fiber production, fiber quality , tolerance to abiotic stress and / or efficiency in the use of nitrogen from the plant; isolated polynucleotides are also provided comprising a nucleic acid sequence determined in SEQ ID NO: 8096, characterized by the fact that the isolated polynucleotide is able to regulate the expression of at least one connected polynucleotide sequence in an operable fashion.
公开号:BR112012016033B1
申请号:R112012016033-8
申请日:2010-12-22
公开日:2021-03-09
发明作者:Zur Granevitze；Hagai Karchi
申请人:Evogene Ltd.；
IPC主号:

专利说明:

FIELD AND HISTORY OF THE INVENTION
[001] The present patent application, in some of its applications, refers to isolated polynucleotides and polypeptides, nucleic acid constructs including these, transgenic cells including these, transgenic plants exogenously expressing these and, more particularly, but not exclusively, methods of using these to increase yield (eg seed yield, oil yield), biomass, growth rate, vigor, oil content, fiber yield, tolerance to abiotic stress the fiber quality and / or the efficiency in the use of fertilizer (eg efficiency in the use of nitrogen) of a plant.
[002] Abiotic stress conditions (ABS; also referred to as "environmental stress") such as salinity, drought, flood, sub-ideal temperature and toxic-chemical pollution, cause substantial damage to agricultural plants. Most plants have developed strategies to protect themselves against these conditions. However, if the severity and duration of these stress conditions are very large, the effects on the development, growth and production of the crop plants are profound. In addition, most cultivated plants are highly susceptible to abiotic stress and thus require ideal growing conditions for commercial production. Continuous exposure to stress causes major changes in the plant's metabolism, which ultimately leads to cell death and, consequently, production losses.
[003] The global scarcity of water supply is one of the most serious problems in agriculture affecting plant growth and crop yield, so efforts are made to minimize the damaging effects of desertification and salinisation of the world's arable land. Water scarcity is a common component of many plant stresses and occurs in plant cells when the plant's total transpiration rate exceeds water absorption. In addition to drought, other stresses, such as salinity and low temperature, produce cellular dehydration.
[004] Drought is a gradual phenomenon, involving periods of abnormal dry weather that persist long enough to produce serious hydrological imbalances such as crop damage and shortages in water supply. In severe cases, the drought can last for many years and result in devastating effects on agriculture and water supply. In addition, drought is associated with increased susceptibility to various diseases.
[005] For most cultivated plants, the land regions of the world are very arid. In addition, the excessive use of available water results in greater loss of land usable for agriculture (desertification) and the increase in the accumulation of salt in soils contributes to the loss of available water in soils.
[006] High salinity, high salt levels, affect one in five hectares of irrigated land. It is expected that this condition will only worsen, thereby reducing the availability of arable land and crop production, as none of the five main food crops, that is, wheat, corn, rice, potatoes and soybeans, can tolerate the excess salt. Harmful effects of salt on plants result both from water scarcity, which leads to osmotic stress (similar to drought stress), as well as from the effect of excess sodium ions in critical biochemical processes. As well as freezing and drought, excess salt causes water shortages; and the presence of excess salt makes it difficult to extract water from its environment to the roots of the plants. Soil salinity is therefore one of the most important variables that determine whether a plant can flourish. In many parts of the world, considerable areas of land are not arable due to the naturally high salinity of the soil. Thus, the salinisation of soils that are used for agricultural production is a significant and growing problem in regions that depend heavily on agriculture, and is made worse by overuse, overuse of fertilization and water scarcity, typically caused by climate change. and the demands of the growing population. Tolerance to salt is of particular importance at the beginning of the plant's life cycle, since evaporation from the soil surface causes an upward movement of the water, and salt is accumulated in the upper layer of the soil where the seeds are placed. On the other hand, germination normally takes place under a salt concentration greater than the measured level of salt in the entire soil profile.
[007] The germination of many crops is sensitive to temperature. A gene that would increase germination in warm conditions would be useful for crops that are planted late in the season or in hot climates. In addition, seedlings and mature plants that are exposed to excess heat can suffer from thermal shock, which. it can appear in several organs, including leaves and, in particular, fruits, when breathing is insufficient to overcome heat stress. Heat also damages cellular structures, including organelles and cytoskeletons, and impairs membrane functions. Heat shock can produce a decrease in the general synthesis of proteins, followed by the expression of heat shock proteins, for example chaperones, which are involved in the renaturation of proteins denatured by heat.
[008] Heat stress usually accompanies conditions of low water availability. The heat itself is seen as an interactive stress and adds to the harmful effects caused by water deficiency conditions. Evaporation of water increases with increasing temperature during the day and can result in high rates of transpiration and low water potentials of the plant. Damage caused to pollen by high temperature almost always occurs in conjunction with drought stress, and rarely occurs under conditions with plenty of water. Combined stress can alter the plant's metabolism in several ways; therefore, understanding the interaction between different stresses can be important for the development of strategies to improve tolerance to stress through genetic manipulation.
[009] Excessive cold conditions, eg low temperatures, but above zero, affect crops of tropical origin, such as soybeans, rice, corn and cotton. Cold damage usually includes wilting, necrosis, chlorosis or ion leakage in cell membranes. The underlying mechanisms of cold sensitivity are not yet fully understood, but they probably involve the level of membrane saturation and other physiological deficiencies. For example, photoinhibition of photosynthesis (interruption of photosynthesis due to high light intensities) usually occurs under clear atmospheric conditions following cold late summer / autumn nights. In addition, cold can lead to loss of production and poor product quality through delayed ripening of corn.
[0010] The signal transduction of salt stress and drought consists of signaling pathways for ionic and osmotic homeostasis. The ionic aspect of salt stress is signaled through the SOS pathway where a calcium-responsive SOS3-SOS2 protein kinase complex controls the expression and activity of ion transporters, such as SOS1. The osmotic component of salt stress involves complex plant reactions that overlap with cold and / or drought stress responses.
[0011] Common aspects of the response to drought, cold and salt stress [Revised in Xiong and Zhu (2002) Plant Cell Environ. 25: 131-139] including: (a) temporary changes in cytoplasmic calcium levels at the start of the signaling event; (b) signal transduction by means of mitogen-activated and / or calcium-dependent protein kinases (CDPKs) and protein phosphatases; (c) increases in abscisic acid levels in response to triggering a subset of responses by stress; (d) inositol phosphates as signal molecules (at least a subset of transcriptional changes responsive to stress); (e) activation of phospholipases, which in turn generate a diverse array of second messenger molecules, some of which can regulate the activity of stress-responsive kinases; (f) induction of abundant genes in late embryogenesis (LEA) including COR / RD genes responsive to CRT / DRE; (g) increased levels of compatible antioxidants and osmolytes, such as proline and soluble sugars; and (h) accumulation of reactive oxygen species, such as superoxide, hydrogen peroxide and hydroxyl radicals. The biosynthesis of abscisic acid is regulated by osmotic stress in multiple stages. The ABA-dependent and independent osmotic stress signals first constitutively modify the expressed transcription factors, leading to the expression of transcriptional activators of early response, which then activate the descendant genes of the stress tolerance effect.
[0012] Several genes that increase tolerance to cold or salt stress can also improve protection against drought stress, these include, for example, the AtCBF / DREBI transcription factor, OsCDPK7 (Saijo et al. 2000, Plant J 23: 319-327) or AVP1 (a vacuolar pyrophosphatase proton pump, Gaxiola et al. 2001, Proc. Natl. Acad. Sci. USA 98: 11444-11449).
[0013] The development of stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of these problems. However, traditional plant breeding strategies used to develop new lines of plants that exhibit ABS tolerance are relatively inefficient as they are tiring, time consuming and have unpredictable results. In addition, limited germinal plasma resources for stress tolerance and incompatibility in crossbreeding between distantly related plant species represent significant problems encountered in conventional breeding. In addition, the cellular processes leading to ABS tolerance are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways.
[0014] Genetic engineering efforts to provide tolerance to abiotic stress in transgenic crops have been described in several publications [Apse and Blumwald (Curr Opin Biotechnol. 13: 14.6-150 2002), Quesada et al. (Plant Physiol. 130: 951-963, 2002), Holmstrom et al. (Nature 379: 683-684, 1996), Xu et a1. (Plant Physiol 110: 249-257, 1996), Piion-Smits and Ebskamp (Plant Physiol 107; 125-130, 1995) and Tarczynski et al. (Science 259: 508510, 1993)].
[0015] Several patents and patent applications disclose genes and proteins that can be used to increase the tolerance of plants to abiotic stresses. These include, for example, US Patent numbers 5,296,462 and 5,356,816 (for increasing tolerance to cold stress); American patent n. 6,670,528 (to increase ABST); American Patent No. 6,720,477 (to increase ABST); Request, American Ser. No. 10/231035 (for ABST increase); W02004 / 104162 (to increase ABST and biomass); W02007 / 020638 (to increase ABST, biomass, vigor and / or yield); W02007 / 049275 (to increase ABST, biomass, vigor and / or yield); W02010 / 076756 (to increase ABST, biomass and / or yield); W02009 / 083958 (to increase efficiency in the use of water, efficiency in the use of fertilizer, tolerance to biotic / abiotic stress, yield and / or biomass); W02010 / 020941 (to increase efficiency in the use of nitrogen, tolerance to abiotic stress, yield and / or biomass); W02009 / 141824 (to increase the utility of the plant); W02010 / 049897 (to increase plant yield).
[0016] The optimized nutrients (macro and micronutrients) affect the growth and development of the plant throughout its life cycle. One of the essential macronutrients for the plant is Nitrogen. Nitrogen is responsible for the biosynthesis of amino acids and nucleic acids, prosthetic groups, plant hormones, plant chemical defenses, and the like. Nitrogen is generally an element in limiting the growth rate of the plant and all crops in the field have a fundamental dependence on inorganic nitrogenous fertilizers. Since fertilizer is quickly removed from most soil types, it must be supplied to growing crops two or three times during the growing season. Other important macronutrients are phosphorus (P) and potassium (K), which have a direct correlation with the yield and the general tolerance of the plant.
[0017] Vegetable or seed oils are the main source of energy and nutrition in the human and animal diet. They are also used in industrial production, for example, paints and lubricants. In addition, plant-based oils represent renewable sources of long-chain hydrocarbons that can be used as fuel. Since the fossil fuels currently used are finite resources and are being gradually depleted, fast-growing biomass crops can be used as alternative fuels or for energy stocks for industrial processes and can reduce dependence on fossil energy supplies. However, the main problem for increasing consumption of plant oils as biofuels is the price of oil, which is still higher than fossil fuel. In addition, the rate of production of plant-based oils is limited by the availability of agricultural soil and water.
[0018] Thus, the increase in yields of plant-based oils from the same cultivation area can efficiently overcome the scarcity of space for production and can, at the same time, increase the prices of vegetable oil.
[0019] Studies aimed at increasing the yields of plant-based oils emphasize the identification of genes involved in oil metabolism, as well as genes capable of increasing plant and seed yields in transgenic plants. Genes known to be involved in increasing the yields of plant-based oils include those that participate in the synthesis or capture of fatty acid, for example, desaturase [ie DELTA6, DELTA12 or acyl-ACP (Ssi2; Source of Arabidopsis Information (TAIR; Hypertext Transfer Protocol: // World Wide Web (dot) arabidopsis (dot) org /), TAIR No. AT2G43710) 1, OleosinA (TAIR No. AT3G01570) or FAD3 (TAIR No. AT2G29980), and several transcription factors and activators, such as Lecl [TAIR No. AT1G21970, Lotan et al. 1998. Cell. 26; 93 (7): 1195-205], Lec2 [TAIR No. AT1G28300, Santos Mendoza et al. 2005, FEBS Lett. 579 (21): 4666-70], Fus3 (TAIR No. AT3G26790), ABB [TAIR No. AT3G24650, Lara et al. 2003. J Biol Chem. 278 (23): 21003-11] and Wril [TAIR No. AT3G54320, Cernac and Benning, 2004. Plant J. 40 (4): 575-85], Genetic engineering efforts to increase the oil content in plants (for example, example, in seeds) include upward regulation of endoplasmic reticulum (FAD3) and plastid unsaturated fatty acids (FAD7) in potatoes (Zabrouskov V., et al., 2002; Physiol Plant. 116: 172-185); overexpression of transcription factors GmDof4 and GmDofll (Wang HW et al., 2007; Plant J. 52: 716-29); overexpression of a glycerol-3-phosphate dehydrogenase yeast under the control of a specific seed promoter (Vigeolas H, et al. 2007, Plant Biotechnol J. 5: 431-41; American Patent Application No. 20060168684); using the genes of Arabidopsis FAE1 and yeast SLC1-1 for improvements in erectic acid and oil content in rapeseed (Katavic V, et al., 2000, Biochem Soc Trans. 28: 935-7).
[0020] Several patents and patent applications disclose genes and proteins that can increase the oil content in plants. These include, for example, American Patent Application No. 20080076179 (lipid metabolism protein); American Patent Application No. 20060206961 (polypeptide Ypr140w); American Patent Application No. 20060174373 [rotein for improving the synthesis of triacylglycerols (TEP)]; American Patent Application numbers 20070169219, 20070006345, 20070006346 and 2.0060195943 (disclosure of transgenic plants as better efficiency in the use of nitrogen, which can be used for conversion into fuel or chemical raw materials); W02008 / 122980 (polynucleotides for increasing the oil content, growth rate, biomass, yield and / or vigor of a plant.
[0021] Cotton and cotton by-products provide raw materials that are used to produce a rich variety of consumer-based products, in addition to textiles including cotton food products, livestock feed, fertilizers and paper. The production, marketing, consumption and sale of cotton-based products generates an excess of $ 100 billion annually. in the US alone, making cotton the number one crop in added value.
[0022] Although 90% of the value of cotton as a live crop in the fiber (row), the yield and quality of the fiber decreased due to general erosion in the genetic diversity of cotton varieties, and an increased vulnerability of the harvest to environmental conditions.
[0023] There are several varieties of cotton plant, from which cotton fibers with a variety of characteristics can be obtained and used for different applications. Cotton fibers can be characterized according to a variety of properties, some of which are considered highly desirable within the textile industry for the production of growing high quality products and optimized exploitation of moderator spinning technologies. Commercially desired properties include length, length uniformity, fineness, maturity ratio, decreased cotton fiber production, micronaire, bundle strength and single fiber strength. Many efforts have been made to improve the characteristics of cotton fibers, focusing mainly on the length of the fiber and the fineness of the fiber. In particular, there is a high demand for cotton fibers of specific lengths.
[0024] A cotton fiber is composed of a single cell that is differentiated from an epidermal cell in the seed coating, with development in four stages, namely, initiation, elongation, secondary cell wall thickness and maturation. More specifically, the elongation of a cotton fiber begins in the epidermal cell of the egg immediately after flowering, after which the cotton fiber quickly stretches for approximately 21 days. The elongation of the fiber is then completed and a secondary cell wall is formed and grows through maturation to become a mature cotton fiber.
[0025] Several candidate genes that are associated with the elongation, formation, quality and yield of cotton fibers have been disclosed in several patent applications, such as American Patent No. 5,880,100 and American Patent applications No. of Ser 08 / 580,545, 08 / 867,484 and 09 / 262,653 (describing genes involved in the cotton fiber elongation stage); W00245485 (improving fiber quality by modulating sucrose synthesis); American Patent No. 6,472,588 and W00117333 (increasing the quality of the fiber by transformation with a DNA encoding sucrose phosphate synthesis); W09508914 (using a fiber specific promoter and a coding sequence encoding cotton peroxidase); W09626639 (using an ovarian-specific promoter sequence to express plant growth by modifying hormones in cotton egg tissue, to change fiber quality characteristics, such as fiber size and strength); American Patent No. 5,981,834, American Patent No. 5,597,718, American Patent No. 5,620,882, American Patent No. 5,521,708 and American Patent No. 5,495,070 (coding sequences for changing fiber characteristics of plants producing transgenic fibers); U.S. Patent No. 2002049999 and U.S. 2003074697 (expressing a gene encoding for endoxyloglucan transferase, catalase or peroxidase to improve the characteristics of the cotton fiber); WO 01/40250 (improvement in the quality of cotton fiber by modulating the expression of the transcription factor gene); WO 96/40924 (a transcriptional regulatory region of the associated cotton fiber that is expressed in cotton fiber); EP0834566 (a gene that controls the mechanism of fiber formation in the cotton plant); W02005 / 121364 (improvement in the quality of the cotton fiber by the cotton plant of the modulated gene); W02008 / 075364 (improvement in fiber quality, yield / biomass / vigor and / or tolerance to plant abiotic stress).
[0026] A promoter is a nucleic acid sequence approximately 200-1500 base pairs in length and is usually located upstream of the coding sequences. A promoter works in the direction of transcribing an adjacent coding sequence and therefore acts as a switch for gene expression in an organism. Therefore, all cellular processes are basically governed by the activity of the promoters, making such regulatory elements important commercial and research tools.
[0027] Promoters are typically used for heterologous gene expression in commercial expression systems, gene therapy and a variety of research applications.
[0028] The choice of the promoter sequence determines when, where and with what strength the heterologous gehe of choice is expressed. Thus, when a constitutive expression throughout an organism is desired, a constitutive promoter is preferably used. On the other hand, when triggered gene expression is desired, an inductive promoter is preferred. Similarly, when an expression has to be confined to a particular tissue, or to a particular physiological or developmental stage, a tissue-specific promoter or a stage-specific promoter is respectively preferred.
[0029] Constitutive promoters are active through the cell cycle and have been used to express heterologous genes in transgenic plants in order to allow the expression of characteristics encoded by heterologous genes throughout the plant at all times. Examples of known constitutive promoters commonly used for plant transformation include the cauliflower heat shock protein (hsp80) promoter, the cauliflower mosaic virus promoter, the nopaline synthase (nos) promoter, octopine ( ocs), the Agrobacterium promoter and the Agrobacterium promoter of the mannopine synthase (mas).
[0030] Inducible promoters can be linked by an inducing agent and are typically active while they are exposed to the inducing agent. The inducing agent can be a chemical agent, such as a metabolite, growth regulator, herbicide or phenolic compound or a physiological stress directly imposed on the plant, such as cold, heat, salt, toxins, or through the action of a pathogen microbial or a plague. In this way, inducible promoters can be used to regulate the expression of desired characteristics, such as genes that control insect pest or microbial pathogens, considering that the protein is only produced shortly after infection or the insect's first bites in a transient manner. in order to decrease the selective pressure for resistant insects. For example, plants can be transformed to express insecticidal or fungicidal characteristics, such as Bacillus thuringiensis (Bt) toxins, virus coating proteins, glucanases, chitinases or phytoalexins. In another example, plants can be transformed to tolerate herbicides by overexpression, under exposure to a herbicide, the acetohydroxy acid synthase enzyme, which neutralizes multiple types of herbicides [Hattori, J. et a /., Mol. General. Genet. 246: 419 (1995)].
[0031] Several specific fruit promoters have been described, including an isolated apple Thi promoter (American Patent No. 6,392,122); an isolated strawberry promoter (American Patent No. 6,080,914); promoters E4 and E8 isolated from tomato (American Patent No. 5,859,330); a polygalacturonase promoter (American Patent No. 4,943,674); and the tomato gene promoter 2A11 [Van H.aaren et al., Plant Mol. Biol. 21: 625-640 (1993)]. Such specific fruit promoters can be used, for example, to modify the ripeness of the fruit by regulating the expression of ACC deaminase that inhibits ethylene biosynthesis. Other gene products that may be desired for expression in fruit tissues include genes encoding taste or color characteristics, such as sucrose phosphate synthase, cyclase or thaumatin.
[0032] Specific seed promoters have been described in US Patent numbers 6,403,862, 5,608,152 and 5,504,200; and in American Patent Application No. Ser. 09/998059 and 10/137964. Such specific seed promoters can be used, for example, to alter the levels of saturated or unsaturated fatty acids; to increase the levels of lysine - or sulfur - containing amino acids, or to modify the amount of starch contained in the seeds.
[0033] Several promoters that regulate gene expression specifically during the germination stage have been described, including the almost isolated promoters of cystatin-1 and a-glucuronidase (American Patent No. 6,359,196), and the hydrolase promoter [Skriver etal., Proc. Natl. Acad. Sci. USA, 88: 7266-7270 (1991)].
[0034] W02004 / 081173 discloses new regulatory sequences derived from plants and constructions and methods of using these to direct the expression of exogenous polynucleotide sequences in plants. SUMMARY OF THE INVENTION
[0035] According to an aspect of some functions of the present invention, a method is provided to increase production, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress, and / or efficiency in the use of nitrogen from a plant, comprising expressing within the plant an exogenous polynucleotide comprising a sequence encoding a polypeptide at least 80% identical to SEQ ID NO: 488-813, 4852-5453, 5460, 5461, 5484, 546-5550, 5553, 5558-8090 or 8091, thereby increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or efficiency in nitrogen use of the plant.
[0036] In accordance with an aspect of some functions of the present invention, a method is provided to increase yield, biomass, growth rate, vigor, oil content, fiber production, tolerance to abiotic stress, and / or efficiency in the use of nitrogen from a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NO: 488-813, 4852-5453, 5460, 5461, 5484, 5486- 5550, 5553, 5558-8091, 5454-5459, 5462-5469, 54715475, 5477-5480, 5482, 5483, 5485, 5551, 5552, and 55545557, thus increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or efficiency in the use of plant nitrogen.
[0037] According to one aspect of some applications of the present invention, a method is provided for increasing the content of. oil, fiber yield and / or fiber quality of a plant, comprising expressing within the plant An exogenous polynucleotide comprising the nucleic acid sequence encoding a polypeptide at least 80% identical to SEQ ID NO: 5470, 5476, or 5481 thereby increasing the oil content, fiber yield and / or plant fiber quality.
[0038] According to one aspect of some applications of the present invention, a method is provided to increase yield, biomass, growth rate, vigor, oil content, fiber production, tolerance to abiotic stress, and / or efficiency in use of nitrogen from a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least 80% identical to SEQ ID NO: 1487, 814-1598, 1600- 1603, 1605-1626, 1632-1642, 1645 -4850 or 4851, thus increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or efficiency in the use of plant nitrogen.
[0039] According to one aspect of some applications of the present invention, a method is provided to increase yield, biomass, growth rate, vigor, oil content, fiber production, tolerance to abiotic stress, and / or efficiency in the use of nitrogen from a plant, comprising expressing within the plant an exogenous polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 1-487, 814-1598, 1600-1603, 1605-1626, 1632-1642, 1645-4851, 1599, 1604, 1628, 1630, and 1644, thereby increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or efficiency in the use of nitrogen from the plant.
[0040] In accordance with an aspect of some applications of the present invention, a method is provided to increase the oil content, fiber yield and / or fiber quality of a plant, comprising expressing within the plant an exogenous polynucleotide comprising the sequence nucleic acid comprising a polypeptide at least 80% identical to SEQ ID NO: 1627, 1629, or 1631, thereby increasing the oil content, fiber yield and / or plant fiber quality.
[0041] According to one aspect of some applications of the present invention, an isolated polynucleotide comprising a sequence of. nucleic acid encoding a polypeptide comprising the amino acid sequence at least 80% homologous to the amino acid sequence described in SEQ ID NO: 488-813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5553, 5558- 8090 or 8091, characterized by the fact that such a sequence of. acid is able to increase yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or the efficiency of nitrogen use of the plant.
[0042] According to one aspect of some applications of the present invention, an isolated polynucleotide is provided comprising a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 488 -813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5553, 55588091, 5454-5459, 5462-5469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5551, 5552, and 55545557.
[0043] According to an aspect of some applications of the present invention, an isolated polynucleotide is provided comprising a nucleic acid sequence at least 80% identical to SEQ ID NO: 1-487, 8141598, 1600-1603, 1605-1626, 1632-1642, 1645- 4850 or 4851, characterized by the fact that the acid sequence is capable of increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance abiotic stress and / or efficiency. in the use of nitrogen from the plant.
[0044] According to one aspect of some applications of the present invention, an isolated polypeptide is provided comprising the nucleic acid sequence selected from the group consisting of SEQ ID Nas: 1487, 814-1598, 1600-1603, 1605-1626 , 1632-1642, 1645-4851, 1599, 1604, 1628, 1630, and 1644.
[0045] In accordance with an aspect of some applications of the present invention, there is provided a nucleic acid construct, comprising the isolated polynucleotide of the invention, and a promoter of direct transcription of the nucleic acid sequence in a host cell.
[0046] According to an aspect of some applications of the present invention, an isolated polynucleotide is provided comprising an amino acid sequence at least 80% homologous to SEQ ID NO: 488-813, 4852-5453, 5460, 5461, 5484, 5486 -5550, 5553, 5558-8090 or 8091, characterized by the fact that such an acid sequence is capable of increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or nitrogen use efficiency of the plant.
[0047] According to one aspect of some applications of the present invention, an isolated polypeptide is provided comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 488813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5558-8091, 5454-5459, 54625469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5551,5552, and 5554-5557.
[0048] According to one aspect of some applications of the present invention, a plant cell is provided which exogenously expresses the polynucleotide of the invention, or the nucleic acid construct of the invention.
[0049] According to one aspect of some applications of the present invention, a plant cell is provided which exogenously expresses the polypeptide of the invention.
[0050] According to one aspect of some applications of the present invention, a transgenic plant is provided which exogenously expresses the polypeptide of some applications of the invention.
[0051] According to an aspect of some applications of the present invention, a transgenic plant comprising the nucleic acid construct of some applications of the invention is provided.
[0052] According to an aspect of some applications of the present invention, an isolated polynucleotide is provided comprising an acid sequence described by SEQ ID NO: 8096.
[0053] In accordance with an aspect of some applications of the present invention, a nucleic acid construct comprising the polynucleotide isolated from some applications of the invention is provided.
[0054] In accordance with an aspect of some applications of the present invention, a transgenic cell is provided comprising the nucleic acid construct of some applications of the invention.
[0055] According to an aspect of some applications of the present invention, a transgenic plant comprising the nucleic acid construction of some applications of the invention is provided.
[0056] According to one aspect of some applications of the present invention, a method of producing a transgenic plant is provided, comprising transforming a plant with the polynucleotide isolated from some applications of the invention or with building nucleic acid from some applications of the invention.
[0057] In accordance with an aspect of some applications of the present invention, a method of expressing a polypeptide of interest in a cell comprising transforming the cell with a nucleic acid construct comprising a polynucleotide sequence encoding the polypeptide of interest operably connected to the polynucleotide isolated from some applications of the invention, thereby expressing the polypeptide of interest in the cell.
[0058] According to some applications of the invention, the nucleic acid sequence encodes the amino acid sequence selected from the group consisting of SEQ ID NO: 488-813, 4852-5453, 5460, 5461, 5484, 5486- 5550, 5553, 5558-8091, 5454-5459, 54625469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5551, 5552, and 5554-5557.
[0059] According to some applications of the invention, the nucleic acid sequence is selected from the group consisting of SEQ ID NO: s: -1-487, 814-1598, 1600-1603, 1605-1626, 1632-1642 , 1645-4851, 1599, 1604, 1628, 1630, e1644.
[0060] According to some applications of the invention, the polynucleotide consists of the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-487, 814-1598, 1600-1603, 16051626, 1632-1642, 1645-4851 , 1599, 1604, 1628, 1630, and 1644.
[0061] According to some applications of the invention, the nucleic acid sequence encodes the amino acid sequence selected from the group consisting of SEQ ID NOs: 488-813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5553, 5558-8091, 5454-5459, 54625469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5551, 5552, and 5554-5557.
[0062] According to some applications of the invention, the plant cell is part of a plant.
[0063] According to some applications of the invention, the promoter is heterologous to the isolated polynucleotide and / or the host cell.
[0064] According to some applications of the invention, the method further comprises the growth of the plant that expresses the exogenous polynucleotide under abiotic stress.
[0065] According to some applications of the invention, abiotic stress is selected from a group consisting of salinity, drought, water deprivation, flood, estiolamento, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency , excess of nutrients, atmospheric pollution and UV irradiation.
[0066] According to some applications of the invention, the yield comprises seed yield or oil yield.
[0067] According to some applications of the invention, the promoter is determined by SEQ ID NO: 8096.
[0068] According to some applications of the invention, the construction of the nucleic acid, further comprising at least one operable heterologous polynucleotide linked to the isolated polynucleotide.
[0069] According to some applications of the invention, the at least one heterologous polynucleotide is a reporter gene.
[0070] According to some applications of the invention, the construction of the nucleic acid, further comprising a heterologous polynucleotide operably linked to the isolated polynucleotide.
[0071] According to some applications of the invention, the heterologous polynucleotide consists of the nucleic acid sequence selected from the group consisting of SEQ ID N's: 1-487, 814-1598, 16001603, 1605-1626, 1632-1642, 1645 -4851, 1599, 1604, 1628, 1630, and 1644.
[0072] According to some applications of the invention, the transgenic cell of some applications of the invention, being a plant cell.
[0073] According to some applications of the invention, the polypeptide of interest comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 488-813, 4852-5453, 5460, 5461, 5484, 5486- 5550, 5553, 5558-8091, 5454-5459, 5462-5469, 54715475, 5477-5480, 5482, 5483, 5485; 5551, 5552, and 5554-5557.
[0074] According to some applications of the invention, the polynucleotide encoding the polypeptide of interest comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 1-487, 814-1598, 1600-1603, 1605 -1626, 1632-1642, 1645-4851, 1599, 1604, 1628, 1630, and 1644.
[0075] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as that commonly understood by the person skilled in the art in which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in practice or to test the invention, suitable methods and materials are described below. In case of conflicts, the patent specification, including definitions, will control. In addition, the materials, methods and examples are illustrative only and are not necessarily intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS
[0076] Some embodiments of the invention are described in the present, just as an example, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is worth noting that the particularities shown serve as an example and for purposes of illustrative discussion of the invention's realizations. In this regard, the description accompanying the drawings makes it apparent to those skilled in the art how the applications of the invention can be practiced.
[0077] In the drawings: Figure 1 is a schematic illustration of the modified binary plasmid pGI containing the new promoter At6669 (SEQ ID NO: 8096) and GUSintron (pQYN_6669) used to express the isolated polynucleotide sequences of the invention. RB - right edge of T-DNA; LB - left edge of the -DNA; MCS - Multiple cloning site; RE - any restriction enzyme; NOS pro = nopaline synthase promoter; NPT-II = neomycin phosphotransferase gene; NOS ter = nopalina synthase terminator; Poli-A signal (polyadenylation signal); Gusintron - the GUS reporter gene (coding sequence and intron). The isolated polynucleotide sequences of the invention were cloned into the vector during the replacement of the GUSintron reporter gene.
[0078] figure 2 is a schematic illustration of the modified binary plasmid pGI containing the new promoter At6669 (SEQ ID NO: 8096) (pQFN or pQFNc) used to express the isolated polynucleotide sequences of the invention. RB right edge of T-DNA; LB - left edge of - DNA; MCS - Multiple cloning site; Any restriction enzyme; NOS pro = nopaline synthase promoter; NPT-II = neomycin phosphotransferase gene; NOS have synthase terminator nopaline; Poli-A signal (polyadenylation signal); Gusintron - the GUS reporter gene (coding sequence and intron). The isolated polynucleotide sequences of the invention were cloned into the vector's MCS.
[0079] Figures 3A-F are images illustrating the visualization of the root development of transgenic plants exogenously expressing the polynucleotide of some applications of the invention when grown on transparent agar plates under normal conditions (FIGS. 3A-B), of asthmatic stress ( 15% PEG, FIGS. 3C-D) or nitrogen limit (FIGS. 3E-F). The different transgenes were grown on transparent agar plates for 17 days (7 days in the nursery and 10 days after transplantation). The plates were photographed every 3-4 days, starting on day 1 after transplantation. Figure 3A - An image of a plant photograph taken 10 days after transplanting on agar plates, when grown under normal conditions (standard). Figure 3B - An image of the root analysis of plants shown in Figure 3A, in which the measured root lengths are represented by arrows. Figure 3C An image of a photograph of plants taken 10 days after transplantation on agar plates, when grown under high osmotic conditions (PEG 15%). Figure 3D - An image of the root analysis of plants shown in Figure 3C, in which the measured root lengths are represented by arrows. Figure 3E - An image of a photograph of plants taken 10 days after transplantation on agar plates, grown under low nitrogen conditions. Figure 3F - An image of the root analysis of plants shown in Figure 3E, in which the measured root lengths are represented by arrows.
[0080] Figure 4 is a schematic illustration of the modified binary plasmid pGI containing the Root Promoter (pQNa_RP) used to express the isolated polynucleotide sequences of the invention. RB - right edge of T-DNA; LB - left edge of - DNA; NOS pro = nopaline synthase promoter; NPT-II - neomycin phosphotransferase gene; NOS ter = nopaline synthase terminator; Poly-A signal (polyadenylation signal); The sequences of isolated polynucleotides, according to some applications of the invention, were cloned into the vector's MCS.
[0081] figure 5 illustrates the sequence alignment between the new promoter sequence (SQE ID NO: 8096) identified in the present from Arabidopsis thaliana and the Arabidopsis At6669 promoter previously disclosed (W02004 / 081173; described by SEQ ID NO: 8093 in the present). Unmatched nucleotides are underlined at positions 270; 484; 867-868; 967; 2295 and 2316-2318 of SEQ ID NO: 8096. New domains are marked with an empty box at positions 862-865; 23922395 and 2314-2317 of SEQ ID NO: 80.96. Note that regulatory element YACT at position 862-865 and regulatory element AAAG at positions 2392-2395 and 2314-2317 of the new promoter sequence (SEQ ID NO: 8096) are missing from the At6669 promoter (SEQ ID NO: 8093) previously disclosed.
[0082] Figure 6 is a schematic illustration of plasmid pQYN.
[0083] Figure 7 is a schematic illustration of plasmid pQFN.
[0084] Figure 8 is a schematic illustration of plasmid pQFYN.
[0085] Figures 9A-D are images that illustrate GUS staining with A. thaliana seedlings with 11 days of life, which were transformed with the GUS intron expression cassette under the new At6669 promoter (SEQ ID NO: 8096). Note that the new promoter sequence p6669 induces the expression GUS (blue color) in A. thaliana seedlings with 11 days of life, especially in roots, cotyledons and leaves. The expression GUS is demonstrated for four independent events (events number 12516, 12515, 12512, 12511).
[0086] Figures 10A-D are images that illustrate GUS staining with A. thaliana seedlings with 20 days of life, which were transformed with the GUS intron expression cassette under the new At6669 promoter (SEQ ID NO: 8096). Note that the new promoter sequence p6669 induces the expression GUS (blue color) in A. thaliana seedlings with 20 days of life, especially in roots, mainly in the tip of the root and in the faults. The expression GUS is shown for four independent events (events number 12516, 12515, 12512, 12511).
[0087] figures 11A-L are images that illustrate the GUS staining with A. thaliana seedlings with 41 days of life, which were transformed with the GUS intron expression cassette under the new At6669 promoter (SEQ ID NO: 8096). Note that the new promoter sequence p6669 induces the expression GUS (blue color) in A. thaliana seedlings with 41 days of life, especially in the stem, mainly in the tip of the root and in the leaves. Strong expression was detected in the flower, leaves and stem leaves. The GUS expression is demonstrated for four independent events: Figures 11A-C - event 12511; Figures 11D-F - event 12516; Figures 11G-I - event 12515; Figures 11J-L - event 12512.
[0088] figure 12 is a schematic illustration of the modified binary plasmid pGI used to express the sequences of the polynucleotide isolated from some applications of the invention. RB - right edge of T-DNA; LB - left edge of the -DNA; NOS pro = nopaline synthase promoter; NPT-II = neomycin phosphotransferase gene; NOS ter = nopaline synthase terminator; RE = any restriction enzyme; Poli-A signal (polyadenylation signal); 35S - the 35S promoter (SEQ ID NO: 809.4). The polynucleotide sequences isolated from some applications of the invention were cloned into the MCS (multiple cloning site) of the vector. DESCRIPTION OF SPECIFIC APPLICATIONS OF THE INVENTION
[0089] The present invention, in some of its applications, is related to isolated polynucleotides and polypeptides, nucleic acid constructions encoding these, cells expressing these, transgenic plants expressing these and methods to use these to increase yield, biomass, vigor, growth rate, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or nitrogen use efficiency of a plant.
[0090] Before explaining at least one embodiment of the invention in detail, it is worth understanding that the invention is not necessarily limited in its application to the details presented in the description below or exemplified by the Examples. The invention is capable of other applications or of being practiced or carried out in various ways.
[0091] The present inventors have identified new polynucleotides and polypeptides that can be used to increase yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or use efficiency of fertilizer (eg, efficiency of nitrogen use) from a plant, and a new latency sequence that can be used to express heterologous genes in host cells, such as in plants.
[0092] Thus, as shown in the Examples section below, the present inventors used bioinformatics tools to identify polynucleotides that increase yield (for example, seed yield, oil yield and oil content), the rate of growth, biomass, vigor, fiber yield, fiber quality, tolerance to abiotic stress and / or efficiency in the use of nitrogen from a plant. The genes that affect the characteristics of interest were identified (Table 27, Example 10) based on the correlation analyzes performed using the Arabidopsis ecotypes (Examples 2 and 3), tomato varieties (Example 4), ecotypes of b. Juncea (Examples 5 and 6), sorghum varieties, corn hybrids (Example 8) and the expression profiles of the genes according to the selected expression applications (eg tissues, developmental stages and stress conditions) (Tables 1-26, Examples 1-9). Homologous polypeptides and polynucleotides having the same function have also been identified (Table 28, Example 11). The identified polynucleotides were cloned into binary vectors (Example 12, Table 29) and transgenic plants overexpressing the identified polynucleotides and polypeptides were generated (Example 13) and tested for the effect of the exogenous gene on the characteristic of interest (eg, greater weight fresh and dry, leaf area, leaf cover and length, leaf area relative growth rate (TCR), leaf cover TCR, leaf length TCR, seed yield, oil yield, dry matter, harvest index, growth rate, rosette area, rosette diameter, leaf number TCR, bed cover TCR, rosette diameter TCR, leaf blade area, percentage of seed oil and weight of 1000 seeds , bed cover, tolerance to abiotic stress conditions and fertilizer limiting conditions; Examples 14-16; Tables 30-48). In addition, as shown below in the Examples section that follows, the present inventors have disclosed a new promoter sequence that can be used to express the gene of interest in a host cell (Example 17, Figures 5, 8-11). Altogether, these results suggest the use of the new polynucleotides and polypeptides of the invention to increase yield (including oil yield, seed yield and oil content), growth rate, biomass, vigor, fiber yield, quality fiber, tolerance to abiotic stress and / or efficiency in the use of nitrogen from a plant.
[0093] Therefore, according to one aspect of some applications of the invention, a method is provided to increase yield, biomass, growth rate, vigor, oil content, fiber yield, tolerance to abiotic stress, and / or efficiency in the use of nitrogen from a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least 80% identical to SEQ ID NO: 1487, 814-1598, 1600-1603, 1605-1626, 1632-1642, 1645-4850 or 4851, thereby increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or efficiency in the use of plant nitrogen.
[0094] As used herein, the phrase "plant yield" refers to the quantity (as determined by weight or size) or quantity (numbers) of tissues or organs produced per plant or per growing season. Therefore, the higher yield could affect the economic benefit that can be obtained from the plant in a given growing area and / or growing season.
[0095] It should be noted that the plant's yield can be affected by several parameters including, among others, the plant's biomass; the vigor of the plant; the growth rate; seed yield; the amount of seeds or grains; the quality of seeds or grains; oil yield; the oil, starch and / or protein content in Organs harvested organs (for example, seeds or vegetative parts of the plant); number of flowers (buds) per panicle (expressed as the ratio between the number of filled seeds and the number of primary panicles); harvest index; number of plants grown per area; number and size of organs harvested per plant and area; number of plants per cultivation area (density); number of organs harvested in the field; total leaf area; carbon assimilation and carbon division (the distribution / allocation of carbon in a plant); shadow resistance; number of harvestable organs (for example, seeds), seeds per pod, weight per seed; and modified architecture [for example, increasing the diameter, thickness or improving the physical properties of the stem (for example, elasticity)].
[0096] As used in this document, the phrase "seed yield" refers to the number or weight of seeds per plant, seeds per pod, or by growing area or the weight of a single seed, or the oil extracted by seed. Therefore, seed yield can be affected by seed dimensions (for example, length, width, perimeter, area and / or volume), number of seeds (filled) and seed fill rate and by seed oil content. Therefore, the increase in seed yield per plant could affect the economic benefit that can be obtained from the plant in a given growing area and / or growing season; and the increase in seed yield per cultivation area can be obtained by increasing the seed yield per plant and / or increasing the number of plants grown in the same area.
[0097] The term "seed" (also called "grain" or "heartwood"), as used herein, refers to a small embryonic plant protected by a covering called a seed coating (usually with some stored food), the product of the mature ovum of gymnosperm and angiosperm plants that occurs after fertilization and some growth within the parent plant.
[0098] The phrase "oil content" as used in this document refers to the amount of lipids in a given plant organ, whether the seeds (seed oil content) or the vegetative part of the plant (vegetative oil content ) and is usually expressed as the percentage of dry weight (10% of the moisture in the seeds) or net weight (for the vegetative part).
[0099] It should be noted that the oil content is affected by the intrinsic oil production of a tissue (for example, seed, vegetative part), as well as the mass or size of the oil-producing tissue per plant or growth period .
[00100] In an application, the increase in the oil content of the plant can be accomplished by increasing the size / mass of tissue (s) of a plant that comprises oil by growing period. Thus, the highest oil content of a plant can be obtained by increasing the yield, the growth rate, the biomass and the vigor of the plant.
[00101] As used herein, the phrase "plant biomass" refers to the amount (eg, measured in grams of air-dried tissue) of a tissue produced from the plant in a growing season, which could also determine or affect the yield of the plant or, the yield by area of cultivation. An increase in the plant's biomass can occur in the whole plant or in parts of it, for example, above-ground (harvestable) parts, vegetative biomass, roots and seeds.
[00102] As used here, the phrase "growth rate" refers to the increase in the size of the organ / tissue of the plant over time (can be measured in cm per day).
[00103] As used herein, the phrase "plant vigor" refers to the amount (measured by weight) of tissue produced by the plant at a given time. Therefore, the increase in vigor could determine or affect the yield of the plant or the yield per growing season or growing area. In addition, early vigor (seed and / or seedling) results in improved field structure. It should be noted that the yield of the plant can be determined under stress (for example, abiotic stress, nitrogen limiting conditions) or stress-free (normal) conditions.
[00104] Improving early vigor is an important objective of modern rice breeding programs in both temperate and tropical cultivars. Long roots are important for the proper anchoring of the soil in atrocious seed with water. When rice is planted in flooded fields, and when plants have to emerge quickly through water, longer shoots are associated with vigor. When a seeder is used, longer mesocotyl and coleoptile are important for good seedling emergence. The ability to perform early engineering on plants would be very important in agriculture. For example, low early vigor has been a limitation on the introduction of corn (Zea mays L.) hybrids based on the Corn Belt germplasm in the European Atlantic.
[00105] As used herein, the phrase "stress-free conditions" refers to growing conditions (for example, water, temperature, light-dark cycles, humidity, salt concentration, fertilizer concentration in the soil, nutrient supply , for example, nitrogen, phosphorus and / or potassium), which do not significantly go beyond the day-to-day climatic conditions and other abiotic conditions that plants may encounter, and which allow for optimal growth, metabolism, reproduction and / or viability of a plant at any stage of its life cycle (for example, in a crop plant from seed to mature plant and back to seed). People with experience in the technique are aware of normal soil conditions and climatic conditions for a certain plant in a certain geographical location. It should be noted that although stress-free conditions may include some slight variations from ideal conditions (which vary from one type / species from one plant to another), these variations do not stop the plant from growing without the ability to resume. growth.
[00106] The phrase "abiotic stress", as used here, refers to any adverse effect on a plant's metabolism, growth, reproduction and / or viability. Thus, abiotic stress can be induced by subideal environmental growth conditions, for example, salinity, water deprivation, floods, frosts, low or high temperature, toxicity to heavy metals, anaerobiosis, nutrient deficiency, atmospheric pollution or UV radiation. The implications of abiotic stress are discussed in the Background section.
[00107] The phrase "abiotic stress tolerance", as used herein, refers to a plant's ability to withstand abiotic stress without undergoing a substantial change in metabolism, growth, productivity and / or viability.
[00108] Plants are subject to a variety of environmental challenges. Many of these, including salt stress, general osmotic stress, drought stress and freezing stress, have the ability to impact the entire availability of cellular and plant water. It is not surprising, therefore, that the plant's responses to this collection of stresses are related. Zhu (2002) Ann. Rev. Plant Biol. 53: 247-273 et al. notes that "most studies on water stress signaling have primarily focused on salt stress, as the plant's responses to salt and drought are closely related and the mechanisms overlap." Many examples of responses and pathways similar to this stress configuration have been documented. For example, CBF transcription factors have been analyzed as being resistant to salt, freezing and drought conditions (Kasuga et al. (1999) Nature Biotech. 17: 287-291). The Arabidopsis rd29B gene is induced in response to both salt stress and dehydration stress, a process that is largely mediated through an ABA signal transduction process (Uno et al. (2000) Proc. Natl. Acad. Sci USA 97: 11632-11637), resulting in altered activity of the transcription factors that are linked to an upstream element within the rd29B promoter. In Mesembryanthemum crystallinum (ice sheet), Patharker and Cushman demonstrated that a calcium-dependent protein kinase (McCDPKI) is induced by exposure to salt and drought stresses (Patharker and Cushman (2000) Plant J. 24: 679- 691). Stress-induced kinase has also been shown to phosphorylate a transcription factor, presumably altering its activity, although the transcription levels of the target transcription factor are not altered in response to salt stress or drought. Similarly, Saijo et. al demonstrated that a drought-induced protein kinase dependent on rice / salt salt (OsCDPK7) conferred greater tolerance to salt and drought when rice was overexpressed (Saijo et al. (2000) Plant J. 23: 319-327) .
[00109] Exposure to dehydration invokes similar survival strategies in plants, as well as freezing stress (see, for example, Yelenosky (1989) Plant Physiol 89: 444-451) and drought stress induces freezing tolerance ( see, for example, Siminovitch et al. (1982) Plant Physiol 69: 250255 ^ and Glár et al. (1992) Plant 188: 265-270). In addition to the induction of cold acclimatization proteins, strategies that allow plants to survive under low water conditions include, for example, reduced surface area, or production of oil or wax on the surface. In another example, the higher solute content of the plant prevents evaporation and water loss due to heat, drought, salinity, osmotic and the like, thus providing better tolerance to the plant for the above stresses.
[00110] It is estimated that some pathways involved in resistance to stress (as described above) will also be involved in resistance to other stresses, regulated by the same genes or by counterparts. In fact, the general pathways of resistance are related, not identical, and therefore all genes controlling resistance to one stress will control and resistance to other stresses. However, if a gene is resistant to the conditions of one of these stresses, it would be apparent to a person skilled in the art to test for resistance to these related stresses. Methods for assessing stress resistance are provided in the Examples section that follows.
[00111] As used in the present, the phrase "water use efficiency (WUE - Water Use Efficiency)" refers to the level of organic matter per unit of water consumed by the plant, that is, the dry weight of the plant in relation to to the use of plant water, for example, the biomass produced by unit transpiration.
[00112] As used in this document, the phrase "fertilizer use efficiency" refers to the metabolic process (s) that lead to increased yield, biomass, vigor, and growth rate by fertilizer unit applied to the plant. The metabolic process can be the capture, dispersion, absorption, accumulation, relocation (in the plant) and use one or more of the organic or mineral halves absorbed by the plant, such as nitrogen, phosphates and / or potassium.
[00113] As used herein, the phrase "conditions limited by fertilizer" refers to growth conditions that include a level (eg, concentration) of an applied fertilizer that is below the level required for metabolism, growth, normal plant reproduction and / or viability.
[00114] As used in this document, the phrase "nitrogen use efficiency (NUE - Nitrogen Use Efficiency)" refers to the metabolic process (s) that lead to an increase in yield, biomass, vigor, and growth rate per unit of nitrogen applied to the plant. The metabolic process can be uptake, dispersion, absorption, accumulation, relocation (in the plant) and use of nitrogen absorbed by the plant.
[00115] As used herein, the phrase "nitrogen-limited conditions" refers to growth conditions that include a level (eg, concentration) of applied nitrogen (eg, ammonia or nitrate) that is below the level necessary for normal plant metabolism, growth, reproduction and / or viability.
[00116] The improved NUE and FUE of the plant are translated into the field either by harvesting similar amounts of yield, while implementing less fertilizers, or improved yields acquired by implementing the same levels of fertilizers. In this way, the improved NUE and FUE have a direct effect on the yield of the plant in the field. Thus, the polynucleotides and polypeptides of some functions of the invention positively affect the yield of the plant and the seed and the biomass of the plant. In addition, the benefit of NUE from plant improvement will certainly improve the quality of the crop and biochemical constituents of the seed, such as protein yield and oil yield.
[00117] It should be noted that the improved ABST will provide plants with improved vigor also under non-stress conditions, resulting in crops with better biomass and / or yield, for example, elongated fibers for the cotton industry, higher oil content .
[00118] The term "fiber" is used even for conductive cells with thick walls, such as vessels and tracheids and for fibrillar assemblies of several individual fiber cells. Thus, the term "fiber" refers to (a) cells with thick conductive and non-conducting xylem cells; (b) fibers of internal origin to the xylem, including those of phloem, suber, plant tissue and epidermis; and (c) fibers from stems, leaves, roots, seeds and inflorescence flowers (such as those from Sorghum vulgare used in the manufacture of brooms and brushes.
[00119] Examples of fiber-producing plants include, but are not limited to, agricultural crops such as cotton, Bombax trees (Kapok, Ceiba pentandra), desert willow, creosote bush, winterfat, raft, kenaf, rosette, jute , sisal abaca, flaxseed, corn, sugar cane, hemp, ramie, mafumeira, cairo, bamboo, Tillandsia usneoides and agave spp. (e.g., sisal).
[00120] As used here, the phrase "fiber quality" refers to at least one fiber parameter that is agriculturally desired, or necessary in the fiber industry (described below). Examples of such parameters include, but are not limited to, fiber length, fiber strength, fiber suitability, fiber weight per unit length, fiber ratio and maturity (described below).
[00121] The quality of the cotton fiber (thread) is usually measured according to the length, strength and quality of the fiber. Thus, the quality of the fiber is considered to be higher when the fiber is longer, stronger and of higher quality.
[00122] As used herein, the phrase "fiber yield" refers to the volume or quantity of fibers produced from the fiber producing plant.
[00123] As used herein, the term "increase" refers to an increase of at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70 %, at least about 80%, in yield, seed yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or nitrogen use efficiency of a plant in comparison to a native plant [that is, an unmodified plant with the biomolecules (polynucleotide or polypeptides) of the invention, for example, an unprocessed plant of the same species that is grown under the same growing conditions).
[00124] The phrase "expressing an exogenous polynucleotide within the plant", as used here, refers to regulating the level of expression of an exogenous polynucleotide within the plant by introducing the exogenous polynucleotide into a plant or plant cell and expressing by recombinant media, as described below.
[00125] As used herein, the term "expressing" refers to the expression in the mRNA and, optionally, at the level of the polypeptide.
[00126] As used herein, the phrase "exogenous polynucleotide" refers to a heterologous nucleic acid sequence that may not be naturally expressed in the plant or whose overexpression in the plant is desired. The exogenous polynucleotide can be introduced into the plant in a stable or temporary manner, in order to produce a ribonucleic acid (RNA) molecule and / or a polypeptide molecule. It should be noted that the exogenous polynucleotide may comprise a nucleic acid sequence that is identical or partially homologous to an endogenous plant nucleic acid sequence.
[00127] The term "endogenous", as used herein, refers to any polynucleotide or polypeptide that is and / or expressed naturally within a plant or cell thereof.
[00128] According to some applications of the invention, the exogenous polynucleotide comprises a nucleic acid that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, for example, 100% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-487, 814-1598, 1600-1603 , 1605-1626, 1632-1642, 1645-4851.
[00129] According to some applications of the invention, homology is a global homology, that is, a homology over the entire amino acid or nucleic acid sequence of the invention and not over its portions.
[00130] According to some applications of the invention, the identity is a global identity, that is, an identity about the entire amino acid or nucleic acid sequence of the invention and not about its portions.
[00131] Identity (ie percent homology) can be determined using any homology comparison software, including, for example, the BlastN software from the National Center of Biotechnology Information (NCBI), for example example, using standard parameters.
[00132] According to some applications of the invention, the exogenous polynucleotide is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, for example, 100% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-487, 814-1598, 1600-1603, 1605-1626, 1632-1642, 1645-4851.
[00133] According to some applications of the invention the exogenous polynucleotide is established by SEQ ID NO: 1-487, 814-1598, 1600-1603, 1605-1626, 1632-1642, 1645- 4851, 1599, 1604, 1628, 1630, or 1644.
[00134] According to one aspect of some applications of the present invention, a method is provided to increase yield, biomass, growth rate, vigor, oil content, fiber yield, tolerance to abiotic stress, and / or efficiency in the use of nitrogen from a plant, comprising expressing within the plant an exogenous polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 1487, 814-1598, 1600- 1603, 1605-1626, 1632-1642, 1645 -4851, 1599, 1604, 1628, 1630, and 1644, thereby increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or efficiency in the use of nitrogen from the plant.
[00135] According to some applications of the invention, the exogenous polynucleotide is defined by the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-487, 814-1598, 16001603, 1605-1626, 1632-1642, 1645-4851, 1599, 1604, 1628, 1630, and 1644.
[00136] According to one aspect of some applications of the invention, a method of increasing the oil content, fiber yield and / or fiber quality of a plant is provided, comprising a nucleic acid sequence of at least about 80 %, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87% at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, for example, 100% identical to the polynucleotide selected from the group consisting of SEQ ID NOs: 1627, 1629, and 1631, thereby increasing the oil content, fiber yield and / or plant fiber quality.
[00137] According to one aspect of some applications of the invention, a method is provided to increase the oil content, fiber yield and / or fiber quality of a plant, comprising expressing within the plant an exogenous polynucleotide comprising the sequence nucleic acid selected from the group consisting of SEQ ID NO: 1627, 1629, and 1631, thereby increasing the oil content, fiber yield and / or plant fiber quality.
[00138] According to some applications of the invention, the exogenous polynucleotide is defined by the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1627, 1629, and 1631.
[00139] As used herein, the term "polynucleotide" refers to a single or double nucleic acid sequence that is isolated and presented in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a sequence of genomic polynucleotide and / or composite polynucleotide sequences (eg, a combination of the above).
[00140] The term "isolated" refers to at least partially separated from the natural environment eg, from a plant cell.
[00141] As used herein, the phrase "complementary polynucleotide sequence" refers to a sequence that results from the reverse transcription of messenger RNA using a reverse transcriptase or any other RNA-dependent DNA polymerase. This sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.
[00142] As used herein, the phrase "polynucleotide genomic sequence" refers to a sequence derived (isolated) from a chromosome and thus represents a contiguous part of a chromosome.
[00143] As used herein, the phrase "sequence composed of polynucleotides" refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exon sequences necessary to encode the polypeptide of the present invention, as well as some intronic sequences that interpose between them. Intronic sequences can be from any source, including other genes, and will typically include conserved splicing signal sequences. These intronic sequences may also include regulatory elements for cis-action expression.
[00144] According to some applications of the invention, the exogenous polynucleotide of the invention encodes a polypeptide comprising an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or up to 100% homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs: 488-813, 4852-5453, 5460, 5461, 5484, 5486 -5550, 5553, and 5558-8091.
[00145] Homology (for example, percentage homology) can be determined using any homology comparison software, including, for example, the BlastP or TBLASTN software from the National Center of Biotechnology Information (NCBI), for example, using standard parameters, when starting from a polypeptide sequence; or the tBLASTX algorithm (available via the NCBI), for example using standard parameters, which compares the conceptual translation products of six frames of a nucleotide queue sequence (both strands) against a protein sequence database.
[00146] Homologous sequences include both orthologous and parologous sequences. The term "parallels" refers to genetic duplications within the genome of a species leading to parallel genes. The term "orthologist" refers to homologous genes in different organisms due to ancestral relationship.
[00147] An option to identify orthologists in monocotyledonous plant species is by doing a reciprocal blast search. This can be accomplished by a first blast involving blasting the sequence of interest against any sequence database, such as the publicly available NCBI database which can be found at: Hypertext Transfer Protocol: // World Wide Web (dot ) ncbi (dot) nlm (dot) nih (dot) gov. If orthologists are sought in rice, the sequence of interest would be blasted against, for example, the 28,469 full-length cDNA clones of Oryza sativa Nipponbare available from the NCBI. The blast results can be filtered. The full-length sequences of either the filtered or unfiltered results are then subjected to a reverse blasting (second blast) against the sequences of the organism from which the sequence of interest is derived. The results of the first and second blasts are then compared. An orthologist is identified when the resultant sequence with the highest score (best option) on the first blast identifies the row sequence (the original sequence of interest) on the second blast as the best option. Using the same justification, a parallel (homologous to a gene in the same organism) is found. In the case of large sequence families, the ClustalW program can be used [Hypertext Transfer Protocol: // World Wide Web (dot) ebi (dot) ac (dot) uk / Tools / clustalw2 / index (dot) html], followed by a neighboring junction tree (Hypertext Transfer Protocol: // en (dot) wikipedia (dot) org / wiki / Neighbor-joining) that helps you visualize clustering.
[00148] According to some applications of the invention, the exogenous polynucleotide of the invention encodes a polypeptide comprising an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or up to 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 488-813, 48525453, 5460, 5461, 5484, 5486-5550 , 5553, and 5558-8091.
[00149] According to some applications of the invention, the exogenous polynucleotide encodes a polypeptide that consists of the amino acid sequence defined by SEQ ID NO: 488-813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5553, 5558 -8091, 5454-5459, 5462-5469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5551, 5552, 55545556 or 5557.
[00150] According to some applications of the invention, the method of increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress, and / or efficiency in the use of nitrogen from a plant, is affected by the expression within the plant of an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or up to 100% homologous to the amino acid sequence selected from group c complying with SEQ ID NOs: 488-813, 4852-5453, 5460, 5461, 5484, 5486, 5550, 5553, and 5558-8091, thereby increasing yield, biomass growth rate, vigor, oil content, yield fiber quality, fiber quality, tolerance to abiotic stress and / or efficiency in the use of plant nitrogen.
[00151] According to some applications of the invention, the method of increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress, and / or efficiency in the use of nitrogen from a plant, is affected by the expression within the plant of an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or up to 100% identical to the amino acid sequence selected from group c complying with SEQ ID NOs: 488-813, 4852-5453, 5460, 5461, 5484, 5486, -5550, 5553, and 5558-8091, thereby increasing yield, biomass growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or efficiency in the use of plant nitrogen.
[00152] According to one aspect of some applications of the invention, the method of increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or efficiency in nitrogen use of a plant, comes into effect by the expression within the plant of an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 488-813, 4852- 5453, 5460, 5461, 5484, 5486-5550, 5553, 5558-8091, 5454-5459, 5462-5469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5550, 5552, and 55545557, thus increasing the yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or nitrogen plant efficiency.
[00153] According to one aspect in some of the applications of the invention, a method is provided to increase yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, the tolerance to abiotic stress and / or the efficiency of nitrogen use of a plant, comprising the expression within the plant of an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NOs: 488- 813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5553, 5558-8091, 5454-5459, 54625469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5551, 5552, and 5554-5557 , thereby increasing the yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or the efficiency in the use of plant nitrogen.
[00154] According to some applications of the invention, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence defined by SEQ ID NO: 488-813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5553, 5558 -8091, 5454-5459, 5462, 5469, 5471-5475, 54775480, 5482, 5483, 5485, 5551, 5552, 5554-5556 or 5557.
[00155] According to an aspect of some applications of the invention, a method of increasing the oil content, fiber yield and / or fiber quality of a plant is provided, comprising the expression within the plant of an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85% at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least minus about 99%, for example, 100% homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs: 5470, 5476, and 5481, increasing, d that mode, the oil content, fiber yield and / or fiber quality of the plant.
[00156] According to one aspect of some applications of the invention, the method for increasing the oil content, fiber yield and / or fiber quality of a plant, comes into effect by expressing within the plant an exogenous polinculeotide comprising a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NOs: 5469, 5476, and 5481, thereby increasing the oil content, fiber yield and / or fiber quality of a plant.
[00157] According to some applications of the invention, the exogenous polynucleotide encodes a polypeptide that consists of the amino acid sequence defined by SEQ ID NO: 5470, 5476, or 5481.
The nucleic acid sequences encoding the polypeptides of the present invention can be optimized for expression. Examples of such sequence modifications include, but are not limited to, an altered G / C content to more closely address that typically found in plant species of interest, and the removal of atypical codons found in plant species commonly referred to as plant optimization. codon.
[00159] The phrase "codon optimization" refers to the selection of DNA nucleotides suitable for use within a structural gene or fragment thereof that approximates the use of the codon within a plant of interest. Therefore, an optimized nucleic acid gene or sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified to use statistically preferred or statistically favored codons within a plant. The nucleotide sequence is typically analyzed at the DNA level and the coding region is optimized for expression in the plant species determined using any suitable procedure, for example, as described in Sardana et al. (1996, Plant Cell Reports 15: 677-681). In this method, the standard deviation of the use of the codon, a measure of the deviation of the use of the codon, can be calculated first by finding the proportional square deviation of the use of each codon of the native gene in relation to that of highly expressed plant genes, followed by calculation of the mean square deviation. The formula used is: 1 SDCU = n = 1 N [(Xn - Yn) / Yn] 2 / N, where Xn refers to the frequency of use of the codon in high-expression plant genes, where Yn refers to the frequency of use of the codon in the gene of interest and N refers to the total number of codons in the gene of interest. A Table of codon usage from highly expressed genes of dicotyledonous plants is compiled using data from Murray et al. (1989, Nuc Acids Res. 17: 477-498).
[00160] A method of optimizing the nucleic acid sequence according to the preferred codon usage for a particular plant cell type is done based on direct use, without performing any additional statistical calculations, from the Optimization Tables codon, such as those provided online in the codon use database of the NIAS DNA bank (National Institute of Agrobiological Sciences) in Japan (Hypertext Transfer Protocol: // World Wide Web ( dot) kazusa (dot) or (dot) jp / codon /). The codon usage database contains codon usage tables for several different species, where each codon usage table has been statistically determined based on data from Genbank.
[00161] Using the Tables above to determine the most preferred or most favored codons for each amino acid in a particular species (for example, rice), a naturally occurring nucleotide sequence that encodes a protein of interest can be the codon optimized for that particular plant species. This is affected by the replacement of codons, which may have a low statistical incidence in the genome of the species in particular with corresponding codons, with respect to an amino acid, which are statistically more favored. However, one or more less favored codons can be selected to exclude existing restriction sites, to create new sites at potentially useful junctions (5 'and 3' terminations to add signal peptide or termination cassettes, internal sites that can be used to cut and join segments to produce a correct full-length sequence), or to eliminate nucleotide sequences that can adversely affect stability or mRNA expression.
[00162] The naturally occurring coding nucleotide sequence may, before any modification, already contain a number of codons that correspond to a statistically favored codon in a particular plant species. Therefore, codon optimization of the native nucleotide sequence can comprise the determination of which codons, within the native nucleotide sequence, are not statistically favored in relation to a particular plant, and the modification of these codons according to a usage table. of the particular plant's codon to produce an optimized codon derivative. A modified nucleotide sequence can be fully or partially optimized for plant codon use as long as the protein encoded by the modified nucleotide sequence is produced at a higher level than the protein encoded by the corresponding naturally occurring or native gene. The construction of synthetic genes by altering the use of codons is described, for example, in PCT patent application 93/07278.
[00163] According to some configurations of the invention, the exogenous polynucleotide is a non-coding RNA.
[00164] As used herein, the phrase "non-coding RNA" refers to an RNA molecule that does not encode an amino acid sequence (a polypeptide). Examples of such non-coding RNA molecules include, but are not limited to, an antisense RNA, a pre-miRNA (precursor to a microRNA), or a precursor to a Piwi-interactive RNA (piRNA).
[00165] Example non-limiting non-encoding RNA polynucleotides are provided in SEQ ID NOs: 211-217, 278-284, 486 and 487.
[00166] Thus, the invention encompasses the nucleic acid sequences described above; their fragments, sequences hybridizable to these, sequences homologous to these, sequences that encode similar polypeptides with the use of different codons, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, both naturally occurring or produced by both randomly and in a targeted manner.
[00167] The invention provides an isolated polynucleotide comprising a nucleic acid sequence of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, eg, 100% identical to the polynucleotide selected from the group consisting of SEQ ID NOs: 1-487, 814-1598, 1600-1603, 1605-1626, 1632-1642 , 1645-4851.
[00168] According to some applications of the invention, the nucleic acid sequence is capable of increasing the yield, the biomass, the growth rate, the vigor, the oil content, the fiber yield, the fiber quality, the tolerance to abiotic stress and / or nitrogen use efficiency of a plant.
[00169] According to some applications of the invention, the isolated polynucleotide comprises the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-487, 814-1598, 16001603, 1605-1626, 1632-1642, 1645-4851 , 1599, 1604, 1628, 1630, and 1644.
[00170] According to some applications of the invention, the isolated polynucleotide is defined by SEQ ID NO: 1-487, 814-1598, 16001603, 1605-1626, 16321642, 1645- 4851, 1599, 1604, 1628, 1630, or 1644.
[00171] The invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence of at least about 80%, at least about 81%, at least about 82%, at least about 83% at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 95%, at least at least about 97%, at least about 98%, at least about 99%, or more, say 100% homologous to the amino acid sequence selected from the group consisting of SEQ ID NO: 488-813, 4852-5453 , 5460, 5461, 5484, 5486-5550, 5553, and 5558-8091.
[00172] According to some applications of the invention, the amino acid sequence is capable of increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or the nitrogen use efficiency of a plant.
[00173] The invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 488-813, 4852-5453, 5460, 5461, 5484, 5486- 5550, 5553, 5558-8091, 5454-5459, 5462-5469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5551, 5552, and 5554-5557.
[00174] The invention provides an isolated polypeptide comprising an amino acid sequence of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, say 100% homologous to an amino acid sequence selected from the group consisting of SEQ ID NO: 488-813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5553, and 5558-8091.
[00175] According to some applications of the invention, the amino acid sequence is capable of increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress and / or the nitrogen use efficiency of a plant.
[00176] According to some applications of the invention, the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 488-813, 4852-5453, 5460,5461, 5484, 5486-5550, 5553, 5558-8091, 5454-5459, 5462-5469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5551, 5552, and 55545557.
[00177] According to some applications of the invention, the polypeptide is determined by SEQ ID NO: 488-813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5553, 5558-8091, 5454-5459, 5462- 5469, 54715475, 5477-5480, 5482, 5483, 5485, 5551, 5552, 5554-5556 or 5557.
[00178] In accordance with an aspect of some embodiments of the invention, a nucleic acid construct, comprising the isolated polynucleotide of the invention, and a promoter of direct transcription of the nucleic acid sequence in a host cell is provided.
[00179] The invention also encompasses fragments of the polypeptides and polypeptides described above having mutations, for example, deletions, insertions or substitutions of one or more amino acids, either naturally occurring or man-made, both randomly and in a targeted manner.
[00180] The term "plant", as used herein, encompasses total plants, ancestors and progeny of plants and parts of plants, including seeds, shoots, stems, roots (including tubers), as well as plant cells, tissues and organs. The plant can be in any form, including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. Plants that are particularly useful in the methods of the invention include all plants that belong to the superfamily Viridiplantae, particularly monocotyledonous and dicotyledonous plants including a forage vegetable, ornamental plants, agriculture, tree or shrubs selected from the list involving Acacia spp., Acer spp ., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp. africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster sp. , Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbe monetary policy, Davallia divaricata,
[00181] Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp., Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp. Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp. Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp. Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissi ma, Petunia spp., Phaseolus 5 spp., Phoenix canadensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopiszeraria, Prosopis cineraria Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp. Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp. pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, brussels sprouts, cabbage, canola, carrots, cauliflower, celery, leaf sprouts, flax, kale, len seed, rapeseed, okra, onion, potato, rice, soy, straw, sugar beet, sugar cane, sunflower, tomato, pumpkin, corn, wheat, barley, rye, oats, peanuts, peas, lentils and alfalfa, cotton , rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, ornamental plant, grass and forage. Alternatively, algae and other non-Viridiplantae species can be used for the methods of the present invention.
[00182] According to some applications of the invention, the plant used by the method of the invention are cultivation plants such as rice, corn, wheat, barley, peanuts, potatoes, sesame, olive, oil palm, banana, soy, sunflower, canola, sugar cane, alfalfa, millet, legumes (beans, peas), flax, lupinus, rapeseed, tobacco, cholpo and cotton.
[00183] According to some applications of the invention, a plant cell is provided which exogenously expresses the polynucleotide of some configurations of the invention, the combination of nucleic acid of some configurations of the invention and / or the polypeptide of some configurations of the invention.
[00184] According to some applications of the invention, the expression of the exogenous polynucleotide of the invention within the plant is performed by transforming one or more cells of the plant with the exogenous polynucleotide, followed by the generation of a mature plant from the transformed cells and cultivation of the mature plant in conditions suitable to express the exogenous polynucleotide within the mature plant.
[00185] According to some applications of the invention, transformation is carried out by introducing into the plant cell a nucleic acid construct that includes the exogenous polynucleotide of some applications of the invention and at least one promoter of direct transcription of the exogenous polynucleotide in a host cell (a plant cell). Further details of the appropriate transformation approach are provided below.
[00186] As mentioned, the nucleic acid construction according to some applications of the invention comprises a promoter sequence and the isolated polynucleotide of the invention.
[00187] According to some applications of the invention, the isolated polynucleotide is operably linked to the promoter sequence.
[00188] A coding nucleic acid sequence is "operably linked" to a regulatory sequence (eg, promoter) if the regulatory sequence is able to exert a regulatory effect on the coding sequence attached to it.
[00189] As used herein, the term "promoter" refers to a region of DNA that lies above the site of the start of transcription of a gene to which RNA polymerase binds to initiate RNA transcription. The promoter controls where (eg, where in a plant) and / or when (ie, at what stage or condition in an organism's life cycle) the gene is expressed.
Any suitable promoter sequence can be used for the nucleic acid construct of the present invention. Preferably, the promoter is a constitutive promoter, a tissue specific promoter or an abiotic stress inducible.
[00191] According to some applications of the invention, the promoter is a promoter plant, which is suitable for expression of the exogenous polynucleotide in a plant cell.
Suitable constitutive promoters include, for example, CaMV 35S promoter (SEQ ID NO: 8094; Odell et al., Nature 313: 810-812, 1985); Arabidopsis At6669 promoter (SEQ ID NO: 8096); Maize ubi (Christensen et al., Plant Sol. Biol. 18: 675689, 1992); rice actin (McElroy et al., Plant Cell 2: 163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81: 581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100: 456462, 1997); GOS2 (by Pater et al, Plant J Nov; 2 (6): 837-44, 1992); ubiquitin (Christensen et al, Plant Mol. Biol. 18: 675-689, 1992); rice cyclophylline (Bucholz et al, Plant Mol Biol. 25 (5): 837-43, 1994); corn histone H3 (Lepetit et al, Mol. Gen. Genet. 231: 276-285, 1992); Actin 2 (An et al, Plant J. 10 (1); 107-121, 1996) and Synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those in U.S. Patent NOs. 5,466,785; 5,399,680; 5,268,463; and 5,608,142.
[00193] Suitable tissue-specific promoters include, but are not limited to, leaf-specific promoters [as described, for example, by Yamamoto et al., Plant J. 12: 255-265, 1997; Kwon et al., Plant Physiol. 105: 357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35: 773-778, 1994; Gotor et al., Plant J. 3: 509-18, 1993; Orozco et al., Plant Mol. Biol. 23: 1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90: 9586-9590, 1993], preferred seed promoters [for example, Napina (originated from Brassica napus, which is characterized by a specific seed promoting activity; Stuitje AR et.al. Plant Biotechnology Journal 1 (4 ): 301-309; SEQ ID NO: 8095), of seed-specific genes (Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), Brazil Nut albumin (Pearson 'et al., Plant Mol. Biol. 18: 235-245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214, 1988), Glutelin (rice) (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts . 221: 43-47, 1987), Zein (Matzke et al Plant Mol Biol, 143) .323-32 1990), napA (Stalberg, et al, Plant 199: 515-519, 1996), Wheat SPA (Albanietal, Plant Cell, 9: 171184, 1997), sunflower oleosin (Cummins, et al., Plant Mol. Biol. 19: 873-10 876, 1992)], endosperm-specific promoters [eg, LMW and HM wheat W, glutenin-1 (Mol Gen Genet 216: 81-90, 1989; NAR 17: 461-2), wheat a, beg gliadins (EMB03: 1409-15, 1984), barley ltrl promoter, barley Bl, C, D hordein (Theor Appl Gen 98: 1253-62, 1999; Plant J 4 : 343-55, 1993; Mol Gen Genet 250: 750-60, 1996), Barley DOF (hena et al, The Plant Journal, 116 (1): 53- 62, 1998), Biz2 (EP99106056.7), promoter synthetic (Vicente- Carbajosa et al., Plant J. 13: 629-640, 1998), NRP33 rice prolamine, rice Glb-1 globulin (Wu et al, Plant Cell Physiology 39 (8) 885- 889, 1998) , rice alpha-globulin REB / OHP-1 (Nakase et al. Plant Mol. Biol. 33: 513S22, 1997), rice ADP-glucose PP (Trans Res 6: 157-68, 1997), ESR gene family maize (Plant J 12: 235-46, 1997), sorghum gamma-kafirine (PMB 32: 1029-35, 1996)], embryo-specific promoters [for example, rice OSH1 (Sato et al, Proc. Nati Acad. Sci. USA, 93: 81178122), KNOX (Postma-Haarsma et al, Plant Mol. Biol. 39: 25771, 1999), rice oleosin (Wu et at, J. Biochem., 123: 386, 1998 )], and specific flower promoters [. e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al Mol. Gen Genet. 217: 240245; 1989) , apetala-3] and root promoters such as the ROOTP promoter [SEQ ID NO: 8097].
[00194] Abiotic stress-inducible promoters include, among others, salt-inducible promoters such as RD29A (Yamaguchi- Shinozalei et al., Mol. Gen. Genet. 236: 331-340, 1993); drought-inducible promoters such as the corn rabl7 gene promoter (Pla et. al, Plant Mol. Biol. 21: 259-266, 1993), corn rab28 gene promoter (Busk et. al., Plant J. 11 : 1285-1295, 1997) and promoter of the corn Ivr2 gene (Pelleschi et. Al, Plant Mol. Biol. 39: 373-380, 1999); heat-inducible promoters such as the tomato hsp80 heat promoter (U.S. Patent No. 5,187,267).
[00195] As mentioned above and further described in Example 15 of the Examples section below, the present inventors have disclosed a new promoter sequence (nucleic acid regulatory sequences) that can be used to express a polynucleotide of interest in a plant.
Therefore, according to one aspect of some applications of the invention, an isolated polynucleotide is provided comprising an acid sequence described by SEQ ID NO:
[00197] According to some applications of the invention, the isolated polynucleotide is capable of regulating the expression of the heterologous polynucleotide in a host cell.
[00198] According to some applications of the invention, the heterologous polynucleotide is operably linked to the regulatory nucleic acid sequence described by SEQ ID NO: 8096.
[00199] According to one aspect of some applications of the invention, a nucleic acid construct comprising the isolated polynucleotide described by SEQ ID NO: 8096 is provided.
[00200] According to some applications of the invention, the construction of the nucleic acid, further comprising at least one operable heterologous polynucleotide linked to the isolated polynucleotide.
[00201] According to some embodiments of the invention, the nucleic acid regulatory sequence of the invention varies in length from approximately 500 nucleotides to approximately 4000 nucleotides and includes one or more sequence regions that are capable of recognizing and binding to RNA polymerase II and other proteins (trans action transcription factors) involved in transcription.
[00202] According to some configurations of the invention, the regulatory sequence is positioned 1-500 bp above the ATG codon of the nucleic acid coding sequence, although it is appreciated that regulatory sequences can also exert their effect when positioned elsewhere in with respect to the nucleic acid coding sequence (for example, within an intron).
[00203] As is clearly illustrated in the Examples section below, the new At6669 promoter sequence of some applications of the invention is able to regulate the expression of a nucleic acid coding sequence (for example, a reporter gene, for example, GUS, luciferase) operationally linked to them (see Example 17 in the Examples section below).
[00204] According to some configurations of the invention, the nucleic acid regulatory sequences of the invention are modified to create variations in the sequences of the molecule, for example, to intensify its promotion activities, using methods known in the art, for example, modification of DNA based on PCR or standard DNA mutagenesis techniques or by chemical synthesis of the modified polynucleotides.
[00205] In this way, the regulatory sequence of the nucleic acid of the invention (eg SEQ ID NO: 8096) can be cut or deleted and can still maintain the ability to direct the transcription of a heterologous DNA sequence operably linked. The minimum length of a promoter region can be determined by systematically removing the 5 'and 3' termination sequences from the isolated polynucleotide by standard methods known in the art, including, but not limited to, removing restriction enzyme fragments or digestion with nuclear. Consequently, any sequence fragments, parts or regions of the polynucleotide promoter sequences disclosed in the invention can be used as regulatory sequences. It will be appreciated that the modified sequences (coutadas, truncadas and the like) can acquire different transcription properties, such as the direction of different pattern of gene expression in comparison to the unmodified element.
[00206] Optionally, the sequences defined in SEQ ID NOs: 8096 can be modified, for example, for expression in a variety of plant systems. In another approach, new hybrid promoters can be designed or genetically modified using a variety of methods. Many promoters contain ascending sequences that activate, enhance or define the potency and / or specificity of the promoter, as described, for example, by Atchison [Ann. Rev. Cell Biol. 4: 127 (1988)]. T-DNA genes, for example, contain "TATA" boxes that define the transcription start site and other parent elements located above the transcription start site modulate transcription levels [Gelvin In: Transgenic plants (Kung, S.-D and Us, R., Eds, San Diego: Academic Press, pp.49-87, (1988)]. Another chimeric promoter combined an octopine synthase activator (OCS) trimer to the mannopine synthase activator (but) more the promoter and reported an increase in the expression of a reporter gene [Min Ni et al. The Plant Journal 7: 661 (1995)]. The upward regulatory sequences of the polynucleotide promoter sequences of the invention can be used to construct these chimeric promoters or Methods for building variant promoters include, but are not limited to, combining control elements from different promoters or duplicating parts or regions of a promoter (see, for example, United States Patent Nos. 5,110,732 and 5,097,025). technical in the subject they are familiar with the specific conditions and with the procedures for the construction, manipulation and isolation of macromolecules (for example, DNA molecules, plasmids, etc.), generation of recombinant organisms and the screening and isolation of genes, [see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, (1989); Mailga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press, (1995); Birren et al., Genome Analysis: volume 1, Analyzing DNA, (1997); volume 2, Detecting Genes, (1998); volume 3, Cloning Systems, (1999); and volume 4, Mapping Genomes, (1999), Cold Spring Harbor, N.Y].
[00207] According to some applications of the invention, the heterologous polynucleotide, which is regulated by the nucleic acid sequence described by SEQ ID NO: 8096, comprises a nucleic acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88 %, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, for example, 100% identical to the polynucleotide selected from the group consisting of in SEQ ID NOs: 1487, 814-1598, 1600-1603, 1605-1626, 1632-1642, 1645-4851, 1599, 1604, 1628, 1630, and 1644.
[00208] According to some applications of the invention, the heterologous polynucleotide, which is regulated by the nucleic acid sequence described by SEQ ID NO: 8096, comprises a nucleic acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88 %, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, for example, 100% identical to the polynucleotide selected from the group consisting of SEQ ID NOs: 488-813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5553, 55588091, 5454-5459, 5462-5469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5551 , 5552, 5554-5556 or 5557 .
[00209] According to some applications of the invention, the heterologous polynucleotide, which is regulated by the nucleic acid sequence described by SEQ ID NO: 8096, comprises a nucleic acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88 %, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, for example, 100% identical to the polynucleotide selected from the group consisting of in SEQ ID NOs: 1627, 1629 and 1631.
[00210] According to some applications of the invention, the heterologous polynucleotide, which is regulated by the nucleic acid sequence described by SEQ ID NO: 8096, comprises a nucleic acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88 %, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, for example, 100% identical to the polynucleotide selected from the group consisting of in SEQ ID NOs: 5470, 5476 and 5481.
[00211] According to some applications of the present invention, the method for increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress, and / or efficiency in using nitrogen from a plant, comes into effect by expressing within the plant a nucleic acid construct comprising the nucleic acid sequence described in SEQ ID NO: 8096 and a heterologous polynucleotide sequence comprising an acid sequence nucleic acid at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, for example, 100% identical to SEQ ID NOs: 1487, 814-1598, 1600-1603, 1605-1626, 1632-1642, 1645-4851, 1599, 1604 , 1628, 1630, and 1644, characterized by the fact that the nucleic acid sequence is able to regulate the expression of the heterologous polynucleotide in a host cell.
[00212] According to some applications of the present invention, the method for increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress , and / or efficiency in using nitrogen from a plant, comes into effect by expressing within the plant a nucleic acid construct that comprises the nucleic acid sequence described in SEQ ID NO: 8096 and a heterologous polynucleotide sequence that encodes a sequence of nucleic acid at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, for example, 100% homologous to SEQ ID NOs: 488813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5553, 55588091, 5454-5459, 5462 -5469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5551, 5552, 5554-5556 or 5557, characterized by the fact that the nucleic acid sequence is able to regulate the expression of the heterologous polynucleotide in a host cell .
[00213] According to some applications of the present invention, the method for increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress , and / or efficiency in using nitrogen from a plant, comes into effect by expressing within the plant a nucleic acid construct comprising the nucleic acid sequence described in SEQ ID NO: 8096 and a heterologous polynucleotide sequence comprising a sequence of nucleic acid at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97% , at least about 98%, at least about 99%, for example, 100% identical to SEQ ID NOs: 1627, 1629 or 1631, characterized by the fact that the nucleic acid sequence is able to regulate the expression of the polynucleotide heterologous in a host cell.
[00214] According to some applications of the present invention, the method for increasing yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, tolerance to abiotic stress , and / or efficiency in using nitrogen from a plant, comes into effect by expressing within the plant a nucleic acid construct that comprises the nucleic acid sequence described in SEQ ID NO: 8096 and a heterologous polynucleotide sequence that encodes a sequence of nucleic acid at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, for example, 100% homologous to SEQ ID NOs: 5470, 5476 or 5481, characterized by the fact that the nucleic acid sequence is able to regulate the expression of the heterologous polynucleotide in a host cell.
[00215] The nucleic acid construct of some applications of the invention may further include a suitable selectable marker and / or an origin of replication. According to some applications of the invention, the nucleic acid construct used is a bridge vector, which can propagate both in E. coli (where the construct comprises a suitable selectable marker and an origin of replication) and can be compatible with propagation in cells. The construction according to the present invention for being, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
[00216] The nucleic acid construct of some applications of the invention can be used to transform plant cells in a stable or temporary manner. In stable transformation, the exogenous polynucleotide is integrated into the plant's genome and thus represents a stable and inherited characteristic. In the temporary transformation, the exogenous polynucleotide is expressed by the transformed cell, however it is not integrated into the genome and, in this way. represents a temporary feature.
[00217] There are several methods of introducing foreign genes to monocot and dicot plants (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42: 205-225; Shimamoto et al., Nature (1989) 338: 274-276).
[00218] The basic methods for achieving the stable integration of exogenous DNA into the plant's genomic DNA include two main approaches: (i) Agrobacterial-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38: 467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L.K, Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S, and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93- 112.
[00219] 00 Direct DNA uptake: Paszkowski et al., In Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct DNA absorption in protoplasts, Toriyama, K. et al. (1988) Bio / Technology 6: 1072-1074. DNA uptake induced by rapid electric shock from plant cells: Zhang et al. Plant Cell Rep. (1988) 7: 379-384.
[00220] Fromm et al. Nature (1986) 319: 791-793. Injection of DNA into plant cells or tissues by particle bombardment, Klein et al. Bio / Technology (1988) 6: 559563; McCabe et al. Bio / Technology (1988) 6: 923-926; Sanford, Physiol. Plant. (1990) 79: 206-209; for the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75: 30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79: 213-217; transformation of fiberglass or silicon carbide from cell cultures, embryos or callus tissue, United States Patent No. 5,464,765 or by direct DNA incubation with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83: 715-719.
[00221] The Agrobacterium system includes the use of plasmid vectors that contain defined segments of DNA that integrate into the plant's genomic DNA. The methods of inoculating plant tissue vary depending on the species of plant and the release system of the agrobacterium. A widely used approach is the leaf disc procedure that can be performed with any tissue explant that offers a good source for beginning all plant differentiation. See, for example, Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A complementary approach employs the agrobacterium release system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
[00222] There are several methods of direct DNA transfer in plant cells. In electroporation, protoplasts are rapidly exposed to a strong electric field. In microinjection, DNA is mechanically injected directly into cells using very small micropipettes. In the bombardment of microparticles, DNA is adsorbed onto microprojectiles, for example, magnesium sulfate crystals or tungsten particles, and microprojectiles are physically accelerated in plant cells or tissues.
[00223] After the stable transformation, the plant is propagated. The most common method of plant propagation is by seed. Seed propagation regeneration, however, has a deficiency. Due to heterozygosity, there is an absence of uniformity in cultivation, since the seeds are produced by plants according to the genetic variances governed by Mendel's laws. Basically, each seed is genetically different and each seed will grow with its own specific characteristics. Therefore, it is preferred that the transformed plant is produced in such a way that the regenerated plant has the identical characteristics and the characteristics of the precursor transgenic plant. Thus, it is preferable that the transformed plant is regenerated by micropropagation, which generates rapid and consistent reproduction of the transformed plants.
[00224] Micropropagation is a process of growing new generation plants from a single piece of tissue that has been taken from a selected precursor plant or cultivar. This process allows for the mass reproduction of plants having the preferred tissue that expresses the fusion protein. The new generation plants that are produced are genetically identical and have all the characteristics of the original plant. Micropropagation allows for the mass production of quality plant material in a short period of time and offers a rapid multiplication of cultivars selected to preserve the characteristics of the original transgenic or transformed plant. The advantages of plant cloning are the speed of plant multiplication and the quality and uniformity of the plants produced.
[00225] Micropropagation is a multi-stage procedure that requires changing the culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culture; stage two, multiplication of tissue culture; stage three, differentiation and formation of the plant; and stage four, greenhouse culture and hardening. During stage one, the initial tissue culture, the tissue culture is defined and certified as free from contaminants. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production objectives. During stage three, the tissue samples grown in stage two are divided and grown on small individual plants. In stage four, the small individual transformed plants are transferred to a greenhouse for hardening where the plants' tolerance to light gradually increases, so that it can be grown in the natural environment.
[00226] According to some applications of the invention, transgenic plants are generated by temporary transformation of leaf cells, meristematic cells or the entire plant.
[00227] Temporary transformation can be performed by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.
[00228] Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, tobacco mosaic virus (TMV), bromine mosaic virus (BMV) and common bean mosaic virus (BV or BCMV). Plant transformation using plant viruses described in U.S. Pat. United States No. 4,855,237 (golden bean mosaic virus; BGV), EP-A 67,553 (TMV), Published Japanese Application No. 6314693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV) and Gluzman, Y et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in the expression of foreign DNA in many hosts, including plants, are described in WO 87/06261.
[00229] According to some applications of the invention, the virus used for temporary transformations is non-virulent and thus unable to cause severe symptoms, such as lower growth rate, mosaic, ring stains, leaf curl, yellowing, appearance of streaks, formation of rashes, formation of tumor and formation of holes. A suitable non-virulent virus can be a naturally occurring non-virulent virus or an artificially attenuated virus. The attenuation of the virus can be performed using methods well known in the art including, among others, sublethal heating, chemical treatment or by targeted mutagenic techniques such as those described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4: 259269, 2003), Galon et al. (1992), Atreya et al. (1992) and Huet et al. (1994).
[00230] Suitable strains of viruses can be obtained from available sources, for example, from the American Type Culture Collection (ATCC) or by isolating infected plants. Virus isolation from infected plant tissues can be performed by methods well known in the art, for example, as described by Foster and Tatlor, Eds. "Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998. In short, the tissues of an infected plant believed to have a high concentration of a Suitable viruses, preferably young leaves and flower petals, are based on a buffer solution (eg, phosphate buffered solution) to generate an infected virus sap that can be used in future inoculations.
[00231] The construction of plant RNA viruses for the introduction and expression of exogenous non-viral polynucleotide sequences in plants is demonstrated by the references above, as well as by Dawson, WO et al., Virology (1989) 172: 285-292 ; Takamatsu et al. EMBO J. (1987) 6: 307-311; French et al. Science (1986) 231: 12941297; Takamatsu et al. FEBS Letters (1990) 269: 73-76; and United States Patent 5,316,931.
[00232] When the virus is a DNA virus, appropriate modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid to facilitate the construction of the desired viral vector with the foreign DNA. The virus can then be extracted from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. The transcription and translation of this DNA will produce the coat protein that will encapsulate the viral DNA. If the virus is an RNA virus, the virus is usually cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all the constructions. The RNA virus is then produced by transcribing the plasmid viral sequence and translating the viral genes to produce the coating protein (s) that encapsid the viral RNA.
[00233] In one configuration, a plant viral polynucleotide is provided in which the native coat protein coding sequence has been deleted from a viral polynucleotide, and a non-native plant viral coat protein coding sequence has been inserted and a non-native promoter, preferably the subgenomic promoter of the non-native coating protein coding sequence, capable of carrying out expression in the plant host, packaging the recombinant plant viral polynucleotide, and ensuring a systemic infection of the host by the viral polynucleotide of recombinant plant. Alternatively, the coat protein gene can be inactivated by inserting the non-native polynucleotide sequence into it, so that a protein is produced. The recombinant plant viral polynucleotide may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or polynucleotide sequences in the plant host and unable to recombine with each other and with native subgenomic promoters. Non-native (foreign) polynucleotide sequences can be inserted adjacent to the plant's native viral subgenomic promoter or to the plant's native and non-native viral subgenomic promoters if more than one polynucleotide sequence is included. The sequences of non-native polynucleotides are transcribed or expressed in the host plant under the control of the subgenomic promoter to generate the desired products.
[00234] In a second configuration, a recombinant plant viral polynucleotide is provided as in the first configuration, except that the native coat protein coding sequence is placed adjacent to one of the subgenomic promoters of non-native coat protein in instead of a non-native coating protein coding sequence.
[00235] In a third configuration, a recombinant plant viral polynucleotide is provided in which the native coat protein gene is adjacent to its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral polynucleotide. The inserted non-native subgenomic promoters are able to transcribe or express adjacent genes in a plant host and are unable to recombine with each other and with native subgenomic promoters. Non-native polynucleotide sequences can be inserted adjacent to non-native viral plant subgenomic promoters, so that the sequences are transcribed or expressed in the host plant under the control of the subgenomic promoters to generate the desired product.
[00236] In a fourth configuration, a recombinant plant viral polynucleotide is provided as in the third configuration, except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.
[00237] Viral vectors are encapsulated by the coatings protein encoded by the recombinant plant viral polynucleotide to produce a recombinant plant virus. The recombinant plant viral polynucleotide or recombinant plant virus is used to infect suitable host plants. The recombinant plant viral polynucleotide is capable of replication in the host, systemic diffusion in the host and the transcription or expression of foreign gene (s) (exogenous polynucleotide) in the host to produce the desired protein.
[00238] Techniques for inoculating viruses in plants can be found in Foster and Taylor, eds. "Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998; Maramorosh and Koprowski, eds. "Methods in Virology" 7 vols, Academic Press, New York 1967-1984; Hill, S.A. "Methods in Plant Virology", Blackwell, Oxford, 1984; Walkey, D.G.A. "Applied Plant Virology", Wiley, New York, 1985; and Kado and Agrawa, eds. "Principies and Techniques in Plant Virology", Van Nostrand-Reinhold, New York.
[00239] In addition to the above, the polynucleotide of the present invention can also be introduced into a chloroplast genome, thus allowing the expression of the chloroplast.
[00240] A technique for introducing exogenous polynucleotide sequences into the chloroplast genome is known. This technique involves the following procedures. First, plant cells are chemically treated to reduce the number of chloroplasts per cell to approximately one. Then, the exogenous polynucleotide is introduced by bombarding particles in the cells with the aim of introducing at least one exogenous polynucleotide molecule into the chloroplasts. The exogenous polynucleotides are selected so that they can be integrated into the chloroplast genome via homologous recombination, which is readily made by the enzymes inherent to the chloroplast. For this purpose, the exogenous polynucleotide includes, in addition to a gene of interest, at least one stretch of polynucleotide that is derived from the chloroplast genome. In addition, the exogenous polynucleotide includes a selectable marker, which serves, through sequential selection procedures, to determine that all or substantially all copies of the chloroplast genomes after this selection will include the exogenous polynucleotide. More details related to this technique are found in Pat. United States Nos. 4,945,050 and 5,693,507, which are hereby incorporated by reference. A polypeptide can then be produced by the chloroplast protein expression system and integrate with the internal chloroplast membrane.
[00241] Since the processes that increase the yield, the growth rate, the biomass, the vigor, the efficiency in the use of nitrogen and / or the tolerance to the abiotic stress of a plant can involve multiple genes that act additively or in synergy (see, for example, Quesda et al., Plant Physiol. 130: 951-063, 2002), the present invention also refers to the expression of several exogenous polynucleotides in a single host plant to achieve a superior effect on the oil content, yield, growth rate, biomass, vigor, nitrogen use efficiency and / or tolerance to abiotic stress.
[00242] The expression of a plurality of exogenous polynucleotides in a single host plant can be accomplished by co-introducing multiple nucleic acid constructs, each including a different exogenous polynucleotide, into a single plant cell. The transformed cell can then be regenerated into a mature plant, using the methods described above.
[00243] Alternatively, the expression of several exogenous polynucleotides in a single host plant can be carried out by cointroduction, in a single plant cell of a single nucleic acid construct, including several different exogenous polynucleotides. This construct can be designed with a single promoter sequence that can transcribe a polycistronic messenger RNA that includes all the different exogenous polynucleotide sequences. To allow the co-translation of the different polypeptides encoded by the polycistronic messenger RNA, the polynucleotide sequences can be interconnected by an internal ribosome entry site sequence (IRES) that facilitates the translation of polynucleotide sequences positioned below the IRES sequence. In this case, a transcribed polycistronic RNA molecule that encodes the different polypeptides described above will be translated both from the 5 'closed end and from the two internal IRES sequences of the polycistronic RNA molecule to produce different polypeptides in the cell. Alternatively, the construct can include several promoter sequences, each linked to a different exogenous polynucleotide sequence.
[00244] The plant cell transformed with the construct including several different exogenous polynucleotides can be regenerated in a mature plant, using the methods described above.
[00245] Alternatively, the expression of several exogenous polynucleotides in a single host plant can be done by introducing different nucleic acid constructs, including different exogenous polynucleotides, in different plants. The regenerated transformed plants can then be crossed and result in the selected generation to present superior characteristics of tolerance to abiotic stress, efficiency in the use of water, efficiency in the use of fertilizer, growth, biomass, yield, oil content and / or in force, using conventional plant generation techniques.
[00246] According to some applications of the invention, the method further comprises the growth of the plant that expresses the exogenous polynucleotide under abiotic stress.
[00247] Non-limiting examples of abiotic stress conditions include salinity, drought, water deprivation, excess water (eg flood, flooding), etiolation, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, excess of nutrients, atmospheric pollution and UV irradiation.
[00248] In accordance with an aspect of some applications of the present invention, a method of expressing a polypeptide of interest in a cell is provided, the method takes effect with transforming the cell with a nucleic acid construct comprising a sequence of polynucleotides which encodes the polypeptide of interest operably connected to the isolated polynucleotide described by SEQ ID NO: 8096, thereby expressing the polypeptide of interest in the cell.
[00249] According to some applications of the invention, the polypeptide of interest comprises an amino acid sequence of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, say 100% homologous to the polypeptide selected from the group consisting of SEQ ID NO: 488813, 4852-5453, 5460, 5461, 5484 , 5486-5550, 5553, 5558-8091, 5454-5459, 5462-5469, 5471-5475, 5477-5480, 5482, 5483, 5485, 5551,5552, and 55545557.
[00250] According to some applications of the invention, the polypeptide of interest comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 488-813, 4852-5453, 5460, 5461, 5484, 5486-5550, 5553, 5558-8091, 5454-5459, 5462-5469, 54715475, 5477-5480, 5482, 5483, 5485, 5551, 5552, and 5554-5557.
[00251] According to some applications of the invention, the polypeptide of interest comprises an amino acid sequence of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, say 100% homologous to the polypeptide selected from the group consisting of SEQ ID NO: 5470, 5476 and 5481.
[00252] According to some applications of the invention, the polypeptide of interest comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 5470, 5476 and 5481.
[00253] According to some applications of the invention, the polynucleotide encoding the polypeptide of interest comprises an amino acid sequence of at least about 80%, at least about 81%, at least about 82%, at least about 83% at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least at least about 97%, at least about 98%, at least about 99%, or more, say 100% identical to the polypeptide selected from the group consisting of SEQ ID NO: 1487, 814-1598, 1600- 1603, 1605-1626, 1632-1642, 1645-4851, 1599, 1604, 1628, 1630, and 1644.
[00254] According to some applications of the invention, the polynucleotide encoding the polypeptide of interest comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-487, 814-1598, 1600-1603, 1605-1626 , 16321642, 1645-4851, 1599, 1604, 1628, 1630, and 1644.
[00255] According to some applications of the invention, the polynucleotide encoding the polypeptide of interest comprises an amino acid sequence of at least about 80%, at least about 81%, at least about 82%, at least about 83% at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least at least about 97%, at least about 98%, at least about 99%, or more, say 100% identical to the polypeptide selected from the group consisting of SEQ ID NO: 1627, 1629 and 1631.
[00256] According to some applications of the invention, the polynucleotide encoding the polypeptide of interest comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 1627, 1629 and 1631.
[00257] Thus, the invention covers transgenic cells (eg, transgenic plant cells), plants exogenously expressing the polynucleotide (s) (eg, transgenic plants), the nucleic acid and / or polypeptide constructs (s) of the invention, and methods for generating or producing the same. Once expressed within the plant cell or the entire plant, the level of the polypeptide encoded by the exogenous polynucleotide can be determined by methods well known in the art such as activity assays, Western blots using antibodies capable of specifically binding the polypeptide, Immunosorbent Assay Linked to Enzyme (ELISA), radioimmunological assays (RIA), immunohistochemistry, immunocytochemistry, immunofluorescence and the like.
[00258] Methods for determining, in the plant, the level of RNA transcribed from the exogenous polynucleotide are well known in the art and include, for example, Northern blot analysis, polymerase chain reaction analysis via reverse transcription (RT-PCR ) (including quantitative, semi-quantitative or real-time RT-PCR) and RNA hybridization in situ.
[00259] The sequence information and annotations revealed by the present teachings can be used in favor of classical procreation. Thus, subsequence data for the polynucleotides described above can be used as markers for marker-assisted selection (MAS), in which a marker is used for the indirect selection of a determinant or genetic determinants of a trait of interest (for example, biomass , growth rate, oil content, yield, tolerance to abiotic stress, efficiency in the use of water, efficiency in the use of nitrogen and / or efficiency in the use of fertilizer). The nucleic acid data of the present teachings (DNA or RNA sequence) can contain or be linked to polymorphic sites or genetic markers in the genome, such as restriction fragment length polymorphism (RFLP), microsatellites and single nucleotide polymorphism (E.MP), DNA fingerprint (DFP), amplified fragment length polymorphism (AFLP), expression level polymorphism, encoded polypeptide polymorphism and any other polymorphism in the DNA or RNA sequence.
[00260] Examples of marker-assisted selections include, among others, the selection of a morphological characteristic (for example, a gene that affects the shape, color, male sterility or resistance, such as the presence or absence of edge, color of the leaf sheath, height, grain color, rice aroma); selection of a biochemical characteristic (for example, a gene that encodes a protein that can be extracted and observed; for example, isozymes and storage proteins); selection of a biological trait (for example, pathogenic breeds or insect biotypes based on the interaction of the host pathogen or host parasite, can be used as a marker, since the genetic makeup of an organism can affect its susceptibility to pathogens or parasites).
[00261] The polynucleotides and polypeptides described above can be used in a wide range of economic plants, safely and inexpensively.
[00262] The plant lines that exogenously express the polynucleotide or polypeptide of the invention are selected to identify those that demonstrate the greatest increase in the desired characteristic of the plant.
[00263] The effect of the transgene (the exogenous polynucleotide that encodes the polypeptide) on the tolerance of abiotic stress can be determined using known methods, such as those detailed below in the Example section below.
[00264] Abiotic stress tolerance - Transformed (that is, expressing the transgene) and untransformed (wild type) plants are exposed to an abiotic stress condition, such as lack of water, sub-ideal temperature (low and high temperature) , nutrient deficiency, excess nutrient, salt stress condition, osmotic stress, heavy metal toxicity, anaerobiosis, air pollution and UV irradiation.
[00265] Salinity tolerance test - Transgenic plants with tolerance to very high concentrations of salt may exhibit better germination, vigor or seedling growth with high salinity. Salt stress can be carried out in many ways, such as, for example, irrigating plants with a hyperosmotic solution, growing the plants hydroponically in a hyperosmotic culture solution (eg Hoaglan's solution) or growing the plants in a hyperosmotic culture (eg 50% Murashige-Skoog medium [MS medium]). Since different plants vary considerably in terms of their tolerance to salinity, the concentration of salt in the irrigation water, in the growth solution or in the growth medium can be adjusted according to the specific characteristics of the cultivar or the specific plant variety , in order to impose a mild or moderate effect on plant physiology and / or morphology (for guidelines on appropriate concentration, see Bernstein and Kafkafi, Root Growth Under Salinity Stress In: Plant Roots, The Hidden Half 3rd ed. Waisel Y, Eshel A and Kafkafi U. (editors) Marcel Dekker Inc., New York, 2002, and their references).
[00266] For example, a salinity tolerance test can be performed by irrigating plants at different stages of development with increasing concentrations of sodium chloride (eg 50 mM NaC1, 100 mM, 200 mM, 400 mM) applied from below and from above to ensure a uniform dispersion of salt. After exposure to the stress condition, plants are often monitored until substantial physiological and / or morphological effects appear on wild type plants. Thus, the external phenotypic appearance, degree to which the plant withered and the general success in reaching maturity and result are compared between control and transgenic plants.
[00267] The quantitative parameters of the measured tolerance include, among others, the average wet and dry weight, the growth rate, the leaf size, the leaf cover (general area of the leaf), the weight of the resulting seeds, the size average seed and the number of seeds produced per plant. Transformed plants that do not have substantial physiological and / or morphological effects, or that have greater biomass than wild type plants, are identified as plants tolerant to abiotic stress.
[00268] Asthmatic tolerance test - Osmotic stress tests (including sodium chloride and mannitol tests) are performed to determine whether an osmotic phenotype was specific to sodium chloride or whether it was a phenotype related to general osmotic stress. Plants that are tolerant to osmotic stress may be more tolerant of drought and / or freezing. For salt germination and osmotic stress experiments, the medium is complemented, for example, with 50 mM, 100 mM, 200 mM NaC1 or with 100 mM, 200 mM NaC1 and 400 mM mannitol.
[00269] Drought tolerance test / osmotic test - Drought tolerance is done to identify the genes generating better plant survival after acute water shortage. To analyze whether transgenic plants are more tolerant to drought, an osmotic stress generated by the non-phonic osmolyte sorbitol in the medium can be performed. Control and transgenic plants are germinated and grown on plant agar plates for 4 days, after which they are transferred to plates containing 500mM sorbitol. The treatment causes growth retardation, so the control and transgenic plants are compared, measuring the weight of the plant (wet and dry), the yield and the growth rates measured at the time of flowering.
[00270] Instead, soil-based drought selections are made with plants overexpressing the polynucleotides detailed above. The seeds of Arabidopsis control plants or other transgenic plants that overexpress the polypeptide of the invention are germinated and transferred to pots. Stress to drought is obtained after irrigation ceases, followed by placing the pots on absorbent paper to increase the drying rate of the soil. Transgenic and control plants are compared to each other when most control plants develop severe withering. The plants are watered again after obtaining a significant fraction of the control plants that present severe withering. Plants are classified by comparing controls to each of the two criteria: tolerance to drought conditions and recovery (survival) after rehydration.
[00271] Cold stress tolerance - To analyze cold stress, mature plants (25 days old) are transferred to 4 ° C chambers for 1 or 2 weeks, with elementary light. Subsequently, the plants are returned to the greenhouse. Two weeks later, the damage from the cold period, resulting in growth retardation and other phenotypes, is compared between control and transgenic plants, measuring the weight of the plant (wet and dry) and comparing the growth rates measured at the time of flowering, plant size, yield, among others.
[00272] Tolerance to heat stress - Tolerance to heat stress is achieved by exposing plants to temperatures above 34 ° C for a specified period. The tolerance of the plant is analyzed after the transfer of the plants back to the condition of 22 ° C for recovery and evaluation after 5 days in relation to the internal controls (non-transgenic plants) or plants not exposed to stress by cold or heat.
[00273] Water use efficiency - can be determined as the biomass generated by transpiration unit. To analyze the WUE, the relative water content of the leaf can be measured in transgenic and control plants. The fresh weight (FW) is recorded immediately; then the leaves are soaked for 8 hours in distilled water at room temperature in the dark, and the turgid weight (TW) is recorded. The total dry weight (PS) is recorded after drying the leaves at 6000 in constant weight. The relative water content (TRA) is calculated according to the following formula I: Formula I TRA = [(PF-PS) / (PT-PS) 1x100
[00274] Efficiency of using fertilizer - To analyze whether transgenic plants are more responsive to fertilizers, plants are grown on agar plates or pots with a limited amount of fertilizer, for example, in Yanagisawa et al (Proc Natl Acad Sci USA 2004; 101: 7833-8). The plants are analyzed for their general size, flowering time, yield, protein content of the branch and / or grain. The verified parameters are the total size of the mature plant, its moisture and dry weight, the weight of the seeds produced, the average seed size and the number of seeds produced per plant. Other parameters that can be tested are: the chlorophyll content of the leaves (since the nitrogen status and the degree of greenness of the leaf are correlated), amino acid content and total protein of the seeds or other parts of the plants, such as leaves or branches, oil content, etc. Similarly, instead of supplying nitrogen in limited quantities, phosphate or potassium can be added in high concentrations. Again, the same measured parameters are the same as those listed above. In this way, the efficiency of the use of nitrogen (NUE), the efficiency of the use of phosphate (PUE) and the efficiency of the use of potassium (KUE) are evaluated, verifying the capacity of transgenic plants to flourish under restricted nutrient conditions.
[00275] Nitrogen use efficiency - To check whether Arabidopsis transgenic plants are more responsive to nitrogen, the plants are grown at 0.75-1.5 mM (nitrogen deficient conditions) or 6-10 mM (ideal concentration nitrogen). Plants are allowed to grow for an additional 25 or even seed production. The plants are then analyzed for their total size, flowering time, yield, sprout protein content and / or grain / seed production. The verified parameters can be the total size of the plant, humidity and dry weight, the weight of the seeds produced, the average size of the seed and the number of seeds produced per plant. Other parameters that can be tested are: the chlorophyll content of the leaves (such as the nitrogen status of the plant and the degree of greenness of the leaf are highly correlated), amino acid and the total protein content of the seeds or other parts of the plant such such as leaves or buds and the oil content. Transformed plants have no substantial physiological and / or morphological effects, or have higher levels of measured parameters than wild plants, are identified as nitrogen-efficient plants
[00276] Efficiency study of the use of nitrogen using plants - The study is done according to Yanagisawa-S. et al. with minor modifications ("Metabolic engineering with Dofl transcription factor in plants: Improved nitrogen assimilation and growth under low nitrogen conditions" Proc. Natl. Acad. Sci. USA 101, 7833-7838). Briefly, transgenic plants that are grown for 7 to 10 days in 0.5 x MS [Murashige-Skoog] supplemented with a selection agent, are transferred to two nitrogen limiting conditions: The average MS in which the combined concentration of nitrogen (NH4NO3 and KNO3) was 0.75 mM or 0.05 mM. The plants are allowed to grow for another 30-40 days and then be photographed, removed individually from the Agar (the sprout without the root) and weighed immediately (fresh weight) for further statistical analysis. Constructions for which only T1 seeds are available are sown on selective media and at least 20 seedlings (each representing an independent transformation event) are carefully transferred to the nitrogen limiting site. For constructions for which T2 seeds are available, different transformation events are analyzed. Typically, 20 plants selected at random from each event are transferred to the site with nitrogen limitations, allowed to grow another 3-4 weeks and be weighed at the end of that period. Transgenic plants are compared to control plants grown in parallel under the same conditions. Transgenic plants expressing the uidA reporter gene (GUS) under the same promoter or transgenic plants containing only the same promoter, but without any reporter gene used as a control.
[00277] Nitrogen determination - The procedure for determining the concentration of N (nitrogen) in the structural parts of plants involves the method of digestion with potassium persulfate to convert organic N into NO3 (Purcell and King 1996 Argon. J. 88: 111113 , the modified Cd mediated reduction of NO3 to NO2 (Vodovotz 1996 Biotechniques 20: 390-394) and the measurement of nitrite by the Griess test (Vodovotz 1996, supra) .The absorbance values are measured at 550nm compared to a standard curve The procedure is described in detail in Samonte et al. 2006 Agron. J. 98: 168-176.
[00278] Germination test - Germination tests compare the percentage of seeds from transgenic plants that could complete the germination process with the percentage of seeds from control plants that are treated in the same way. Normal conditions are considered, for example, incubations at 22 ° C under daily cycles of 22 hours of light and 2 hours of darkness. The evaluation of germination and seedling vigor is carried out between 4 and 14 days after planting. The basal medium is 50% MS medium (Murashige and Skoog, 1962 Plant Physiology 15, 473-497).
[00279] Germination is also verified in unfavorable conditions, such as cold (incubating with temperatures below 10 ° C instead of 22 ° C) or using seed inhibiting solutions that contain high concentrations of an osmolyte, such as sorbitol (in concentrations 50 mM, 100 mM, 200 mM, 300 mM, 500 mM and up to 1000 mM) or by applying increasing concentrations of salt (from 50 mM, 100 mM, 200 mM, 300 mM, 500 mM NaCl)
[00280] The effect of the transgene on vigor, growth rate, biomass, yield and / or oil content can be determined using known methods.
[00281] Vigor of the plant - The vigor of the plant can be calculated by increasing the growth parameters, such as the leaf area, the length of the fiber, the diameter of the rosette, the fresh weight of the plant and the like per unit of time.
[00282] Growth rate - The growth rate can be measured using digital analysis of plants in cultivation. For example, images of plants grown in a greenhouse on a bed base can be captured every 3 days and the area of the rosette can be calculated by digital analysis. The growth of the rosette area is calculated using the difference in rosette area between the sampling days divided by the difference in days between the samples.
[00283] The evaluation or growth rate can be made by measuring the biomass of the plant produced, rosette area, leaf size or root length by time (can be measured in cm per day of the leaf area).
[00284] The area of relative growth can be calculated using Formula II. Formula II: Area of relative growth = Area regression coefficient over time
[00285] Thus, the rate of the relative growth area is in units of 1 / day and the growth rate by length is in units of 1 / day.
[00286] Seed yield - The evaluation of seed yield per plant can be done by measuring the quantity (weight or size) or quantity (that is, numerical) of dry seeds produced and harvested from 8 to 16 plants and divided by number of plants.
[00287] For example, the total of seeds from 8 to 16 plants can be collected, weighed using, for example, an analytical balance and the total weight can be divided by the number of plants. Seed yield per cultivation area can be calculated in the same way while taking into account the cultivation area assigned to a single plant. The increase in seed yield per cultivation area could be obtained by increasing the seed yield per plant and / or increasing the number of plants capable of growing in a given area.
[00288] In addition, the seed yield can be determined by the weight of 1000 seeds. The weight of 1000 seeds can be determined as follows: the seeds are spread on a glass tray and photographed. Each sample is weighed and then, using digital analysis, the number of seeds in each sample is calculated.
[00289] The weight of 1000 seeds can be calculated using formula II: Formula III: Weight of 1000 seeds = number of seeds in the sample / weight of the sample X 1000
[00290] The Harvest Index can be calculated using Formula IV Formula IV: Harvest Index = Average seed yield per plant / Average dry weight
[00291] Protein concentration of the grain - The protein content of the grain (g protein of the grain m-2) is estimated as the product of the mass of the grain N (g grain N m-2) multiplied by the ratio of conversion of N / protein of k-5.13 (Musgoe 1990, supra). The protein concentration of the grain is estimated as the ratio of the protein content of the grain per unit / mass of the grain (g grain protein kg-1 grain).
[00292] Fiber length - The fiber length can be measured by fibrography. The fibrography system was used to compute the length in terms of "Upper Half Average" length. The average length of the upper half (UHM) is the average length of the longest half of the fiber distribution. Fibrography measures the length in wide lengths of a certain percentage point (Hypertext Transfer Protocol: // World Wide Web (dot) cottoninc (dot) com / ClassificationofCotton / Pg = 4 # Length).
[00293] According to some applications of the invention, the increased yield of corn can be manifested as one or more of the following: Increase in the number of plants per cultivation area, increase in the number of ears per plant, increase in the number of rows per ear, number of heartwood per row, heartwood weight, thousand heartwood weight (weight-1000), ear length / diameter, increase in oil content by heartwood and increase in starch content by heartwood.
[00294] As mentioned, the increase in plant yield can be determined by several parameters. For example, the increased yield of rice can be manifested by an increase in one or more of the following: the number of plants per cultivation area, number of panicles per plant, number of spikelets per panicle, number of flowers per panicle, increase in seed filling rate, increase in the weight of a thousand heartwoods (weight-1000), increase in the oil content per seed, increase in the starch content per seed, among others. An increase in throughput can also result in modified architecture, or it can occur because of a modified architecture.
[00295] Similarly, the increased soybean yield can be manifested by an increase in one or more of the following: number of plants per cultivation area, number of pods per plant, number of seeds per pod, increase in the seed filling rate , increase in the weight of a thousand heartwoods (weight-1000), reduction in the breaking of pods, increase in the oil content per seed, increase in the protein content per seed, among others. An increase in throughput can also result in modified architecture, or it can occur because of a modified architecture.
[00296] The increased rapeseed yield can be manifested by an increase in one or more of the following: number of plants per cultivation area, number of pods per plant, number of seeds per pod, increase in the seed filling rate, increase in the weight of a thousand heartwoods (weight-1000), reduction in pod breakage, increase in oil content per seed, among others. An increase in throughput can also result in modified architecture, or it can occur because of a modified architecture.
[00297] The increased yield of cotton can be manifested by an increase in one or more of the following: number of plants per cultivation area, number of capsules per plant, number of seeds per capsule, increase in the seed filling rate, increase in the weight of a thousand heartwoods (weight-1000), increase in the oil content per seed, improvement in the fiber length, fiber strength, among others. An increase in throughput can also result in modified architecture, or it can occur because of a modified architecture.
[00298] Oil content - The oil content of a plant can be determined by extracting the oil from the seed or the vegetative part of the plant. Briefly, lipids (oil) can be extracted from the plant (for example, the seed) by grinding the plant tissue in the presence of specific solvents (for example, hexane or petroleum ether) and extracting the oil in a continuous extractor. The indirect analysis of the oil content can be performed using several known methods, such as Nuclear Magnetic Resonance Spectroscopy (NMR), which measures the resonance energy absorbed by the hydrogen atoms in the liquid state of the sample [See, for example, Conway TF. and Earle FR., 1963, Journal of the American Oil Chemists' Society; Springer Berlin / Heidelberg, ISSN: 0003-021X (Print) 1558-9331 (Online)]; by Near Infrared Spectroscopy (NI), which uses the absorption of energy almost in the infrared (1100-2500 nm) by the sample; and a method described in W0 / 2001/023884, which is based on the extraction of oil solvent, evaporation of the solvent in a gas stream that forms oil particles, and directing a light in the gas and oil particle stream that form a detectable reflected light.
[00299] Thus, this invention is of great value to agriculture as it promotes the yield of commercially desired crops (eg, biomass of the vegetative organ such as poplar wood or reproductive organ, such as number of seeds or seed biomass).
[00300] Any of the transgenic plants as described above or parts of these can be processed to produce a food, meal, protein or oil preparation, for example, for ruminant animals.
[00301] The transgenic plants described above, which exhibit an increased oil content can be used to produce vegetable oil (by extracting oil from the plant).
[00302] Herbal oil (including seed oil and / or oil from the vegetative part) produced according to the method of the invention, can be combined with a variety of other components. The specific components included in a product are determined according to the intended use. Examples of products include animal feed, raw material for chemical modification, biodegradable plastic, mixed food product, edible oil, biofuel, cooking oil, lubricant, biodiesel, snacks, cosmetics, and raw material for the fermentation process. Examples of products to be incorporated into plant-based oil include animal feed, human foods such as extruded snacks, breads, as a food binding agent, aquaculture feed, fermentable mixtures, food supplements, sports drinks, nutritional bars, multivitamin supplements, diet drinks and cereals. According to some applications of the invention, the oil comprises a seed oil.
[00303] According to some applications of the invention, the oil comprises a vegetative part oil.
[00304] According to some applications of the invention, the plant cell is part of a plant.
[00305] As used herein, the term "about" refers to ± 10%.
[00306] The terms "involves", "involving", "includes", "including", "have" and their conjugations mean "including, but not limited to".
[00307] The term "consisting of" means "including and limited to".
[00308] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or part do not materially alter the basic and new characteristics of the claimed composition, method or structure.
[00309] As used here, the singular form "one", "one" and "o / a" includes plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures of them.
[00310] Throughout this request, several applications of this invention may be present in a variety of formats. It should be understood that the description in varying format is simply for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Consequently, it should be considered that the description of a variation must have specifically revealed all possible subvariations, as well as the individual numerical values within that variation. For example, it should be considered that the description of a variation such as 1 to 6 has specifically revealed subvariations such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the range of the range.
[00311] Whenever a numerical variation is indicated here, it means that it includes any numeral mentioned (fractional or integral) within the indicated variation. The phrases "varying / varies between" a first indicated number and a second indicated number and "varying / varies from" a first indicated number "to" a second indicated number are used here interchangeably and are intended to include the first and second number indicated and all fractional and integer numbers between them.
[00312] As used herein, the term "method" refers to ways, means, techniques and procedures for carrying out a given task including, but not limited to, those ways, means, techniques and procedures known or readily developed from ways , means, techniques and procedures by practitioners of chemical, pharmacological, biological, biochemical and medical techniques.
[00313] It is estimated that certain characteristics of the invention, which are, for the purpose of clarification, described in the context of separate applications, can also be presented in combination in a single application. Conversely, several features of the invention, which are, for brevity, described in the context of a single application, can also be presented separately or in any suitable subcombination or suitably in any other described application of the invention. Certain characteristics described in the context of various applications should not be considered essential characteristics of those applications, unless the application is not functional without those elements.
[00314] Various applications and aspects of the present invention, as described above and claimed in the claims section below, find experimental support in the following examples. EXAMPLES
[00315] Now, reference is made to the following examples which, together with the above descriptions, illustrate some applications of the invention in a non-limiting way.
[00316] In general, the nomenclature used here and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are explained carefully in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); compliant methodologies described in United States Patent Numbers 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); the available immunoassays are extensively described in the scientific and patent literature, see, for example, United States Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if they were fully defined herein. Other general references are presented throughout this document. The procedures described are believed to be well known in the art and are provided for the convenience of the reader. All information contained in the literature is incorporated by reference here. GENERAL EXPERIMENTAL AND BIOINFORMATIC METHODS
[00317] RNA extraction - Tissues developing under various development conditions were sampled and RNA was extracted using Invitrogen's TRIzole Reagent [Hypertext Transfer Protocol: // World Wide Web (dot) invitrogen (dot) com / content (dot) cfm pageid = 469]. Approximately 30 to 50 mg of tissue was removed from the samples. The heavy tissues were crushed using a pistil and crucible in liquid nitrogen and resuspended in 500 µl of TRIzol Reagent. To the homogenized lysate, 100 µl of chloroform was added, followed by precipitation using isopropanol and two washes with 75% ethanol. The RNA was eluted in 30 pl of water without RNase. The RNA samples were cleaned using Qiagen's RNeasy minikit cleaning protocol according to the manufacturer's protocol (QIAGEN Incs, CA USA). For convenience, each type of microarray expression information fabric has been given an Expression Set ID.
[00318] Correlation analysis - was performed for the selected genes according to some applications of the invention, in which the characterized parameters (parameters measured according to the correlation IDs) were used as "x-axis" for correlation with the transcription of the fabric that was used as a "Y-axis". For each gene and parameter measured, a correlation coefficient R was calculated [using the Pearson Hypertext Transfer Protocol correlation test: // World Wide Web (dot) davidmlane (dot) com / hyperstat / A34739 (dot) html] together with a p-value for the significance of the correlation. When the correlation coefficient (R) between the expression levels of a gene in a certain tissue and a phenotypic performance along ecotypes / variety / hybrid is high in the absolute value (between 0.5-1), there is an association between the gene (specifically the level of expression of such a gene) and the phenotypic character (eg, better efficiency in the use of nitrogen, tolerance to abiotic stress, yield, growth rate and the like). A positive correlation indicates that gene expression in a certain tissue or stage of development and the correlation vector (phenotype performance) are positively associated (both express and phenotypic performance increase or decrease simultaneously) while a negative correlation indicates a negative association (while one increases, another decreases, and vice versa). Genes whose expression in certain tissues correlates significantly with certain characteristics are shown in Table 26, together with their correlation coefficient (R, calculated using Pearson's correlation) and the p-values under the category of the biodiesel ecotype vector set. . EXAMPLE 1 IDENTIFICATION OF GENES AND PROGNOSTICATED PAPER USING BIOINFORMATICS TOOLS
[00319] The present inventors have identified polynucleotides that can increase plant yield, seed yield, oil yield, oil content, biomass, growth rate, fiber yield and / or its quality, tolerance to abiotic stress, efficiency in the use of nitrogen and / or the vigor of a plant, as follows.
[00320] The nucleotide sequence data sets used here were from publicly available databases or from sequences obtained using Solexa technology (for example, Barley and Sorghum). Sequence data from 100 different plant species was entered into a single, comprehensive database. Other information about gene expression, protein annotation, enzymes and pathways has also been incorporated. The main databases used include: Genomes
[00321] Arabidopsis genome [TAIR genome version 8 (Hypertext Transfe Protocol: // World Wide Web (dot) arabidopsis (dot) org /)]; Rice genome [build 6.0 (Hypertext Transfer Protocol: // http: // rice (dot plantbiology (dot) msu (dot) edu / index.shtml]; Poplar [Populus trichocarpa release 1.1 from JGI (assembly release v1.0) (Hypertext Transfer Protocol: // World Wide Web (dot) genome (dot) jgi-psf (dot) org /)]; Brachypodium [JGI 4x assembly, Hypertext Transfer Protocol: // World Wide Web (dot) brachpodium (dot) org)]; Soy [DOE-JGI SCP, version Glyma0 (Hypertext Transfer Protocol: // World Wide Web (dot) phytozome (dot) net /)]; Grape [French-Italian Public Consortium for Grapevine Genome Characterization grapevine genome (Hypertext Transfer Protocol: // World Wide Web (dot) genoscope (dot) cns (dot) fr /)]; Fava beans [TIGR / J Craig Venter Institute 4x assembly [(Hypertext Transfer Protocol: // msc (dot) jcvi ( dot) org / r communis]; Soy [DOE-JGI SCP, Sbil version [Hypertext Transfer Protocol: // World Wide Web (dot) phytozome (dot) net /)]; Partially assembled corn genome [Hypertext Transfer Protocol: / / maizesequence (dot) or g /]; The EST and mRNA sequences expressed were extracted from the following databases: NCBI EST and RNA sequences (Hypertext Transfer Protocol: // World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov / dbEST / ); RefSeq (Hypertext Transfer Protocol: // World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov / RefSeq /); TAIR (Hypertext Transfer Protocol: // World Wide Web (dot) arabidopsis (dot) org /); Uniprot protein and pathway databases [Hypertext Transfer Protocol: // World Wide Web (dot) uniprot (dot) org /].
[00322] AraCyc [Hypertext Transfer Protocol: // World Wide Web (dot) arabidopsis (dot) org / biocyc / index (dot) jsp].
[00323] ENZYME [Hypertext Transfer Protocol: // expasy (dot) org / enzyme /].
[00324] The micromatrix data sets were downloaded from: GEO (Hypertext Transfer Protocol: // World Wide Web.ncbi.nlm.nih.gov/geo/) TAIR (Hypertext Transfer Protocol: // World Wide Web.arabidopsis. org /).
[00325] Data from proprietary microtrips (See W02008 / 122980) and Examples 2-9 below.
[00326] Gramene QTL and SNPs information [Hypertext Transfer Protocol: // World Wide Web (dot) gramene (dot) org / qt1 /].
[00327] Panzea [Hypertext Transfer Protocol: // World Wide Web (dot) PAnzea (dot) org / index (dot) html].
[00328] Database set - was developed to build a wide, rich, reliable and easy to analyze annotated database containing genomic mRNA sequences, publicly available DNA ESTs, data from various cultures, as well as QTLs of data from gene expression, protein annotation and pathways, and other relevant information.
[00329] The database set comprises a toolbox for refining, structuring, annotating and analyzing genes, allowing the construction of a customized database for each gene discovery project. The tools of refining and structuring genes allow to reliably detect connection variants and antisense transcriptions, generating the understanding of several potential phenotypic results of a single gene. The capabilities of the "LEADS" platform of Compugen LTD for analysis of the human genome have been confirmed accepted by the scientific community [see, for example, "Widespread Antisense Transcription", Yelin, et al. (2003) Nature Biotechnology 21, 379-85; "Splicing of Alu Sequences", Lev-Maor, et al. (2003) Science 300 (5623), 1288-91; "Computational analysis of alternative splicing using EST tissue information", Xie H et al. Genomics 2002], also proved to be more efficient in the plant genome. EST clustering and gene assembly - For clustering and gene assembly of organisms with available data on the genome sequence (arabidopsis, rice, beans, broad beans, brachypodium, poplar, soybeans, sorghum), the LEADS genomic version ( GANG) was employed. This tool allows for more accurate clustering of ESTs and mRNA sequences in the genome, and predicts the structure of the gene, as well as alternative events of anti-sense transcription splicing.
[00330] For organisms without complete genome sequence data available, the clustering software "expressed LEADS" was applied.
[00331] Gene annotation - The predicted genes and proteins were noted as follows: a blast search was performed [Hypertext Transfer Protocol: // blast (dot) ncbi (dot) nlm (dot) nih (dot) gov / Blast ( dot) cgi] against all UniProt plant strings [Hypertext Transfer Protocol: // World Wide Web (dot) uniprot (dot) org /]. Open reading frames of each alleged transcript were analyzed and the longest ORF with the largest number of homologues was selected as the predicted transcription protein. The predicted proteins were analyzed by InterPro [Hypertext Transfer Protocol: // World Wide Web (dot) ebi (dot) ac (dot) uk / interpro /].
[00332] Blast against AraCyc proteins and ENZYME databases were used to map the transcriptions planned for the AraCyc pathway.
[00333] Predicted proteins of different species were compared using a blast algorithm [Hypertext Transfer Protocol: // World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov / Blast (dot) cgi] to validate the accuracy of the predicted protein sequence and for the efficient detection of orthologists.
[00334] Gene expression profile - Several data sources have been explored for the gene expression profile that combined micromatrix data and digital expression profile (see below). According to the gene expression profile, correlation analysis was performed to identify genes that are co-regulated at different stages of development and environmental conditions and that are associated with different phenotypes.
[00335] Micromatrix data sets available to the public were downloaded from the TAIR and NCBI GEO websites, renormalized and integrated into the database. The expression profile is one of the most important resource data to identify important genes for yield, biomass, growth rate, vigor, oil content, tolerance to plant abiotic stress and efficiency in the use of nitrogen.
[00336] A digital expression profile summary has been compiled for each cluster according to all keywords included in the sequence records comprising the cluster. Digital expression, also known as electronic Northern Blot, is a tool that displays a virtual expression profile based on the EST sequences that form the gene cluster. The tool provides the expression profile of a cluster in terms of the anatomy of the plant (for example, the tissue / organ in which the gene is expressed), stage of development (the stages of development in which a gene can be found) and profile of treatment (provides the physiological conditions under which a gene is expressed, such as drought, cold, infection by pathogens, etc.). Given a random distribution of ESTs in the different clusters, the digital expression provides a probability value that describes the probability that a cluster will have a total of N ESTs to contain X ESTs from a certain library collection. For the probability calculations, the following is taken into account: a) the number of ESTs in the cluster, b) the number of ESTs from the implicated and related libraries, c) the total number of available ESTs that represent the species. Thus, clusters with low probability values are highly enriched with ESTs from the group of libraries of interest, indicating a specialized expression.
[00337] Recently, the accuracy of this system was demonstrated by Portnoy et al., 2009 (Analysis Of The Melon Fruit Transcriptome Based On 454 Pyrosequencing) in: Plant & Animal Genomes XVII Conference, San Diego, CA. Recently, the accuracy of this system was demonstrated by Portnoy et al., 2009 (Analysis Of The Melon Fruit Transcriptome Based On 454 Pyrosequencing) in: Fourteen double-stranded cDNA samples obtained from two genotypes, two fruit tissues (pulp and peel ) and four stages of development have been sequenced. Pyrosequencing GS FLX (Roche / 454 Life Sciences) of non-normalized and purified cDNA samples resulted in 1,150,657 expressed sequence tags that were assembled in 67,477 unigenes (32,357 singletons and 35,120 contigs). The analysis of the data obtained in comparison to the Cucurbit Genomics database [Hypertext Transfer Protocol: // World Wide Web (dot) icugi (dot) org /] confirmed the accuracy of the sequencing and assembly. The expression patterns of selected genes corresponded well to their qRT-POR data. EXAMPLE 2 PRODUCTION OF ARABIDOPSIS TRANSCRIPTOM AND HIGH CORRELATION ANALYSIS OF YIELD, BIOMASS AND / OR VIGOR PARAMETERS USING THE 44K ARABIDOPSIS INTEGRAL OLIGONUCLEOTIDE MICROMATRON
[00338] To produce a high production correlation analysis, the present inventors used an Arabidopsis thaliana micro-array produced by Agilent Technologies [Hypertext Transfer Protocol: // World Wide Web (dot) chem. (dot) agilent (dot) com / Scripts / PDS (dot) asp 1Page = 50879]. The matrix oligonucleotide represents approximately 40,000 A. thaliana genes and transcripts designed based on data from the TIGR ATH1 v.5 database and the Arabidopsis MPSS databases (University of Delaware). To define correlations between RNA expression levels and parameters related to yield, biomass or vigor, several plant characteristics of 15 different Arabidopsis ecotypes were analyzed. Among them, nine ecotypes covering the observed variance were selected for the analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test.
[00339] Experimental procedures RNA extraction - Five tissues in different stages of development including the root, leaf, flower in anthesis, seed 5 days after flowering (DAF) and seed 12 DAF, representing different characteristics of the plant, were sampled and the RNA was extracted as described in Example 3 above. Expression sets (eg, root, leaf, etc.) are shown in Table 26 below.
[00340] Yield components and evaluation of parameters related to vigor - eight out of nine Arabidopsis ecotypes were used in each of the 5 repetitive blocks (namely, A, B, C, D and E), each containing 20 plants per construction site. The plants were grown in a greenhouse under controlled conditions at 22 ° C, the fertilizer N: P: K [20:20:20; weight relationships; nitrogen (N), phosphorus (P) and potassium (K)] was added; During that time, data was collected, documented and analyzed. Additional data were collected during the seedling stage of plants grown in tissue culture on transparent agar plates with vertical growth. Most of the chosen parameters were analyzed by digital image.
[00341] Digital image in tissue culture assays - A laboratory image acquisition system was used to capture images of plants sown on square agar plates. The laboratory image acquisition system consists of a digital reflex camera (Canon EOS 300D) with a 55 mm focal length lens (Canon EF-S series), mounted on a reproduction device (Kaiser RS) that included 4 light units (4 lamps of 150 Watts) and located in a dark room.
[00342] Digital image in greenhouse tests - The image capture process was repeated every 3 to 4 days, starting on the 7th until the 30th. The same camera with a 24 mm focal length lens (Canon EF series ), placed on a custom iron easel, was used to capture images of larger plants enclosed in white pots in a greenhouse with a controlled environment. The white vases were square in shape, measuring 36 x 26.2 cm and 7.5 cm deep. During the capture process, the pots were placed under the iron easel, avoiding direct sunlight and shadows. This process was repeated every 3 to 4 days for up to 30 days.
[00343] An image analysis system was used, which consisted of a desktop personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.37, a Java-based image processing program, which was developed in US National Institutes of Health and freely available on the Internet at Hypertext Transfer Protocol: // rsbweb (dot) nih (dot) gov /. The images were captured in a resolution of 6 Mega Pixels (3072 x 2048 pixels) and stored in a low compression JPEG format (Joint Photographic Experts Group standard). Then, the analyzed data were saved in text files and processed using the statistical analysis software JMP (instituto SAS).
[00344] Sheet analysis - Using digital analysis, the sheet data was calculated, including the number, area, perimeter, length and width of the sheet. On day 30, 3-4 representative plants were chosen from each bed in blocks A, B and C. The plants were dissected, each leaf was separated and inserted between two glass trays, a photo of each plant was taken and the various parameters (such as, total sheet area, laminar length, etc.) were calculated from the images. The circularity of the limbus was calculated as the laminar width divided by the laminar length.
[00345] Root analysis - For 17 days, the different ecotypes were grown on transparent agar plates. The plates were photographed every 2 days, starting on the 7th in the photography room and the development of the roots was documented (Figures 3A-F). The growth rate of the roots was calculated according to Formula V. Formula V: Relative growth rate of the root cover = Coefficient of the root cover over time.
[00346] Analysis of the vegetative growth rate - was calculated according to Formula VI. The analysis was concluded with the appearance of overlapping plants. Formula VI: Area of relative vegetative growth rate = Coefficient of vegetative area regression over time.
[00347] For comparison between ecotypes, the calculated rate was normalized using the plant's development stage as represented by the number of real leaves. In cases where plants with 8 leaves were sampled twice (for example, on the 10th and 13th), only the largest sample was chosen and added to the Anova comparison.
[00348] Seeds in silica analysis - On day 70, 15 to 17 silicas were collected from each bed in blocks D and E. The chosen silicas were light brown, but still intact. The silicas of each bed were opened in the photography room and the seeds were spread on a glass tray and photographed using a high resolution digital camera. Using the images, the number of seeds per silica was determined.
[00349] Average seed weight - At the end of the experiment, all seeds from the beds in blocks A-C were collected. An average weight of 0.02 grams was measured for each sample, the seeds were spread on a glass tray and a picture was taken. Using digital analysis, the number of seeds in each sample was calculated.
[00350] Percentage of oil in the seeds - At the end of the experiment, all seeds from the beds in blocks A-C were collected. Columbia seeds from 3 beds were mixed and crushed and then mounted in the extraction chamber. 210 ml of n-Hexane (Cat. No. 080951 Biolab Ltd.) was used as a solvent. The extraction was carried out for 30 hours with an average temperature of 50 ° C. Once the extraction was finished, the n-Hexane was evaporated using the evaporator under 35 ° C and vacuum conditions. The process was repeated twice. The information obtained from the Soxhlet extractor (Soxhlet, F. Die gewichtsanalytische Bestimmung des Milchfettes, Politechnisches J. (Dingler's) 1879, 232, 461) was used to create a calibration curve for Low Resonance NMR.
[00351] oil content of all seed samples was determined using the Low Resonance NMR (MARAN Ultra-Oxford Instrument) and its MultiQuant software package.
[00352] Silica length analysis - On day 50 after sowing, 30 silicas from different plants in each bed were sampled in block A. The silicas chosen were yellow-green and were collected from the lower parts of a stem of the cultivated plant. A digital photograph was taken to determine the length of the silica.
[00353] Dry weight and seed yield - On day 80 after sowing, the plants in blocks A-C were harvested and allowed to dry at 30 ° C in a drying chamber. The biomass and seed weight of each bed were separated, measured and divided by the number of plants. Dry weight = total weight of the vegetative portion above the soil (excluding the roots) after drying at 20 ° C in a drying chamber; Seed yield per plant = total seed weight per plant (g). Oil yield - The oil yield was calculated using Formula IX. Formula VII: Seed oil yield = Seed yield per plant (g) *% oil in the seed.
[00354] Harvest index - The harvest index was calculated using Formula IV as described above [Harvest index = average seed yield per plant / average dry weight]. Experimental results
[00355] Nine different Arabidopsis ecotypes were grown and characterized for 18 parameters (named as correlation vectors in Table 26). The measured parameters are present in Tables 1 and 2 below. Correlations of gene expression in various tissues with these phenotypic measurements are shown in Table 26, as "Arabidopsis 1" in the "Vector Set" column. Table 1

[00356] Table 1. The values of each of the parameters measured in ecotypes of Arabidopsis are presented: Seed yield per plant (grass); oil yield per plant (mg); % of oil per seed; weight of 1000 seeds (gr); dry matter per plant (gr); harvest index; total leaf area per plant (cm); seeds per silica; silica length (cm), "gr" = grams; "mg" = milligrams; "cm" = centimeters. "Table 2

[00357] Table 2. The values of each of the parameters measured in ecotypes of Arabidopsis: Cresc are shown. Veg. = Vegetative growth rate (cm2 / day) up to 8 real leaves; relative root growth = relative root growth (cm / day); root length, day 7 (cm); root length, day 13 (cm); fresh weight per plant (g) in the sprouting stage; blade length = blade length (cm); Blade width = Blade width (cm); Sheet width / length; circularity of the limbus. EXAMPLE 3 PRODUCTION OF ARABIDOPSIS TRANSCRIPTOM AND HIGH CORRELATION ANALYSIS OF YIELD PARAMETERS AND LIMITING NITROGEN CONDITIONS USING THE 44K ARABIDOPSIS INTEGRAL MICROMATRIZ OF ARABIDOPSIS
[00358] To produce a high production correlation analysis, the present inventors used an Arabidopsis oligonucleotide microarray, produced by Agilent Technologies [Hypertext Transfer Protocol: // World Wide Web (dot) agilent (dot) chem (dot) com / Scripts / PDS (dot) asp 1Page = 50879]. The matrix oligonucleotide represents about 44,000 Arabidopsis genes and transcripts. To define correlations between RNA expression levels and parameters related to yield, biomass or vigor, several plant characteristics of 14 different Arabidopsis ecotypes were analyzed. Among them, ten ecotypes covering the observed variance were selected for the analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test. Experimental procedures
[00359] RNA extraction - two plant tissues [leaves and stems] grown at two different levels of nitrogen fertilization (1.5 mM Nitrogen or 6 mM Nitrogen) were sampled and the RNA was extracted as described above. Expression sets (eg, root, leaf, etc.) are shown in Table 26 below.
[00360] Evaluation of Arabidopsis yield components and parameters related to vigor under different levels of nitrogen fertilization - 10 Arabidopsis adhesions in 2 repetitive beds, each containing 8 plants per bed, were grown in the greenhouse. The cultivation protocol used was: Seeds sterilized on the surface were sown in Eppendorf tubes containing 0.5 x Murashige-Skoog medium basal salt and grown at 23 ° C under daily cycles of 12 light hours and 12 dark hours for 10 days. Then, seedlings of similar size were carefully transferred to beds filled with a mixture of peat and perlite in a 1: 1 ratio. Constant nitrogen limit conditions were achieved by irrigating plants with a solution containing 1.5 mM inorganic nitrogen in the form of KNO3, supplemented with 2 mM CaC12, 1.25 mM KH2PO4, 1.50 mM MgSO4, 5 mM KC1, 0.01 mM H3B03 and microelements, while the normal irrigation condition (normal Nitrogen condition) was achieved by applying a 6 mM inorganic nitrogen solution also in the form of KNO3, supplemented with 2 mM and CaC12, 1.25 mM KH2PO4, 1.50 mM MgSO4, 0.01 mM H3B03 and microelements. To follow the plant's growth, the trays were photographed on the day that the nitrogen limiting conditions were initiated and, subsequently, every 3 days for an additional 15 days. The area of the rosette plant was then determined from digital photographs. The ImageJ software was used to quantify the plant size from digital photographs [Hypertext Transfer Protocol: // rsb (dot) info (dot) nih (dot) gov / ij /], using proprietary scripts designed to analyze the size of the plant. rosette area from individual plants with a function of time. The image analysis system included a desktop personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.37 (Java-based image processing program, which was developed at the US National Institutes of Health and freely available on the internet at Hypertext Transfer Protocol: // rsbweb (dot) nih (dot) gov /). Then, the analyzed data were saved in text files and processed using the statistical analysis software JMP (instituto SAS).
[00361] The data parameters collected are summarized in Table 26, below.
[00362] NUE analysis, yield of components and parameters related to vigor - Ten Arabidopsis ecotypes were grown in trays, each containing 8 plants per cover, in a greenhouse under controlled temperature for approximately 12 weeks. The plants were irrigated with different concentrations of hydrogen, as described above, depending on the treatment applied. During this time, data was collected, documented and analyzed. Most of the chosen parameters were analyzed by digital image. Digital image - greenhouse analysis.
[00363] An image acquisition system, consisting of a digital reflex camera (Canon EOS 400D) coupled with 55 mm long focal lenses (Canon EF-S series) placed in a custom structure made of aluminum, was used for capturing images of plants grown in containers within a controlled greenhouse environment. The image capture process was repeated every 2-3 days, starting on 9-12 until 16-19 (respectively) after transplantation.
[00364] An image processing system was used, which consists of a desktop personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.37, Java-based image processing software, developed at the US National Institutes of Health and is freely available on the Internet at http://rsbweb.nih.gov/. The images were captured in 10 megapixel resolution (3888x2592 pixels) and stored in low compression JPEG format (Joint Photographic Experts Group standard). Next, the image processing output data was saved in text files and analyzed using JMP statistical analysis software (SAS institute).
[00365] Leaf analysis - Using digital analysis, the leaf data was calculated, including the number, leaf limb area, bed cover, rosette diameter and leaf rosette area.
[00366] Relative growth rate area: The growth rate and the relative growth rate of the rosette and leaves were calculated according to the following formulas VIII and IX:
* 41 It is the current day of the analyzed image subtracted from the initial day (meaning that the growth rate of the area is measured in units of cm / day and the growth rate of the length is measured in units of cm / day).
[00367] * EU While the examples shown here are for area growth rate parameters, length growth rate parameters are calculated using similar formulas.
[00368] Seed yield and weight of 1000 seeds - At the end of the experiment, all seeds from all beds were collected and weighed to measure seed yield per plant in terms of total seed weight per plant (gr). To calculate the weight of 1000 seeds, an average weight of 0.02 grams was measured for each sample, the seeds were spread on a glass tray and a picture was taken. Using digital analysis, the number of seeds in each sample was calculated.
[00369] Dry weight and seed yield - At the end of the experiment, the plants were harvested and allowed to dry at 30 ° C in a drying chamber. The biomass was separated from the seeds, weighed and divided by the number of plants. Dry weight = total weight of the vegetative part above the ground (except the roots) after drying at 30 ° C in a drying chamber.
[00370] Harvest index - The harvest index was calculated using Formula IV (Harvest index = Average seed yield per plant / average dry weight).
[00371] T50 days to flowering - Each of the plants was monitored by date of flowering. The flowering days were calculated from the day of cultivation until 50% of the beds were flowering.
[00372] Nitrogen level of the plant - The chlorophyll content of the leaves is a good indicator of the nitrogen status of the plant, since the degree of greenness of the leaf is highly correlated to this parameter. The chlorophyll content was determined using a Minolta SPA 502 chlorophyll meter and the measurement was performed at the time of flowering. The SPAD meter readings were taken on fully developed young leaves. Three measurements per sheet were taken per bed. Based on this measure, parameters such as the ratio of seed yield per unit of nitrogen [seed yield / level N = seed yield per plant [gr] / SPAD unit], plant DW per unit of nitrogen [DW / level N = plant biomass per plant [g] / unit of SPAD], and level of nitrogen per gram of biomass [level N / DW = unit of SPAD / plant biomass per plant (gr)] were calculated.
[00373] Percentage of reduction in seed yield - measures the amount of seeds obtained from plants when grown under nitrogen limiting conditions compared to the yield of seeds produced at normal levels of nitrogen expressed in%. Table 3

[00374] Table 3. The values of each of the parameters measured in ecotypes of Arabidopsis are presented: N 1.5 mM Rosette area on the 8th (measured in cm2); N 1.5 mM Rosette area on the 10th (measured in cm2); N 1.5 mM Leaf number on day 10; N 1.5 mM Leaf Limbo Area on the 10th (measured in cm2); N 1.5 mM TCR of the Rosette area on day 3; N 1.5 mM Flowering t50 (measured in days); "cm" = centimeters ". Table 4.

[00375] The values of each of the parameters measured in ecotypes of Arabidopsis are presented: N 1.5 mM Dry Weight (measured in grams); N 1.5 mM Seed Yield (measured in gr / plant); N 1.5 mM Harvest Index; N 1.5 mM Weight of 1000 seeds (measured in grams); N 1.5 mM seed yield per rosette area on day 10 (measured in gr / plant * cm2); N 1.5 mM seed yield per leaf blade (measured in gr / plant * cm2); Table 5

[00376] Table 5. The values of each of the parameters measured in ecotypes of Arabidopsis are presented: N 6 mM Rosette area on the 8th; N 6 mM Rosette area on the 10th; N 6 mM Rosette number on the 10th; N 6 mM Leaf Limbo Area day 10. Table 6 Additional parameters measured in Arabidopsis ecotypes

[00377] Table 6. The values of each of the parameters measured in ecotypes of Arabidopsis are presented: N 6 mM TCR of the Rose Area on day 3; N 6 mM Flowering t50 (measured in days); N 6 mM Dry Weight (measured in gr / plant); N 6 mM Seed Yield (measured in gr / plant); N 6 mM Harvest index; N 6 mM Weight of 1000 seeds (measured in gr); "gr." = grams; "mg" = milligrams; "cm" = centimeters ". Table 7 Additional parameters measured in Arabidopsis ecotypes

[00378] Table 7. The values of each of the parameters measured in ecotypes of Arabidopsis are shown: N 6 mM seed yield / rosette area day 10 (measured in gr / plant * cm2); N 6 mM seed / leaf blade yield (measured in gr / plant * cm2); Table 8 Additional parameters measured in Arabidopsis ecotypes

[00379] Table 8. The values of each of the parameters measured in ecotypes of Arabidopsis are shown: N 6 mMSpad / Fresh Weight; N 6 mM Dry Weight / SPAD (biomass / community); N 6 mM spad / Dry Weight (gN / g plant); N 6 mM Seed Yield / N unit (measured in gr / N units); N 1.5 mM Spad / Fresh Weight (measured in 1 / gr); N 1.5 mM SPAD / Dry Weight (measured in 1 / gr); N 1.5 mM Dry Weight / SPAD (measured in 1 / gr); N 1.5 mM seed / spad yield (measured in grain); Experimental results
[00380] Ten different Arabidopsis adhesions (ecotypes) were cultivated and characterized for 33 parameters, as described above (Tables 3-8). The average of each of the measured parameters was calculated using the JMP software. The subsequent correlation analysis was performed between the parameters characterized in the Arabidopsis ecotypes (which are used as an x-axis for correlation) and tissue transcription, and genes exhibiting a significant correlation for selected characteristics (classified using the correlation vector) are presented. in Table 26 below, together with their correlation values (R, calculated using Pearson's correlation) and p values under the category of the Arabidopsis 2 NUE and Arabidopsis 2 vector sets. EXAMPLE 4 TOMATO PRODUCTION AND HIGH CORRELATION ANALYSIS OF PRODUCTION USING THE TOMATO OLIGONUCLEOTIDE MICROMATRIZ
[00381] To produce a high production correlation analysis, the present inventors used a Barley oligonucleotide microarray, produced by Agilent Technologies [Hypertext Transfer Protocol: // World Wide Web (dot) chem. (dot) agilent (dot) com / Scripts / PDS (dot) asp 1Page = 50879]. The matrix oligonucleotide represents about 44,000 genes and transcripts from Tomato. To define correlations between the levels of RNA expression with ABST, parameters related to yield or vigor, several plant characteristics of 18 different Tomato ecotypes were analyzed. Among them, 10 varieties covering the observed variance were selected for the analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test. I. Correlation of Tomato varieties along the ecotype grown under 50% irrigation conditions Experimental procedures
[00382] Cultivation Procedure - The tomato variety was grown under normal conditions (4-6 liters / m per day) until the flowering stage. At that time, irrigation was reduced to 50% compared to normal conditions.
[00383] RNA extraction - Two tissues with different stages of development [flower and leaf], representing different characteristics of the plant, were sampled and the RNA was extracted, as described above. Expression sets (eg, root and leaf) are shown in Table 26 below.
[00384] Parameters related to the components of yield and vigor of tomatoes under water irrigation evaluation with 50% - 10 varieties of tomatoes in 3 repetitive blocks (named A, B, C and D), each containing 6 plants per bed were grown in the greenhouse. The plants were phenotyped on a daily basis following the standard tomato descriptor (Table 11, below). The harvest was carried out while 50% of the fruits were red (ripe). The plants were separated into their vegetative parts and fruits, of which, two nodes were analyzed for additional inflorescent parameters such as size, number of flowers and inflorescent weight. The fresh weight of all vegetative material was measured. The fruits were separated into colors (red x green) and according to the size of the fruit (small, medium and large). Then, the analyzed data were saved in text files and processed using the statistical analysis software JMP (instituto SAS).
[00385] The data parameters collected are summarized in Table 9 below. Table 9 Correlated parameters of tomatoes (vectors)

[00386] Table 9. The correlated parameters of the tomato are provided, "gr" = grams; "SPAD" = chlorophyll levels; Fruit Weight (grams) - At the end of the experiment, [when 50% of the fruits were ripe (red)] all the fruits from the A-C blocks were collected. The total of fruits was counted and weighed. The average weight of the fruits was calculated by dividing the total weight of the fruits by the total number of fruits.
[00387] Vegetative Weight of the Fruit (grams) - At the end of the experiment, [when 50% of the fruits were ripe (red)] all the fruits from the A-C blocks were collected. Fresh weight was measured (grams). Inflorescence Weight
[00388] (grams) - At the end of the experiment, [when 50% of the fruits were ripe (red)] all the fruits from the flowerbeds in blocks A-C were collected. The weight of the inflorescence (gr) and the number of flowers per inflorescence were counted.
[00389] SPAD - The chlorophyll content was determined by using a Minolta SPA 502 chlorophyll meter and the measurement was performed at the time of flowering. The SPAD meter readings were taken on fully developed young leaves. Three measurements per sheet were taken per bed. Water use efficiency
[00390] (WUE) - can be determined as the biomass produced by unit transpiration. To analyze the WUE, the relative content of the leaf content can be measured in transgenic and control plants. Fresh weight (FW - Fresh Weight) was recorded immediately; the leaves were then soaked for 8 hours in distilled water at room temperature in the dark, and the turgid weight (TW) was recorded. The total dry weight (DW) was recorded after drying the leaves at 60 ° C at constant weight. The Relative Water Content (CRA) was calculated according to Formula I (PF - PS / PT - PS) x 100] as described above.
[00391] Plants that maintain a high relative water content (TRA) compared to control lines were considered more tolerant to drought than those that demonstrate reduced relative water content. Experimental results
[00392] Ten different Arabidopsis adhesions (ecotypes) were cultivated and characterized for 23 parameters, as described below. The mean for each of the measured parameters was calculated using the JMP software and the values were summarized in Tables 10, 11 and 12 below. The subsequent analysis of the correlation between the expression of the selected genes in different transcriptom expression sets and the parameters measured in tomato adhesions (Tables 10-12) was performed, and the results were integrated into the database and provided in Table 26 below under the category of vector sets, Normal tomato vector field, Dry tomato vector field. Table 10 Parameters measured in tomato adhesions

[00393] Table 10: The parameters related to the measured components of yield and vigor under normal irrigation or 50% water are provided for tomato adhesions (Varieties) according to the Correlation ID numbers (described in Table 9 above) as follows: 2 [50% Irrigation; Fruit per plant (gr.)]; 10 [Normal irrigation; Fruit per plant (gr.)]; 1 [50% Irrigation; Fresh Vegetative Weight (gr.)]; 9 [Normal Irrigation; Fresh Vegetative Weight (gr.)]; 7 [50% Irrigation; Average weight of ripe fruits (gr.)]; 15 [Normal irrigation; Average weight of ripe fruits (gr.)]; 18 [50% Irrigation; Fruit per plant (gr.) / Normal irrigation; Fruit per plant (gr.)]; 17 [50% Irrigation; Fresh vegetative weight (gr.) / Normal irrigation; fresh vegetative weight (gr.)]. Table 11 Additional parameters measured in tomato adhesions

[00394] Table 11: Parameters related to the measured components of yield and vigor under irrigation of 50% water are provided for tomato adhesions (Varieties) according to the Correlation ID numbers (described in Table 9 above) as follows: 22 [50% Irrigation; Average weight of ripe fruit (gr.) / Normal irrigation; Average weight of ripe fruit (gr.)]; 8 [50% Irrigation: SPAD]; 16 [Normal Irrigation; SPAD]; 5 [50% Irrigation; relative efficiency of water use]; 13 [Normal irrigation; relative efficiency of water use]; 23 [50% Irrigation: SPAD / Normal Irrigation; SPAD], Table 12 Additional parameters measured in Tomato adhesions

[00395] Table 12: Parameters related to the measured components of yield and vigor under irrigation of 50% water are provided for tomato adhesions (Varieties) according to the Correlation ID numbers (described in Table 9 above) as follows: 21 [50% Irrigation; relative efficiency of water use / Normal Irrigation; Water use efficiency]; 4 [50% Irrigation; number of flowers]; 12 [Normal irrigation; number of flowers]; 3 [50% Irrigation; Inflorescence weight (gr.)]; 11 [Normal irrigation; Inflorescence weight (gr.)]; 20 [50% Irrigation; number of flowers / Normal Irrigation; number of flowers]; 19 [50% Irrigation; Inflorescence weight (gr.) / Normal irrigation; Inflorescence weight (gr.)]. II. Correlation of Tomato varieties under stress built under 50% irrigation conditions Experimental procedures
[00396] Cultivation Procedure - Tomato varieties were grown under normal conditions (4-6 liters / m per day) until the flowering stage. At that time, irrigation was reduced to 50% compared to normal conditions. Tissue samples were taken during a period of stress developed every two days.
[00397] RNA extraction - All 10 selected tomato varieties were sampled for each treatment. Two tissues [leaves and flowers] being grown under normal conditions or 50% irrigation were sampled and RNA was extracted using Invitrogen's Hypertext Transfer Protocol: // World Wide Web (dot) invitrogen (dot) com / content (dot) cfriapageid = 469]. Expression sets (eg, root and leaf) are shown in Table 26 below. The extraction of RNA from the tissues was performed as described in Example 2 above.
[00398] Correlation of early characteristics throughout the collection of tomato ecotypes under high salinity concentration - Ten tomato varieties were grown in three repetitive beds, each containing 17 plants, in a greenhouse under semi-hydroponic conditions. Briefly, the growth protocol was as follows: The tomato seeds were sown in trays filled with a mixture of vermiculite and peat in a 1: 1 ratio. After germination, the trays were transferred to high salinity culture conditions (100 mMM NaCl solution) or to normal growing conditions [Hogland campleoto; KNO3 - 0.808 grams / liter, MgSO4 - 0.12 grams / liter, KH2PO4 - 0.172 grams / liter and 0.01% (volume / volume) of micro elements of 'Super coratin "(Ferro-EDDHA [ethylenediamine-N, N '-bis (2-hydroxyphenylacetic acid)] - 40.5 grams / liter; Mn - 20.2 grams / liter; Zn 10.1 grams / liter; Co 1.5 grams / liter, and Mo 1.1 grams / liter ), the pH of the solution should be 6.5 - 6.8].
[00399] Parameters related to tomato vigor under 100 mM NaC1 - After 5 weeks of cultivation, the plant was harvested and analyzed for number of leaves, plant height and plant weight (the data parameters are summarized in Table 13) . Then, the analyzed data were saved in text files and processed using the statistical analysis software JMP (instituto SAS). Table 13 Correlated parameters of tomato (vectors)

[00400] Table 13. The correlated parameters of tomato are presented (IDs numbers 1-7). Experimental results
[00401] Ten different varieties of Tomato were grown and characterized for 7 parameters, as described below (Table 13). The mean for each of the measured parameters was calculated using the JMP software and the values were summarized in Table 14 below. The subsequent correlation analysis between the expression of the selected genes in various transcriptom expression sets and the measured mean parameters was conducted and the results were integrated into the database and provided in Table 26 below, under the vector sets: Vector bath of Normal tomato, and bath of tomato Salinity vectors. Table 14 Parameters measured in tomato adhesions

[00402] Table 14. The parameters related to the measured components of yield and vigor under normal conditions or 100 mM NaC1 are provided for tomato adhesions (Varieties) according to the Correlation ID numbers (described in Table 13 above) as follows: 1 [100 mM NaCl: Number of leaves], 4 [Normal: Number of leaves]; 2 [100 mM NaCl: Height of the plant]; 5 [Normal: Height of the Plant]; 3 [100 mM NaCl: Plant Biomass]; 6 [100 mM NaCl: Number of leaves / Normal: Number of leaves]; 7 [100 mM NaCl: Plant height / Normal Plant height]. EXAMPLE 5 TRANSCRIPTOM B. JUNCEA PRODUCTION AND HIGH CORRELATION ANALYSIS WITH PERFORMANCE PARAMETERS USING 44KB. MICROMATRIZES OF THE OLIGONUCLEOTIDE JUNCEA
[00403] To produce a high production correlation analysis, the present inventors used a micro-array of B. Juncea oligonucleotide, produced by Agilent Technologies [Hypertext Transfer Protocol: // World Wide Web (dot) chem. (dot) agilent (dot) com / Scripts / PDS (dot) asp 1Page = 50879]. The matrix oligonucleotide represents about 60,000 B. juncea genes and transcripts. In order to define correlations between the levels of RNA expression with parameters related to vigor or yield components, several plant characteristics of 11 different ecotypes of B. juncea were analyzed and used for analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test.
[00404] Correlation of expression levels of B. juncea genes with phenotypic characteristics throughout the ecotype Experimental procedures Eleven varieties of B. juncea were cultivated in three repetitive beds, in the field.
[00405] Briefly, the growth protocol was as follows: B. juncea seeds were sown in soil and cultivated under normal conditions until harvest. In order to define correlations between the levels of RNA expression with parameters related to vigor or yield components, the eleven different varieties of B. juncea were analyzed and used for analysis of gene expression.
[00406] RNA extraction - All eleven B. juncea varieties selected were sampled for each treatment. Plant tissues [leaf, stem, lateral meristem and flower] grown under normal conditions were sampled and RNA was extracted, as described above. The expression sets (eg, leaf, stem, lateral meristem and flower) are present in Table 26 below.
[00407] The data parameters collected were as follows: Fresh weight (bed-harvest) [gr / plant] - total fresh weight per bed at the time of harvest normalized to the number of plants per bed.
[00408] Seed Weight [milligrams / plant] - the total of seeds from each bed was extracted, weighed and normalized to the number of plants in each bed.
[00409] Harvest index - The harvest index was calculated: seed weight / fresh weight
[00410] Days until sifting / flowering - number of days up to 50% of sifting / flowering for each bed.
[00411] SPAD - The chlorophyll content was determined by using a Minolta SPA 502 chlorophyll meter and the measurement was performed at the time of flowering. The SPAD meter readings were taken on fully developed young leaves. Three measurements per sheet were taken for each bed.
[00412] Main branch - average node length - total length / total number of nodes in the main branch.
[00413] Lateral branch - average node length - total length / total number of nodes in the lateral branch.
[00414] Main branch - 20 ° length- the length of the plant at the 20th node of the tip of the main branch.
[00415] Lateral branch - 20 ° length- the length of the plant at the 20th node of the tip of the lateral branch.
[00416] Main branch - 20 ° number of seeds - the length of the plant in the 20 ° node of the tip of the main branch.
[00417] Lateral branch - 20 ° number of seeds - the length of the plant in the 20 ° node of the tip of the side branch.
[00418] Number of lateral branches - total number of lateral branches, average of three plants per bed.
[00419] Height of the main branch [cm] - total length of the main branch.
[00420] Position min. lateral branch - the lowest node in the main branch that developed the lateral branch.
[00421] Position max. lateral branch - highest node in the main branch that developed the lateral branch.
[00422] Maximum number of nodes on the side branch - the highest number of nodes that a side branch had per plant.
[00423] Maximum length of the lateral branch [cm] - the largest length of the lateral branch per plant.
[00424] Maximum diameter of the lateral branch [mm] - the largest base diameter that a lateral branch had per plant.
[00425] Oil Content - The indirect analysis of the oil content was performed using Nuclear Magnetic Resonance Spectroscopy (NMR), which measures the resonance energy absorbed by the hydrogen atoms in the liquid state of the sample [See, for example, Conway TF. and Earle FR., 1963, Journal of the American Oil Chemists' Society; Springer Berlin / Heidelberg, ISSN: 0003-021X (Print) 1558-9331 (Online)].
[00426] Fresh weight (single plant) (gr / plant) - average fresh weight of three plants per bed measured in the middle of the season. Base diameter of the main branch [mm] - the base diameter of the main branch, average of three plants per bed.
[00427] 1000 Seeds [gr] - weight of 1000 seeds per bed. Experimental results
[00428] Eleven different varieties of B. juncea (ie seeds ID 646, 648, 650, 657, 661, 662, 663, 664, 669, 670, 671) were grown and characterized in 23 parameters, as specified above. The mean for each of the measured parameters was calculated using the JMP software and the values were summarized in Table 15 below. The subsequent analysis of the correlation between the different sets of transcriptom and the average parameters was carried out. The results were then integrated into the database and the selected correlations are shown in Table 26 below, under the vector set column, Juncea ecotype vector. Table 15 Parameters measured in adhesions of B. juncea

[00429] Table 15: The values of each of the parameters (as described above) are measured in adhesions of B. juncea (seed ID) under normal conditions. EXAMPLE 6 TRANSCRIPTOM B. JUNCEA PRODUCTION AND HIGH CORRELATION ANALYSIS WITH YIELD PARAMETERS CULTIVATED UNDER VARIOUS POPULATION DENSITIES USING 44KB OLIGONUCLEOTIDE MICROMATRIZES. JUNCEA
[00430] To produce an analysis of high production correlation, the present inventors used a micro-array of B. Juncea oligonucleotide, produced by Agilent Technologies [Hypertext Transfer Protocol: // World Wide Web (dot) chem. (dot) agilent (dot) com / Scripts / PDS (dot) asp 1Page = 50879]. The matrix oligonucleotide represents about 60,000 B. juncea genes and transcripts. In order to define correlations between the levels of RNA expression with parameters related to vigor or yield components, several plant characteristics of two different varieties of B. juncea cultivated under seven different population densities were analyzed and used for analysis of expression of RNA. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test.
[00431] Correlation of expression levels of B. juncea genes with phenotypic characteristics over seven population densities for two ecotypes Experimental procedures
[00432] Two varieties of B. juncea (646 and 671) were grown in a field under seven population densities (10, 60, 120, 160, 200, 250 and 300 plants per m) in two repetitive beds. Briefly, the growth protocol was as follows: B. juncea seeds were sown in soil and grown under normal conditions until harvest. In order to define correlations between the levels of RNA expression with parameters related to vigor or yield components, the two different varieties of B. juncea grown under various population densities were analyzed and used for analysis of gene expression. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test for each ecotype independently.
[00433] RNA extraction - the two varieties of B. juncea grown under seven population densities were sampled for each treatment. Plant tissues [lateral meristem and flower] grown under normal conditions were sampled and RNA was extracted, as described above. For convenience, each type of microarray expression information fabric has been assigned a Set ID. Expression sets (eg, Flor and Lateral Meristem) are included in Table 26 below.
[00434] The data parameters collected were as follows: Fresh weight (bed-harvest) [gr / plant] - total fresh weight per bed at the time of harvest normalized to the number of plants per bed.
[00435] Seed Weight [gr / plant] - the total of seeds from each bed was extracted, weighed and normalized to the number of plants in each bed.
[00436] Harvest index - The harvest index was calculated: seed weight / fresh weight
[00437] Days until sifting / flowering - number of days up to 50% of sifting / flowering for each bed.
[00438] SPAD - The chlorophyll content was determined by using a Minolta SPA 502 chlorophyll meter and the measurement was performed at the time of flowering. The SPAD meter readings were taken on fully developed young leaves. Three measurements per sheet were taken for each bed.
[00439] Main branch - average node length - total length / total number of nodes in the main branch.
[00440] Lateral branch - average node length - total length / total number of nodes in the lateral branch.
[00441] Main branch - 20 ° length- the length of the plant at the 20th node of the tip of the main branch.
[00442] Lateral branch - 20 ° length- the length of the plant in the 20 ° node of the tip of the lateral branch.
[00443] Main branch - 20 ° number of seeds - the length of the plant in the 20 ° node of the tip of the main branch.
[00444] Lateral branch - 20 ° number of seeds - the length of the plant in the 20 ° node of the tip of the side branch.
[00445] Number of lateral branches - total number of lateral branches, average of three plants per bed.
[00446] Height of the main branch [cm] - total length of the main branch.
[00447] Position min. lateral branch - the lowest node in the main branch that developed the lateral branch.
[00448] Position max. lateral branch - highest node in the main branch that developed the lateral branch.
[00449] Maximum number of nodes on the side branch - the highest number of nodes that a side branch had per plant.
[00450] Maximum length of the lateral branch [cm] - the largest length of the lateral branch per plant.
[00451] Maximum diameter of the lateral branch [mm] - the largest base diameter that a lateral branch had per plant.
[00452] Oil Content - The indirect analysis of the oil content was performed using Nuclear Magnetic Resonance Spectroscopy (NMR), which measures the resonance energy absorbed by the hydrogen atoms in the liquid state of the sample [See, for example, Conway TF. and Earle FR., 1963, Journal of the American Oil Chemists' Society; Springer Berlin / Heidelberg, ISSN: 0003-021X (Print) 1558-9331 (Online)]; Fresh weight (single plant) (gr / plant) - average fresh weight of three plants per bed measured in the middle of the season. Base diameter of the main branch [mm] - the base diameter of the main branch, average of three plants per bed.
[00453] 1000 Seeds [gr] - weight of 1000 seeds per bed. Total number of pods in the main branch - total number of pods in the main branch, average of three plants per bed. Main branch dist. 1-20 - the length between the youngest pod and pod number 20 of the main branch, average of three plants per bed.
[00454] Total number of pods on the side branch - total number of pods on the side branch, average of three plants per bed. Lateral branch dist. 1-20 - the length between the youngest pod and pod number 20 of the lateral branch, average of three plants per bed.
[00455] Dry weight / plant - weight of total plants per bed at harvest after three days in an oven under 60 ° C normalized for the number of plants per bed.
[00456] Total leaf area - The total leaf area per bed was calculated based on three random plants and normalized for the number of plants per bed.
[00457] Total Perimeter - the total leaf perimeter was calculated based on three random plants and normalized for the number of plants per bed. Experimental results
[00458] Two varieties of B. juncea were grown under seven different population densities and characterized by 29 parameters, as specified below. The mean for each of the measured parameters was calculated using the JMP software and the values were summarized in Table 16 below. Subsequent analysis of the correlation between the expression of selected genes in various sets of transcriptom expression and the mean parameters was conducted. The results were then integrated into the database and provided in Table 26 below, under the column vector sets, population densities Juncea. Table 16 Parameters measured in adhesions of B. juncea at various densities of

[00459] Table 16: The values of each of the parameters (as described above) measured in adhesions of B. juncea (seed ID) in seven population densities (Population Density) under normal conditions are provided. Stop. = parameter. EXAMPLE 7 PRODUCTION OF SORGHUM TRANSCRIPTOM AND HIGH CORRELATION ALISE WITH PARAMETERS RELATED TO YIELD, NUE AND ABST MEASURED IN FIELDS USING 44K SORGHUM MICROWAYS
[00460] To produce a high production correlation analysis between the plant phenotype and the level of gene expression, the present inventors used a sorghum oligonucleotide microarray, produced by Agilent Technologies [Hypertext Transfer Protocol: // World Wide Web ( dot) chem. (dot) agilent (dot) com / Scripts / PDS (dot) asp 1Page = 50879]. The matrix oligonucleotide represents about 44,000 Sorghum genes and transcripts. To define correlations between the levels of RNA expression with ABST, parameters related to yield or vigor, several plant characteristics of 17 different sorghum hybrids were analyzed. Among them, 10 hybrids covering the observed variance were selected for the analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test.
[00461] Correlation of sorghum varieties along ecotypes grown under low nitrogen, regular growth and severe drought conditions Experimental procedures
[00462] Seventeen sorghum varieties were grown in three repetitive beds, in the field. Briefly, the growth protocol was as follows: Regular cultivation conditions: sorghum plants were grown in the field using commercial fertilization and irrigation protocols.
[00463] Low nitrogen fertilization conditions: sorghum plants were fertilized with 50% less nitrogen in the field than the amount of nitrogen applied in the regular growth treatment. All fertilizer was applied before flowering.
[00464] Drought stress: sorghum seeds were sown in the soil and grown under normal conditions until about 35 days after sowing, close to V8. At that point, irrigation stopped and severe drought stress developed. To define correlations between the levels of RNA expression with parameters related to NUE, drought and yield components, the 17 different sorghum varieties were analyzed. Among them, 10 varieties covering the observed variance were selected for the analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test.
[00465] Analyzed Sorghum Tissues - All 10 selected Sorghum hybrids were sampled for each treatment. Plant tissues [Flor da Bandeira, Meristema da flor and Flor] being grown under conditions of low nitrogen, severe drought stress and plants grown under normal conditions were sampled and RNA was extracted, as described above.
[00466] The following parameters were collected using a digital imaging system: Average Grain Area (cm) - At the end of the cultivation period, the grains were separated from the 'Head' of the Plant. A sample of 200 grains was weighed, photographed and the images were processed using the image processing system described above. The grain area was measured from the images and divided by the number of grains
[00467] Average Grain Length (cm) - At the end of the cultivation period, the grains were separated from the 'Head' of the Plant. A sample of 200 grains was weighed, photographed and the images were processed using the image processing system described above. The sum of the grain lengths (longest axis) was measured from the images and divided by the number of grains.
[00468] Average Head Area (cm2) - At the end of the cultivation period, five 'Heads' were photographed and the images processed using the described image processing system. The 'Head' area was measured from the images and divided by the number of 'Heads'. Average Head Length
[00469] (cm2) - At the end of the cultivation period, five 'Heads' were photographed and the images processed using the described image processing system. The length of the 'Head' was measured from the images and divided by the number of 'Heads'.
[00470] The image processing system was used, which consists of a desktop personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.37, a Java-based image processing program, which was developed at the US National Institutes of Health and freely available on the Internet at Hypertext Transfer Protocol: // rsbweb (dot) nih (dot) gov /. The images were captured in a resolution of 10 Mega Pixels (3888x2592 pixels) and stored in a low compression JPEG format (Joint Photographic Experts Group standard). Then, the image processing output data for the seed area and seed length were saved in text files and processed using the JMP statistical analysis software (SAS institute).
[00471] Additional parameters were collected by sampling five plants per site or by measuring parameters across all plants within the site.
[00472] Average seed weight per head (gr) - At the end of the experiment ('Heads' of plants), the heads of the beds within blocks A-C were collected. 5 heads were threshed separately and the beans were weighed, all additional heads were threshed together and weighed. The average weight of the grains per head was calculated by dividing the total weight of the grains by the number of total heads per bed (based on the bed). In the case of five heads, the total weight of the five head grains was divided by 5.
[00473] Fresh Head Weight per Plant gr - At the end of the experiment (when the heads were harvested) total and five heads selected per bed within blocks A-C were collected separately. The heads (total and five) were weighed (gr.) Separately and the average fresh weight per plant was calculated for the total (Fresh Head Weight / Plant gr based on the bed) and for the five (Fresh Head Weight / Plant gr based on five plants).
[00474] Height of the plants - The plants were characterized as to their height during the period of cultivation in five moments. In each measurement, plant heights were measured using a tape measure. The height was measured from the floor level to the top of the longest leaf.
[00475] Number of leaves of the plants - The plants were characterized in terms of their number of leaves during the period of cultivation in five moments. In each measurement, the number of leaves of the plants was measured, counting all the leaves of the three plants selected per bed.
[00476] The Relative Growth Rate was calculated using Formulas X and XI, as follows: Formula X Relative growth rate of plant height = Plant height regression coefficient over the course of time. Formula XI Relative growth rate of the number of leaves of the plant = Coefficient of regression of the number of leaves of the plant over the course of time.
[00477] SPAD - The chlorophyll content was determined by using a Minolta SPA 502 chlorophyll meter and the measurement was performed 64 days after sowing. The SPAD meter readings were taken on fully developed young leaves. Three measurements per sheet were taken per bed.
[00478] Vegetative dry weight and heads - At the end of the experiment, (when the inflorescence was dry) all vegetative material and the inflorescences of the beds inside blocks A-C were collected. The biomass and the weight of the heads of each site were separated, measured and divided by the number of Heads.
[00479] Dry weight = total weight of the vegetative part above the ground (except the roots) after drying at 70 ° C in an oven for 48 hours.
[00480] Harvest index (CI) - The harvest index was calculated using Formula XII. Formula XII: Harvest Index = Average dry weight of grains per head / (Average dry vegetative weight per head + Average dry weight of head)
[00481] Fresh Head Weight (Fresh Head Weight + Fresh Plant Weight) - The total fresh head weight and its respective plant biomass was measured on the day of harvest. The weight of the heads was divided by the sum of the weights of the heads and the plants. Experimental results
[00482] 17 different varieties of sorghum hybrids were grown and characterized for different parameters: The mean for each of the measured parameters was calculated using the JMP software (Tables 1721) and a subsequent correlation analysis was performed (Table 26 below) under the sets of vectors "Normal Sorghum Vectors Field" or "NUE Sorghum Vectors Field" .Table 17 Parameters related to Sorghum (vectors)

[00483] Table 17. The correlated parameters of Sorghum (vectors), "gr" = grams are provided; "SPAD" = chlorophyll levels; "FW" = Fresh Weight of the Plant; "DW" = Dry weight of the plant; "normal" = normal growth conditions. Table 18 Parameters measured in sorghum adhesions under normal conditions

[00484] Table 18: The values of each of the parameters (as described above) measured in sorghum adhesions (seed ID) are given under normal conditions. Growth conditions are specified in the experimental procedure section. Table 19 Parameters measured in sorghum adhesions under low nitrogen conditions

[00485] Table 19: The values of each of the parameters (as described above) measured in sorghum adhesions (seed ID) under low nitrogen conditions are provided. Growth conditions are specified in the experimental procedure section. Table 20 Additional parameters measured in sorghum adhesions under low nitrogen growth conditions

[00486] Table 20: The values of each of the parameters (as described above) measured in sorghum adhesions (seed ID) under low nitrogen conditions are provided. Growth conditions are specified in the experimental procedure section. Table 21 Parameters measured in sorghum adhesions under drought conditions

[00487] Table 21: The values of each of the parameters (as described above) measured in sorghum adhesions (seed ID) under drought conditions are provided. Growth conditions are specified in the experimental procedure section. EXAMPLE 8 PRODUCTION OF MAIZE TRANSCRIPTOM AND HIGH CORRELATION ANALYSIS WITH YIELD RELATED PARAMETERS USING 44K MAIZE OLIGONUCLEOTIDE MICRO-MATERIALS
[00488] In order to produce a high production correlation analysis between the plant phenotype and the level of gene expression, the present inventors used a corn oligonucleotide microarray, produced by Agilent Technologies [Hypertext Transfer Protocol: // World Wide Web ( dot) chem. (dot) agilent (dot) com / Scripts / PDS (dot) asp 1Page = 50879]. The matrix oligonucleotide represents about 44,000 corn genes and transcripts. In order to define correlations between the levels of RNA expression with parameters related to vigor or yield components or NUE, several plant characteristics of twelve different corn hybrids were analyzed. Among them, 10 hybrids covering the observed variance were selected for the analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test.
[00489] Correlation of corn hybrids along ecotypes grown under regular growing conditions Experimental procedures
[00490] Twelve corn hybrids were grown in three repetitive beds, in the field. The corn seeds were plants and the plantations were grown in the field using fertilization and commercial irrigation protocols. In order to define correlations between the levels of RNA expression with parameters related to vigor or yield components and NUE, the twelve different varieties of maize hybrids were analyzed. Among them, 10 hybrids covering the observed variance were selected for the analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test.
[00491] Analyzed Sorghum Tissues - All 10 selected corn hybrids were sampled for each treatment. Plant tissues [Flag flower, Flower meristem, Grain, Ear, Internal Knots] grown under normal conditions were sampled and RNA was extracted, as described above.
[00492] The following parameters were collected using a digital imaging system: Grain Area (cm) - At the end of the cultivation period, the grains were separated from the ear. A sample of 200 grains was weighed, photographed and the images were processed using the image processing system described above. The grain area was measured from the images and divided by the number of grains.
[00493] Grain Length and Width (cm) - At the end of the cultivation period, the grains were separated from the ear. A sample of 200 grains was weighed, photographed and the images were processed using the image processing system described above. The sum of the lengths / widths of the grain (longest axis) was measured from the images and divided by the number of grains.
[00494] Ear Area (cm) - At the end of the cultivation period, five ears were photographed and the images processed using the described image processing system. The ear area was measured from the images and divided by the number of ears.
[00495] Ear Length and Width (cm) - At the end of the cultivation period, five ears were photographed and the images processed using the described image processing system. The length and width of the ear (longest axis) was measured from the images and divided by the number of ears.
[00496] The image processing system was used, which consists of a desktop personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.37, a Java-based image processing program, which was developed at the US National Institutes of Health and freely available on the Internet at Hypertext Transfer Protocol: // rsbweb (dot) nih (dot) gov /. The images were captured in a resolution of 10 Mega Pixels (3888x2592 pixels) and stored in a low compression JPEG format (Joint Photographic Experts Group standard). Then, the image processing output data for seed area and length were saved to text files and analyzed using the statistical analysis software JMP (SAS institute). Additional parameters were collected by sampling six plants per bed or by measuring the parameter across all plants within the bed.
[00497] Normalized Grain Weight per plant (gr) - At the end of the experiment, all ears of the beds inside blocks A-C were collected. 6 ears were threshed separately and the beans were weighed, all additional ears were threshed together and weighed. The average weight of the grains per ear was calculated by dividing the total weight of the grains by the number of total ears per bed (based on the bed). In the case of six ears, the total weight of the grains of six ears was divided by 6.
[00498] Fresh Cob Weight (gr.) - At the end of the experiment (when the ears were harvested) total and six ears selected per bed within blocks A-C were collected separately. The plants (total and with six) were weighed (gr.) Separately and the average ear per plant was calculated as to the total (Fresh Weight of the Ear per bed) and as for six (Fresh Weight of the Ear per plant).
[00499] Height of the plant and Height of the ear - The plants were characterized as to their height during harvest. In each measurement, the heights of six plants were measured using a tape measure. The height was measured from the ground level to the top of the plant below the tassel. The height of the ear was measured from the ground level to the place where the main ear is located
[00500] Number of leaves per plant - The plants were characterized in terms of their number of leaves during the period of cultivation in five moments. In each measurement, the number of leaves of the plants was measured, counting all the leaves of the three plants selected per bed.
[00501] The Relative Growth Rate was calculated using Formulas X and XI (described above).
[00502] SPAD - The chlorophyll content was determined by using a Minolta SPA 502 chlorophyll meter and the measurement was performed 64 days after sowing. The SPAD meter readings were taken on fully developed young leaves. Three measurements per sheet were taken per bed. Data were collected after 46 and 54 days after sowing (DPS)
[00503] Dry weight per plant - At the end of the experiment, (when the inflorescence was dry) all vegetative material from the beds inside blocks A-C were collected.
[00504] Dry weight = total weight of the vegetative part above the ground (except the roots) after drying at 70 ° C in an oven for 48 hours. Harvest index (CI)
[00505] (Maize) - The harvest index was calculated using the Formula XIII Formula: Harvest Index = Average dry weight of grains per Ear / (Average dry vegetative weight per Ear + Average dry weight of Ear)
[00506] Ear Fill Percentage [%] - was calculated as a percentage of the Ear area with total ear grains.
[00507] Ear diameter [cm] - The diameter of the ear without grains was measured using a ruler.
[00508] Number of Rows of Heartwood per Ear - The number of rows on each ear has been counted. Experimental results
[00509] Twelve different varieties of corn hybrids were cultivated and characterized for different parameters: The mean for each of the measured parameters was calculated using the JMP software (Tables 2225) and a subsequent correlation analysis was performed (Table 26 below) using "Normal Corn Vectors". Table 22 Correlated corn parameters (vectors)

[00510] Table 22. SPAD 46DPS and SPAD 54DPS: Chlorophyll level after 46 and 54 days after sowing (DPS). Table 23 Parameters measured in Corn adhesions under normal conditions

[00511] Table 23. The values of each of the parameters (as described above) measured in corn adhesions (seed ID) under normal growing conditions are provided. Growth conditions are specified in the experimental procedure section. Table 24 Additional parameters measured in maize adhesions under regular growing conditions

[00512] Table 24. The values of each of the parameters (as described above) measured in corn adhesions (seed ID) under normal growing conditions are provided. Growth conditions are specified in the experimental procedure section. Table 25 Additional parameters measured in maize adhesions under regular growing conditions
[00513] Table 25. The values of each of the parameters (as described above) measured in corn adhesions (seed ID) under normal growing conditions are provided. Growth conditions are specified in the experimental procedure section. EXAMPLE 9 CORRELATION ANALYSIS
[00514] Table 26 below provides representative results of the correlation analyzes described in Examples 2-8 above. Table 26 Correlation analyzes

























[00515] Table 26: Correlation analyzes. EXAMPLE 10 IDENTIFICATION OF GENES AND HOMOLOGISTS THAT INCREASE YIELD, BIOMASS, GROWTH RATE, VIGOR, OIL CONTENT, TOLERANCE TO ABIOTIC PLANT STRESS AND EFFICIENCY IN THE USE OF NITROGEN
[00516] Based on the experimental and bioinformatics tools described above, the present inventors identified 217 genes that have a major impact on yield, seed yield, oil yield, biomass, growth rate, vigor, oil content, yield of fiber, fiber quality, tolerance to abiotic stress and / or efficiency in the use of nitrogen when the present expression is increased in plants. The identified genes (including the genes identified by bioinformatics tools and their cured sequences), and the encoded polypeptide sequences are summarized in Table 27, just below. Table 27 Polynucleotides identified that affect plant yield, seed yield, oil yield, oil content, biomass, growth rate, vigor, fiber yield, fiber quality, tolerance to abiotic stress and / or nitrogen use efficiency of a plant





[00517] Table 27: The identified genes, their annotation, organism and polynucleotide and polypeptide sequence identifiers, "polynucl." = polynucleotide; "polypept." = polypeptide. EXAMPLE 11 IDENTIFICATION OF HOMOLOGICAL SEQUENCES THAT INCREASE SEED YIELD, OIL YIELD, GROWTH RATE, OIL CONTENT, FIBER YIELD, FIBER QUALITY, BIOMASS, VIGOR, ABST AND / OR ABST AND / OR NUE OF A PLANT
[00518] The concepts of ontology and paralogy have recently been applied to characterizations and functional classifications on the scale of comparisons of total genome. Orthologists and parallels are two main types of counterparts: The first evolved from a common ancestor by specialization and the others are related by duplication events. It is assumed that the parallels resulting from events of duplication of ancestors probably diverged in terms of function while the real orthologists are more likely to keep the function identical over the course of evolution.
[00519] To identify alleged orthologs of genes that affect plant yield, oil yield, oil content, seed yield, growth rate, vigor, biomass, tolerance to abiotic stress and / or efficiency in the use of nitrogen, all sequences were aligned using the BLAST (Basic Local Alignment Search Tool). Similar enough strings were tentatively grouped together. These supposed orthologists were also organized under a Filogram - a branching diagram (tree) assumed to be a representation of the evolutionary relationships between the biological rate. The alleged orthological groups were analyzed for their agreement with the filogram and in cases of disagreement these orthological groups were properly broken.
[00520] Expression data was analyzed and EST libraries were classified using a fixed vocabulary of standard terms, such as stages of development (for example, genes that show a similar expression profile through development with upward regulation at a specific stage, such as such as the seed filling stage) and / or plant organ (for example, genes that have a similar profile of expression in all its organs with upward regulation in specific organs, such as the seed). The annotations of all ESTs in clusters for a gene were analyzed statistically by comparison with their frequency in the cluster versus their abundance in the database, allowing the construction of a numerical and graphic expression profile of that gene, which is called "digital expression" . The justification for using these two complementary methods with methods of studying phenotypic association of QTLs, SNPs and phenotype expression correlation is based on the assumption that real orthologists are likely to maintain the identical function over the course of evolution. These methods provide different sets of indications of function similarities between two homologous genes, similarities at the sequence level - identical amino acids in the protein domains and similarity in expression profiles.
[00521] The search and identification of homologous genes involves sorting the available sequence information, for example, in public databases, such as the DNA database of Japan (DDBJ), Genbank, and the Database of Nucleic Acid Sequence from the European Molecular Biology Laboratory (EMBL) or versions of these or the MIPS database. Several different search algorithms have been developed, including, among others, the set of programs called BLAST programs. There are five implementations of BLAST, three designed for nucleotide sequence queues (BLASTN, BLASTX and TBLASTX) and two designed for protein sequence queues (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology: 76-80, 1994; Birren et al ., Genome Analysis, I: 543, 1997). These methods involve aligning and comparing strings. The BLAST algorithm calculates the percent sequence identity and performs the statistical analysis of the similarity between the two sequences. The software for performing the BLAST analysis is available to the public through the National Center for Biotechnology Information. Other software or algorithms are GAP, BESTFIT, FASTA and TFASTA. GAP uses the Needleman and Wunsch algorithm (J. Mol. Biol. 48: 443-453, 1970) to discover the alignment of two complete strings that increase the number of matches and reduce the number of gaps.
[00522] These homologous genes may belong to the same gene family. Analysis of a gene family can be performed using sequence similarity analysis. To perform this analysis, standard programs for multiple alignments can be used, for example, Clustal W. A neighborhood junction tree of proteins homologous to the genes in this invention can be used to provide an overview of structural and ancestral relationships. The sequence identity can be calculated using an alignment program as described above. Other plants are expected to carry a gene of similar function (ortholog) or a family of similar genes and these genes will provide the same preferred phenotype as the genes presented here. Advantageously, such family members can be useful in the methods of the invention. Examples of other plants included here, among others, are barley (Hordeum vulgare), Arabidopsis (Arabidopsis thaliana), corn (Zea mays), cotton (Gossypium), turnip seed oil (Brassica napus), rice (Oryza sativa), sugar cane (Saccharum officinarum), sorghum (Sorghum bicolor), soy (Glycine max), sunflower (Helianthus annuus), tomato (Lycopersicon esculentum), wheat (Triticum aestivum).
The sequence homology analyzes mentioned above can be performed in a full length sequence, but they can also be based on a comparison of certain regions, for example, conserved domains. The identification of these domains would also be within the sphere of knowledge of the technician in the subject and would involve, for example, a computer-readable format of the nucleic acids of the present invention, the use of alignment software programs and the use of publicly available information on domains of protein, topicals and preserved boxes. This information is available from the PRODOM (Hypertext Transfer Protocol: // World Wide Web (dot) biochem (dot) ucl (dot) ac (dot) uk / bsm / dbbrowser / protocol / prodomqry (dot) html) database, PIR (Hypertext Transfer Protocol: // pir (dot) Georgetown (dot) edu /) or Pfam (Hypertext Transfer Protocol: // World Wide Web (dot) sanger (dot) ac (dot) uk / Software / Pfam /). Sequence analysis programs designed for topic search can be used for the identification of conserved fragments, regions and domains as mentioned above. Preferred computer programs include, but are not limited to, MEME, SIGNALSCAN and GENESCAN.
[00524] A person skilled in the art can use the homologous sequences provided here to find similar sequences in another species and in other organisms. Homologues of a protein include peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and / or insertions in relation to the unmodified protein in question and having biological and functional activity similar to that of the unmodified protein from which they are derived. To produce these homologues, the amino acids in the protein can be replaced by other amino acids having similar properties (conservative changes, such as hydrophobicity, hydrophilicity, antigenicity, similar propensity to form or break a-helical structures or 3-leaf structures). Conservative substitution tables are well known in the art (see, for example, Creighton (1984) Proteins. W.H. Freeman and Company). Nucleic acid homologues encompass nucleic acids having nucleotide substitutions, deletions and / or insertions in relation to the unmodified nucleic acid in question and having biological and functional activity similar to those of the unmodified nucleic acid from which they are derived.
[00525] Table 28, below, lists a summary of orthologous and homologous sequences of the polynucleotide and polypeptide sequences presented in table 27, above, which were identified from databases using the NCBI BLAST software (for example, using the Blastp and tBlastn algorithms) and needle (EMBOSS package) as being at least 80% homologous to the selected polypeptides and polynucleotides, which are expected to increase plant yield, seed yield, oil yield, oil content, the growth rate, fiber yield, fiber quality, biomass, vigor, ABST and / or NUE of a plant. Table 28 Polynucleotides and homologous polypeptides that can increase plant yield, seed yield, oil yield, oil content, growth rate, fiber yield, fiber quality, biomass, vigor, the ABST and / or the NUE of a plant






































































[00526] Table 28: Polynucleotides (Polynuc.) And polypeptides (Polypept.) Are presented that are homologous to the identified polynucleotides or polypeptides in Table 27. Homol. = homologous; "Algor." = Algorithm; EXAMPLE 12 GENE CLONING AND GENERATION OF BINARY VECTORS FOR PLANT EXPRESSION
[00527] To validate its role in improving oil content, plant yield, seed yield, oil content, biomass, growth rate, fiber yield, fiber quality, ABST, NUE and / or vigor, the selected genes were overexpressed in plants, as follows. Cloning strategy
[00528] The genes mentioned in Examples 10 and 11 here just above were cloned into binary vectors for the generation of transgenic plants. For cloning, the full-length open reading frame (ORF) was first identified. In the case of ORF-EST clusters and in some cases already published, the mRNA sequences were analyzed to identify the entire open reading frame by comparing the results of several translation algorithms to proteins known from another plant species. To clone the full-length cDNAs, reverse transcription (RT) followed by the polymerase chain reaction (PCR; RT-PCR) was performed on the total RNA extracted from leaves, flowers, silica or other plant tissues, grown under normal conditions . The total RNA was extracted as described in "GENERAL EXPERIMENTAL AND BIOINFORMATIC METHODS" above. CDNA production and PCR amplification is performed using standard protocols described (Sambrook J., EF Fritsch, and T. Maniatis. 1989. Molecular Cloning. A Laboratory Manual., 2nd Ed. Cold Spring Harbor Laboratory Press, New York. ) and well known to those skilled in the art. PCR products are purified using the PCR purification kit (Qiagen). In the event that the entire coding sequence was not found, the Invitrogen RACE kit (RACE = R apid A ccess to cDNA E nds) [Quick Access to cDNA Ends] was used to access the complete cDNA transcription of the gene a from the RNA samples described above. The RACE products were cloned into a high copy vector followed by sequencing or sequencing directly.
[00529] The information from the RACE procedure was used to clone the entire ORF length of the corresponding genes.
[00530] If the genomic DNA has been cloned, the genes were amplified by direct PCR in genomic DNA extracted from the leaf tissue using the DNAeasy kit (Qiagen Cat. No. 69104).
[00531] In general, 2 sets of primers are synthesized for the amplification of each gene of a cDNA or of a genomic sequence; an external set of primers and an internal set (nest PCR primers). When necessary (for example, when the first PCR reaction does not result in a satisfactory product for sequencing), another primer (or two others) from the nest PCR primers was used.
[00532] To facilitate the cloning of cDNAs / genomic sequences, an extension of 8-12 bp was added to the 5 'of each primer. The primer extension includes an endonuclease restriction site. The restriction sites were selected using two parameters: (a). The site does not exist in the cDNA sequence; and (b). The restriction sites on the forward and reverse primers were designed so that the digested cDNA is inserted into the sense formation in the binary vector used for the transformation.
[00533] Each digested PCR product was inserted into a high copy vector pBlue-script KS plasmid vector [Blue-script KS plasmid vector, http: // www (dot) stratagene (dot) com / manuals / 212205 (dot) pdf ] or pUC19 (New England BioLabs Inc], or plasmids from these vectors. In some cases, the undigested PCR product was inserted into the pCR-Blunt II-TOPO (Invitrogen). In the case of the high copy vector pBlue-script KS plasmid (pGXN), the PCR product was inserted above the high copy plasmid of the NOS terminator (SEQ ID NO: 8092) originating from the binary vector pBI 101.3 (Acquisition of GenBank No. U12640, nucleotides 4356 to 4693) and below 35S promoter.
[00534] The sequencing of the amplified PCR products was performed using the ABI 377 sequencer (Amersham Biosciences Inc). In some cases, after confirming the sequence of the cloned genes, the cloned cDNA accompanied or not with the NOS terminator was introduced into a modified binary vector pGI containing the At6669 promoter or the 35S promoter (SEQ ID NO: 8094), via digestion with the appropriate restriction endonucleases. In any case, the insertion was followed by a simple copy of the NOS terminator (SEQ ID NO: 8092). The digested products and the linearized plasmid vector are ligated using the enzyme T4 DNA ligase (Roche, Switzerland).
[00535] High copy plasmids containing the cloned genes were digested with restriction endonucleases (New England Biolabs Inc) according to the designated locations on the primers and cloned into binary vectors according to Table 29, below. Several DNA sequences of the selected genes have been synthesized by a commercial supplier, namely, GeneArt, GmbH [Hypertext Transfer Protocol: // World Wide Web (dot) geneart (dot) com /)]. The synthetic DNA was designed in silico. Suitable enzyme restriction sites have been added to the cloned sequences at the 5 'and 3' ends to allow for later cloning into the binary pQFN vectors (Figure 2) downstream of the At6669 promoter (SEQ ID NOs: 8093 and 8096).
[00536] Binary vectors used for cloning: The plasmid pPI is constructed by inserting a sequence of synthetic poly (A) signals from the basic plasmid vector pGL3 (Promega, Acc No U47295; bp 4658-4811) at the site of HindilI restriction of the binary vector pBI101.3 (Clontech, Acc. No. U12640). PGI (pBXYN) is similar to pPI, however the original gene in the backbone, the GUS gene, is replaced by the GUS-Intron gene followed by the NOS terminator (SEQ ID NO: 8092) (Vancanneyt. G, et al MGG 220, 245-50, 1990). PGI has been used in the past to clone polynucleotide sequences, initially under the control of the 35S promoter [Odell, JT, et al. Nature 313, 810 - 812 (28 February 1985); SEQ ID NO: 8094].
[00537] The modified pGI vector (pQXYNc in Figure 12, or pQFN and pQFN in Figure 2, or pQYN_6669 in Figure 1) are modified versions of the pGI vector in which the cassette is inverted between the left and right edges, so that the gene and its corresponding promoter are close to the right border and the NPTII gene is close to the left border.
[00538] At6669, the promoter sequence of Arabidopsis thaliana (SEQ ID NO: 8096) is insertion in the modified binary vector pGI, upstream of the cloned genes, followed by DNA binding and extraction of E. coli positive colony plasmid, as described above.
[00539] Colonies are analyzed by PCR using primers covering the insert, which are designed to encompass the introduced promoter and gene. Positive plasmids are identified, isolated and sequenced.
[00540] The genes that have been cloned by the present inventors are shown in Table 29 below. Table 29 Cloned genes in high copy number plasmids



Table 29. EXAMPLE 13 PRODUCTION OF ARABIDOPSIS TRANSGENIC PLANTS EXPRESSING POLYNUCLEOTIDES OF SOME APPLICATIONS OF THE INVENTION Experimental Methods
[00541] Production of agrobacterium tumefaciens cells harboring the binary vectors according to some applications of the invention - Each of the binary vectors described in Example 12 above was used to transform the Agrobacterium cells. Two additional binary constructs, having only the 35S or At6669 promoter or no additional promoter were used as negative controls.
[00542] Binary vectors were introduced to Agrobacterium tumefaciens GV301, or competent cells LB4404 (about 109 cells / mL) by electroporation. Electroporation was performed using a MicroPulser electroporator (BioRad), 0.2 cm cuvettes (BioRad) and EC-2 electroporation program (BioRad). The treated cells were cultured in liquid LB medium at 28 ° C for 3 hours, then placed on LB agar supplemented with gentamicin (50 mg / L; for strains of Agrobacterium GV301) or streptomycin (300 mg / L; for strains of Agrobacterium LB4404) and kanamycin (50 mg / L) at 28 ° C for 48 hours. Colonies of Abrobacterium that were developed in the selective medium, were also analyzed by PCR using primers designed to cover the sequence inserted in the plasmid pPI. The resulting PCR products were isolated and sequenced to verify that the correct polynucleotide sequences of the invention were introduced correctly into Agrobacterium cells.
[00543] Preparation of Arabidopsis plants for transformation - Arabidopsis thaliana var Columbia (To plants) were transformed according to the Floral Dip procedure [Clough SJ, Bent AF. (1998) Floral dip: a simplified method for transformation in Agrobacterium medium into Arabidopsis thaliana. Plant J. 16 (6): 73543; and Desfeux C, Clough SJ, Bent AF. (2000) Female reproductive tissues are the main targets for transformation into Agrobacterium into Arabidopsis by the floral-dip method. Plant Physiol. 123 (3): 895-904] with minor modifications. In summary, the Arabidopsis thaliana Columbia (Co10) To plants were sown in 250 ml pots filled with a moist peat-based culture mixture. The pots were covered with foil and plastic sheeting, kept at 4 ° C for 3 to 4 days, and then discovered and incubated in an 18-24 growth chamber in 16/8 hour light / dark cycles. . TO plants were ready for transformation six days before anthesis.
[00544] Preparation of Agrobacterium carrying the binary vectors for transformation into Arabidopsis plants - Single colonies of Agrobacterium carrying the binary vectors harboring the genes of some applications of the invention were grown in LB medium supplemented with kanamycin (50 mg / L) and gentamicin ( 50 mg / L). The cultures were incubated at 28 ° C for 48 hours under intense agitation and centrifuged at 4000 rpm for 5 minutes. The pellets comprising Agrobacterium cells were resuspended in a transformation medium, which contains half the dose (2.15 g / L) -Murashige Skoog (Duchefa); 0.044 gM benzylaminopurine (Sigma); 112 gg / L vitamin B5 Gamborg (Sigma); 5% sucrose and 0.2 ml / L Silwet L-77 (specialists OSI, CT) in double distilled water, with a pH of 5.7.
[00545] Transformation of Arabidopsis plants with agrobacterium. The plant transformation was performed by inversion of each plant in a suspension of Agrobacterium so that the plant tissue above the ground was submerged for 3 to 5 seconds.
[00546] Each inoculated To plant was immediately placed in a plastic tray, and then covered with a transparent plastic lid to maintain moisture and was kept in the dark at room temperature for 18 hours to facilitate infection and transformation. The transformed (transgenic) plants were then discovered and transferred to a greenhouse for recovery and maturation. The transgenic To plants were grown in a greenhouse for 3 to 5 weeks until the silicas were brown and dry, and then the seeds were harvested from the plants and kept at room temperature until sowing.
[00547] Generation of transgenic plants Ti and T2 - To generate transgenic plants T1 and T2 containing the genes, the seeds collected from transgenic To plants had the surface sterilized by soaking in 70% ethanol for 1 minute, followed by soaking in hypochlorite. 5% sodium and 0.05% triton for 5 minutes. The seeds with a sterile surface were completely washed in sterile distilled water and then placed in culture plates containing Murashig-Skoog (Duchefa) half power; 2% sucrose; 0.8% plant agar; 50 mM kanamycin; and 200 mM carbenicillin (Duchefa). The culture plates were incubated at 4 ° C for 48 hours and then transferred to a culture room at 25 ° C for an additional week of incubation. The vital Arabidopsis T1 plants were transferred to fresh culture plates for another week of incubation. After incubation, T1 plants were removed from the culture plates and planted in a growth mix contained in 250 ml pots. The transgenic plants were allowed to grow in a greenhouse until maturity. The seeds harvested from T1 plants were cultivated and grew to maturity as well as T2 plants under the same conditions of cultivation and growth as T1 plants. EXAMPLE 14 ASSESSMENT OF THE NUE OF TRANSGENIC ARABIDOPSIS UNDER LOW OR NORMAL NITROGEN CONDITIONS USING IN VITRO TESTS (TISSUE CULTURE, T1 AND T2 PLANTS)
[00548] Test I: plant growth under levels of low and favorable concentration of nitrogen.
[00549] The seeds sterilized on the surface were sown in basal medium [50% Murashige-Skoog medium (MS) supplemented with 0.8% plant agar as a solidifying agent] in the presence of kanamycin (used with a selection agent). After sowing, the plates were transferred for 2-3 days for stratification at 4 ° C and then cultured at 25 ° C in daily cycles of 12 hours of light and 12 hours of darkness for 7 to 10 days. At that moment, the seedlings chosen at random were carefully transferred to plates containing V2 MS medium (15 mM N) for the treatment of normal nitrogen concentration and 0.75mM of nitrogen for treatments of low nitrogen concentration. For experiments carried out on a T2 line, each plate contained 5 seedlings from the same transgenic event and 3 to 4 different plates (replicates) for each event. For each polynucleotide of the invention, at least four or five independent transformation events were analyzed for each construct. For experiments performed in Ti line, each plate contained 5 seedlings of 5 independent transgenic events and 3 to 4 different plates (replicates) were planted. In total, for the Ti line, 20 independent events were evaluated. The plants expressing the polynucleotides of the invention were compared to the average measurement of the control plants (empty vector or GUS reporter gene under the same promoter) used in the same experiment.
[00550] Digital image - A laboratory image acquisition system, consisting of a digital reflex camera (Canon EOS 300D) with a 55 mm focal length lens (Canon EF-S series), mounted on a scanning device reproduction (Kaiser RS) which includes 4 units of light (4 lamps of 150 Watts) and located in a dark room, was used to capture images of small plants sown on square agar plates.
[00551] The image capture process was repeated every 3-4 days starting on day 1 through day 10 (see, for example, the images in Figures 3A-F). An image analysis system was used, which consists of a personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.39 [Java-based image processing program that was developed at the National Institutes of Health from the USA and is available for free on the Internet at http: // rsbweb (dot) nih (dot) gov /]. The images were captured in a resolution of 10 Mega Pixels (3888 x 2592 pixels) and stored in a low compression JPEG format (Joint Photographic Experts Group standard). Then, the analyzed data were saved in text files and processed using the statistical analysis software JMP (instituto SAS).
[00552] Seedling analysis - Through digital analysis, seedling data was calculated, including leaf area, root coverage and root length.
[00553] The relative growth rate for the various seedling parameters was calculated according to the following formulas XIV, V (described above) and XV. Formula XIV Relative growth rate of leaf area = Regression coefficient of leaf area over time. Formula XV: Rate of relative growth of root length = Coefficient of regression of root length over time.
[00554] At the end of the experiment, the small plants were removed from the medium and weighed to determine the fresh weight of the plant. The seedlings were then dried for 24 hours at 60 ° C and weighed again to measure the dry weight of the plant for further statistical analysis. Dry and fresh weights are provided for each Arabidopsis plant. The growth rate was determined by comparing the leaf area coverage, root coverage and root length between each pair of sequential photographs, and the results are used to determine the effect of the gene introduced on the plant's vigor under ideal conditions . Similarly, the effect of the introduced gene on the accumulation of biomass, under ideal conditions, was determined by comparing the fresh and dry weight of the plant to that of the control plants (containing an empty vector or the GUS reporter gene under the same promoter). From each building created, 3 to 5 independent transformation events are examined in replicates.
[00555] Statistical analysis - To identify the genes giving significantly greater tolerance to the vigor of the plant or greater root architecture, the results obtained from transgenic plants were compared to those obtained from control plants. To identify the genes and constructs with superior performance, the results of the independent transformation events tested were analyzed separately. To assess the effect of a gene event under control, data were analyzed using Student's t-test and the p-value was calculated. The results were considered significant for p <0.1. The JMP statistical software package was used (Version 5.2.1, SAS Institute Inc., Cary, NC, USA). Experimental Results:
[00556] The genes presented in Table 30 demonstrated a significant improvement in the plant's NUE since they produced greater biomass in the plant (dry and fresh weights of the plant) in the T2 generation when grown under limit conditions of growth in nitrogen, compared to plants of control. The genes were cloned under the regulation of a constitutive promoter (At6669, SEQ ID NO: 8096) or Ca35S (SEQ ID NO: 8094). The evaluation of each of the genes was carried out by testing the performance of a different number of events. Some of the genes have been evaluated in more than one tissue culture assay. The results obtained in these second experiments were also significantly positive. Table 30 Genes demonstrating improved plant performance under Low nitrogen conditions under regulation of the 6669 promoter


[00557] Table 30. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. Values are provided by plant.
[00558] The genes presented in Tables 31 and 32 demonstrated a significant improvement in the plant's NUE, since they produced a greater leaf biomass (leaf area) and root biomass (length and root cover) (Table 31) and a higher relative growth rate of leaf area, cover and root length (Table 32) when grown under nitrogen-limited growth conditions compared to other plants. Plants that produce more root biomass have a better chance of absorbing a large amount of nitrogen from the soil. Plants that produce higher leaf biomass are better able to produce assimilates). The genes were cloned under the regulation of the constitutive promoter (At6669) or the preferred root promoter (RaizP). The evaluation of each of the genes was carried out by testing the performance of a different number of events. Some of the genes have been evaluated in more than one tissue culture assay. This second experiment confirmed the significant improvement in leaf and root performance. Events with p values <0.1 were considered statistically significant. Table 31 Genes demonstrating improved plant performance under Low nitrogen conditions under regulation of the 6669 promoter


[00559] Table 31. "CONT." - Control; "Avg." - Average; "% Increase." =% Increase; "p-val." - p-value, L- p <0.01. Values are provided by plant. Table 32 Genes demonstrating improved plant performance under Low nitrogen conditions under regulation of the 6669 promoter

[00560] Table 32. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. Values are provided by plant.
[00561] The genes presented in Table 33 demonstrated a significant improvement in the performance of the plant since they produced greater biomass in the plant (dry and fresh weights of the plant) in the T2 generation when grown under normal conditions of growth in nitrogen, compared to plants of control. The genes were cloned under the regulation of a constitutive promoter (At6669, SEQ ID NO: 8096) or 35S (SEQ ID NO: 8094). The evaluation of each of the genes was carried out by testing the performance of a different number of events. Some of the genes have been evaluated in more than one tissue culture assay. The results obtained in these second experiments were also significantly positive. Table 33 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter







[00562] Table 33. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. Values are provided by plant
[00563] The genes presented in Tables 34 and 35 demonstrated a significant improvement in plant performance, since they produced a greater leaf biomass (leaf area) and root biomass (length and root cover) (Table 34) and a higher relative growth rate of leaf area, cover and root length (Table 35) when grown under normal growth conditions under nitrogen, compared to other plants. Plants that produce more root biomass have a better chance of absorbing a large amount of nitrogen from the soil. Plants that produce higher leaf biomass are better able to produce assimilates). The genes were cloned under the regulation of the constitutive promoter (At6669) or the preferred root promoter (RaizP). The evaluation of each of the genes was carried out by testing the performance of a different number of events. Some of the genes have been evaluated in more than one tissue culture assay. This second experiment confirmed the significant improvement in leaf and root performance. Events with p values <0.1 were considered statistically significant. Table 34 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter










[00564] Table 34. "CONT." - Control; "Avg." - Average; "% Increase." =% Increase; "p-val." - p-value, L- p <0.01. Values are provided by plant. Table 35 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter









[00565] Table 35. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. Values are provided by plant. Results of T1 plants
[00566] The genes presented in Tables 36-39 demonstrated a significant improvement in plant biomass and root development, as they produced greater root and leaf biomass (coverage and root length) (Table 36), greater biomass of root and leaf (leaf area, leaf length and cover; Table 37), a higher relative growth rate of leaf area, cover and leaf length (Table 38), and greater fresh and dry weight (Table 39 ) when grown under standard conditions or with low nitrogen, compared to other plants. Plants that produce more root biomass have a better chance of absorbing a large amount of nitrogen from the soil. Plants that produce higher leaf biomass are better able to produce assimilates). The genes were cloned under the regulation of the constitutive promoter (At6669; SEQ ID NO: 8096) or the preferred root promoter (RaizP). The evaluation of each of the genes was carried out by testing the performance of a different number of events. Some of the genes have been evaluated in more than one tissue culture assay. This second experiment confirmed the significant improvement in leaf and root performance. Events with p values <0.1 were considered statistically significant. Table 36 Genes demonstrating improved plant performance under Low nitrogen conditions under promoter regulation

[00567] Table 36. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. * - measurement on day 9 of the plantation Table 37 Genes demonstrating improved performance under standard growth conditions (IT generation) under the regulation of the At6669 promoter

[00568] Table 37. "CONT." - Control; "Avg." - Average; "% Aum." = increase; "p-val." - p value. * - measurement on day 5 of planting Table 38 Genes showing improved growth rate under standard growth conditions (IT generation) under the regulation of the At6669 promoter
*
[00569] Table 38. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value. Table 39 Genes demonstrating improved plant performance under Low nitrogen conditions under 6669 promoter regulation

[00570] Table 39. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value. EXAMPLE 15 ASSESSMENT OF THE NUE, YIELD AND GROWTH RATE OF THE TRANSGENIC ARABIDOPSIS PLANT UNDER NORMAL OR LOW NITROGEN FERTILIZATION IN A GREENHOUSE TEST
[00571] Test I: Efficiency in the use of nitrogen: The biomass of the plant of the seed yield and the growth rate of the plant with ideal and limited concentration of nitrogen under greenhouse conditions - This test follows the production of the seed yield, the formation of biomass and the growth of the rosette area of plants grown in the greenhouse under nitrogen conditions for limiting and non-limiting growth. The Arabdopsis transgenic seeds were sown on an agar medium supplemented with V2 MS medium and a selection agent (kanamycin). The transgenic T2 seedlings were then transplanted to 1.7-liter trays with peat and perlite in a 1: 1 ratio. The trays were irrigated with a solution containing nitrogen limiting conditions, which were achieved by irrigating the plants with a solution containing 1.5 mM inorganic nitrogen in the form of KNO3 supplemented with 1 mM KH2PO4, 1 mM MgSO4, 3, 6 mM KC1, 2 mM CaC12 and microelements, while normal nitrogen levels were achieved by applying a 6 mM inorganic nitrogen solution also in the form of KNO3 with 1 mM KH2PO4, 1 mM MgSO4, 2 mM CaC12 and microelements. All plants were grown in the greenhouse until the seeds ripened. The seeds were harvested, extracted and weighed. The remaining biomass of the plant (the above-ground tissue) was also harvested and weighed immediately or after drying in the oven at 50 ° C for 24 hours.
[00572] Each building was validated as its T2 generation. Transgenic plants transformed with a construction formed by an empty vector carrying the 35S promoter and the selectable marker were used as controls.
[00573] The plants were analyzed for general size, growth rate, flowering, seed yield, weight of 1000 seeds, dry matter and harvest index (CI - seed yield / dry matter). The performance of transgenic plants is compared to that of control plants grown in parallel under the same conditions. Mock- transgenic plants expressing the uidA reporter gene (GUS-Intron) or even with no genes, were used as controls under the same promoter.
[00574] The experiment was planned in a distribution of lots in randomized nested niches. For each gene of the invention 3 out of 5 independent transformation events were analyzed from each construct.
[00575] Digital image - A laboratory image acquisition system, which consists of a digital reflex camera (Canon EOS 300D) accompanied by a 55 mm focal length lens (Canon EF-S series), installed in a digital imaging device reproduction (Kaiser RS), which includes four light units (4 x 150 Watt lamps) was used to capture images of plant samples.
[00576] The image capture process was repeated every two days from the 1st day after transplanting until the 15th. The same camera, positioned in a customized iron frame, was used to capture images of larger sawn plants in white vats in an environmentally controlled greenhouse. The vats are square in shape and include 1.7 liter trays. During the capture process, the pots are placed under the iron easel, avoiding direct sunlight and shadows.
[00577] An image analysis system was used, which consists of a personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.39 [Java-based image processing program that was developed at National US Institutes of Health and is freely available on the Internet at http: // rsbweb (dot) nih (dot) gov /]. The images are captured in a resolution of 10 Mega Pixels (3888 x 2592 pixels) and stored in a low compression JPEG format (Joint Photographic Experts Group standard). Then, the analyzed data were saved in text files and processed using the statistical analysis software JMP (instituto SAS).
[00578] Leaf analysis - Using digital analysis, leaf data was calculated, including leaf number, rosette area, rosette diameter, leaf leaf area.
[00579] Vegetative growth rate: the relative growth rate (TCR) of the leaf number [formula XI (described above)], rosette area (formula XVI), bed cover (formula XVII) and the harvest index ( formula IV) was calculated with the indicated formulas. Formula XVI: Rosacea area relative growth rate = rosacea area regression coefficient over time. Formula XVII Flat cover relative growth rate = flat cover regression coefficient over time.
[00580] Average seed weight - At the end of the experiment, all seeds are collected. The seeds were spread on a glass tray and a picture was taken. Using digital analysis, the number of seeds in each sample was calculated.
[00581] Dry weight and seed production - Around the 80th day of sowing, the plants are harvested and left to dry at 30 ° C in a drying chamber. The biomass and seed weight of each lot is measured and divided by the number of plants in each lot. Dry weight = total weight of the vegetative portion above the soil (excluding the roots) after drying at 30 ° C in a drying chamber; Seed production per plant = total seed weight per plant (g). Weight of 1000 seeds (the weight of 1000 seeds) (gr.).
[00582] The harvest index (CI) was calculated using Formula IV, as described above.
[00583] Percentage of oil in the seeds - At the end of the experiment, all seeds from each bed were collected. Seeds from 3 beds are mixed and crushed and then assembled in the extraction chamber. 210 ml of n-Hexane (Cat. No. 080951 Biolab Ltd.) is used as the solvent. The extraction was carried out for 30 hours with an average temperature of 50 ° C. Once the extraction was finished, the n-Hexane was evaporated using the evaporator under 35 ° C and vacuum conditions. The process was repeated twice. The information obtained from the Soxhlet extractor (Soxhlet, F. Die gewichtsanalytische Bestimmung des Milchfettes, Politechnisches J. (Dingler's) 1879, 232, 461) was used to create a calibration curve for Low Resonance NMR. The oil content of all seed samples was determined using the Low Resonance NMR (MARAN Ultra-Oxford Instrument) and its MultiQuant software package.
[00584] Silica length analysis - On the 50th day after sowing, 30 silicas of different plants in each bed are sampled in block A. The chosen silicas are green-yellow and are collected from the lower parts of a stem of the cultivated plant. A digital photograph was taken to determine the length of the silica.
[00585] Statistical analysis - To identify the genes that confer a significantly improved tolerance to abiotic stresses, the results obtained with transgenic plants are compared with those obtained from control plants. To identify the top performing genes and constructs, the results of the tested independent transformation events are analyzed separately. The data were analyzed using the Student's t-test and the results are considered significant if the p-value was less than 0.1. The J1MP statistical software package was used (Version 5.2.1, SAS Institute Inc., Cary, NC, USA). Table 40 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter





[00586] Table 40. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. * - was regulated by the 35S promoter (SEQ ID NO: 8094). Table 41 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter





[00587] Table 41. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. Table 42 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter




[00588] Table 42. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. Table 43 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter





[00589] Table 43. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. * - was regulated by the 35S promoter (SEQ ID NO: 8094). Table 44 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter



[00590] Table 44. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. * - was regulated by the promoter 35S. EXAMPLE 16 EVALUATION OF THE NUE, YIELD AND GROWTH RATE OF THE TRANSGENIC ARABIDOPSIS PLANT UNDER NORMAL OR LOW NITROGEN FERTILIZATION IN A GREENHOUSE STUDY
[00591] Test 2: Efficiency in the Use of Nitrogen measured up to the pRtAain of watering. the plant's biomass of the seed yield and the growth rate of the plant with ideal and limited concentration of nitrogen under greenhouse conditions - This test follows the formation of plant biomass and the growth of the rosette area of plants grown in the greenhouse under conditions of nitrogen for limiting and non-limiting growth. The Arabdopsis transgenic seeds were sown on an agar medium supplemented with V2 MS medium and a selection agent (kanamycin). The transgenic T2 seedlings were then transplanted to 1.7-liter trays with peat and perlite in a 1: 1 ratio. The trays were irrigated with a solution containing nitrogen limiting conditions, which were achieved by irrigating the plants with a solution containing 1.5 mM inorganic nitrogen in the form of KNO3 supplemented with 1 mM KH2PO4, 1 mM MgSO4, 3, 6 mM KC1, 2 mM CaC12 and microelements, while normal nitrogen levels were achieved by applying a 6 mM inorganic nitrogen solution also in the form of KNO3 with 1 mM KH2PO4, 1 mM MgSO4, 2 mM CaC12 and microelements. All plants were grown in the greenhouse until the seeds ripened. The plant's biomass (the above-ground tissue) was weighed directly after the rosette harvest (fresh weight of the plant [Fresh Weight]).
[00592] Then, the plants were dried in an oven at 50 ° C for 48 hours and weighed (dry weight of the plant [Dry Weight]).
[00593] Each building has been validated as its T2 generation. Transgenic plants transformed with a construction formed by an empty vector carrying the 35S promoter and the selectable marker were used as controls.
[00594] The plants were analyzed for their general size, growth rate, fresh weight and dry matter. The performance of transgenic plants is compared to that of control plants grown in parallel under the same conditions. Mock- transgenic plants expressing the uidA reporter gene (GUS-Intron) or even with no genes, were used as controls under the same promoter.
[00595] The experiment was planned in a batch distribution in randomized nested niches. For each gene of the invention 3 out of 5 independent transformation events were analyzed from each construct.
[00596] Digital image - A laboratory image acquisition system, which consists of a digital reflex camera (Canon EOS 300D) accompanied by a 55 mm focal length lens (Canon EF-S series), installed in a digital imaging device reproduction (Kaiser RS), which includes four light units (4 x 150 Watt lamps) was used to capture images of plant samples.
[00597] The image capture process was repeated every two days from the 1st day after transplanting until the 15th. The same camera, positioned in a customized iron frame, was used to capture images of larger sawn plants in white vats in an environmentally controlled greenhouse. The vats were square in shape and included 1.7 liter trays. During the capture process, the pots were placed under the iron easel, avoiding direct sunlight and shadows.
[00598] An image analysis system was used, which consists of a personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.39 [Java-based image processing program that was developed at National US Institutes of Health and is freely available on the Internet at http: // rsbweb (dot) nih (dot) gov /]. The images were captured in a resolution of 10 Mega Pixels (3888 x 2592 pixels) and stored in a low compression JPEG format (Joint Photographic Experts Group standard). Then, the analyzed data were saved in text files and processed using the statistical analysis software JIMP (SAS institute).
[00599] Leaf analysis - Using digital analysis, the leaf data was calculated, including the number of leaves, the area of the rosette, the diameter of the rosette, the area of the leaf leaf.
[00600] Vegetative growth rate: the relative growth rate (TCR) of the leaf number [Formula XI (described above)], rosette area (formula XVI, described above) and bed cover (formula XVII, described above) is calculated using the formulas indicated.
[00601] Plant Fresh and Dry Weight Around day 80 of sowing, the plants were harvested and weighed directly to determine the plant's fresh weight (FW) and placed in a drying chamber at 50 ° C for 48 hours to drying before weighing to determine the dry weight of the plant (DW).
[00602] Statistical analysis - To identify the genes that confer a significantly improved tolerance to abiotic stresses, the results obtained with transgenic plants were compared with those obtained from control plants. To identify the genes and constructs with superior performance, the results of the independent transformation events tested were analyzed separately. The data were analyzed using the Student's t-test and the results are considered significant if the p-value was less than 0.1. The JMP statistical software package was used (Version 5.2.1, SAS Institute Inc., Cary, NC, USA). Experimental Results:
[00603] The genes listed in Tables 45-48 improved the plant's NUE when grown under normal levels of nitrogen concentration. These genes produced larger plants with a larger photosynthetic area and biomass (fresh weight, dry weight, rosette diameter, rosette area and bed cover) when grown under normal nitrogen conditions. The genes were cloned under the regulation of the constitutive promoter (At6669; SEQ ID NO: 8096) and the preferred root promoter (RaizP). The evaluation of each of the genes was carried out by testing the performance of a different number of events. Events with p values <0.1 were considered statistically significant. Table 45 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter






[00604] Table 45. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. * - measurement on day 9 of the plantation Table 46 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter





[00605] Table 46. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. Table 47 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter





[00606] Table 47. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. Table 48 Genes demonstrating improved plant performance under normal conditions under regulation of the 6669 promoter

[00607] Table 48. "CONT." - Control; "Avg." - Average; "% Aum." =% increase; "p-val." - p value, L- p <0.01. EXAMPLE 17 IDENTIFICATION OF A NEW ARABIDOPSIS PROMOTER
[00608] W02004 / 081173 discloses the At6669 promoter (SEQ ID NO: 8093 at present) which is capable of expressing a heterologous polynucleotide operably connected in a host cell. Experimental Procedures
[00609] Isolation of DNA regulatory elements (DREs): A high production method of cloning DNA regulatory elements (DREs) using a single reaction tube, the method referred to here as "one tube" was used in order to enable large-scale production of transformed DRE plants. Thus, the genomic DNA (gDNA) was extracted from the leaves of Arabidopsis thaliana using the Mini Kit for Easy DNA Removal from Plants (Qiagen, Germany).
[00610] Primers for PCR amplification of DREs were designed using the PRIMER3 software and modified to contain restriction sites absent from the DRE sequence, for insertion of the PCR producer into plasmid pQYN.
[00611] Amplification of the new AT6669 promoter sequence - The promoter was cloned from a genomic DNA of Arabidopsis thaliana using the following primers:
[00612] Advance primer (without location restriction): 5'- TATACCAGTGGAGACGAAAGC (SEQ ID NO: 8098); and Primer reverse (which includes a Sail restriction site): 5'- TAATAAATAGTCGACTCTTTGGGG (SEQ ID NO: 8099).
[00613] Analyzes of the polymerase chair reaction were performed using the Taq Long Expansion Model PCR Kit (Roche), according to the manufacturer's instructions, using as a thermal cycle: 92 ° C / 2 min 10 x [94 ° C / 10 min -> 55 ° C / 30 sec. -> 68 ° C / 5 min] -> 18 x [94 ° C / 10 min - »• 55 ° C / 30 sec. - »• 68 ° C / 5 min (+ 20 sec. Each cycle)] -» • 68 ° C / 7 min.
[00614] The amplified PCR product was digested with the restriction enzymes Hindlll and Sail and was designated as 6669 Cid506.
[00615] Vector pQYN - The starting plasmid is pQYN (Pid # 1468; Figure 5). This plasmid is based on plasmid pBII01 (Clontech, Laboratories, Inc. Mountain View, CA 94043) and contains the following different characteristics from pBII01: (i) the PoliA signal was inserted before the MCS (multi cloning site) ) (upstream of the Hindlll restriction site); (ii) the GUS gene has been replaced by the GUS intron gene; (iii) Originally the pBI101 NPTII expression cassette was close to the right end of the DNA. In pQYN, the region between the left and right ends (not including the ends) was inverted in order to bring the NPTII expression cassette closer to the left end and to bring the intron GUS expression cassette closer to the right end.
[00616] Cloning of the promoter sequence in the pQYN vector - The pQYN vector was digested with HindllUSall. The 6669 Cid506 that was digested in HindllUSall was linked to the plasmid pQYN (Pid # 1468) digested by HindllUSall, creating plasmid pQYN_6669 (Pid # 1996). To facilitate cloning in plasmid pQYN_6669 (Pid # 1996), the expanded MCS + NOS terminator was linked to pQYN 6669 (Pid # 1996) digested with SalUEcoRl, replacing the existing MCS + GUS intron + NOS terminator. There was no change in the sequence of the NOS terminator. The resulting plasmid was designed for pQFN (Pid # 2054) (Figure 6).
[00617] Generation of a nucleic acid construct, including the new promoter and a heterologous coding sequence (eg, a reporter gene). I. GUS Reporter Expression Cassette
[00618] Generation of the 6669 Cid506 expression cassette promoter + GUS intron + NOS terminator (GUS intron expression cassette At6669) - the GUS intron + NOS terminator cassette was extracted from pQXYN (Pid # 1481) digesting with restriction enzymes SmaUEcoRl e linked to pQFN (Pid # 2054), which was also digested with SmaUEcoRl, generating the final plasmid pQFYN (Pid # 2431; Figure 8). There was no change in the sequence of the NOS terminator.
[00619] The transformation of agrobacterium with the At6660-GUS intron expression cassette and also with Arabidopsis thaliana Columbia (for plants) was carried out essentially as described in Example 13 just above using the At6660-GUS intron expression cassette. In addition, the generation of T1 and T2 transgenic plants housing the At6660-GUS intron expression cassette was performed as described in Example 13 just above. Evaluation of the promoter's activity
[00620] Evaluation of the new sequence activity of the AT6669 promoter in transgenic plants.
[00621] The ability of the DRE to promote gene expression in plants was determined based on the expression of the GUS reporter gene. Thus, small Arabdopsis transgenic plants at different stages of development were subjected to GUS assays using a standard GUS staining protocol [Jefferson RA, Kavanagh TA, Bevan MW. 1987. GUS fusions: beta-glucuronidase as a versatile and sensitive gene fusion marker in taller plants. EMBO J. 6 (13): 3901-7], Experimental results
[00622] Identification of a new At6669 promoter with two new regulatory elements -
[00623] The present inventors have surprisingly disclosed that during the cloning procedure a new sequence (described by SEQ ID NO: 8096) that includes some mutations with respect to the previously disclosed At6669 promoter (SEQ ID NO: 8093) was obtained. A comparison of the sequence between the two promoters is provided in Figure 5 with the unmatched nucleotides being underlined. As shown by the sequence alignment (Figure 5), the new promoter identified in the present exhibits three sites of regulatory elements compared to the previously disclosed At669 promoter, as follows: the regulatory element "YACT" (Y can be a thymidine nucleotide or a cytosine ) in position 862-865 of SEQ ID NO: 8096; and two locations of the regulatory element "AAAG" at positions 2392-2395 and 2314-2317 of SEQ ID NO: 8096.
[00624] The new At6669 comprises an additional YACT regulatory element that is capable of targeting a mesophyll expression module - High rates of photosynthesis, greater efficiency in the use of water greater efficiency in the use of nitrogen from C4 plants are attributed to the unique assimilation mode carbon in these plants, which includes strict compartmentalization of assimilatory enzymes into mesophilic cells [which include phosphenol pyruvate carboxylase (ppcAl)] and packet cells
[00625] sheath [including ribulose bisphosphate carboxylase / oxygenase]. The regulatory element "YACT" was considered by Gowik U, et al., 2004 (cis-Regulatory elements for mesophyll-specific gene expression in the C4 plant Flaveria trinervia, the promoter of the C4 phosphoenolpyruvate carboxylase gene; Plant Cell. 16: 10771090 ) as an important component of module 1 of mesophilic expression (Meml) of ppcAl in the dicotyledonous LA F. trinervia. In addition, when used in a heterologous expression system, the YACT regulatory sequence was shown to be necessary and sufficient for high expression of specific mesophilics (3-glucuronide reporter, and as an enhancer that directs the specific expression of the plant's ppcAl promoter mesophilic) C3 F. pringlei (Gowik U, et al., 2004, Supra).
[00626] The new At6669 comprises two additional sites of AAAG regulatory elements, the primary binding site for Dof transcription factors - The AAAG regulatory element is the primary site required for binding of Dof proteins to corn (Z.m.). Dof proteins are DNA-binding proteins, presumably with only a zinc finger, and are unique to plants. There are four known Dof proteins: Dofl, which improves the transcription of both cytosolic orthophosphate kinase (CyPPDK) promoters and a non-photosynthetic PEPC gene; Dof2, which suppresses the C4PEPC promoter; Dof3; and PBF, which is an endosperm-specific Dof protein that binds to the prolamine box [Yanagisawa S, Schmidt RJ, Diversity and similarity among recognition sequences of Dof transcription factors. Plant J, 17: 209-214 (1999)]. The transcription factors Dofl and Dof2 are associated with the expression of multiple genes involved in carbon metabolism in corn [Yanagisawa S, Plant J 21: 281-288 (2000)].
[00627] Together, these results show that the new promoter identified in the present can direct the expression of heterologous polynucleotides in a host cell with high efficiency.
[00628] Characterization of the new sequence of the At6669 promoter (SEQ ID NO: 8096) - The ability of the At6669 promoter to promote gene expression in plants was determined based on the expression of the GUS reporter gene. Several characteristics of the At6669 isolated promoter of some applications of the invention are described in Figures 9A-D, 10A-D and 11A-L. As is evident from the experiments, the At6669 promoter is constitutively expressed in the plant model at various stages of development, including the early vegetative stages of seedling (eg, day 10-11; Figures 9A-D), the stage of sieving in which the plant is developed towards the reproductive stage (eg: day 20, Figures 10A-D), and the mature reproductive stage (eg: day 40-41; Figures 11A-L) in which various tissues express the reporter gene under the new promoter At6669 (SEQ ID NO: 8096), including roots, leaves, stems and flowers, with a strong expression in leaves and flowers.
[00629] Although the invention has been described together in specific applications of it, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Consequently, it intends to involve all alternatives, modifications and variations that are within the spirit and the broad scope of the attached claims.
[00630] All publications, patents and patent applications mentioned in that specification are incorporated herein in full by reference in the specification, as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated here by reference. In addition, the citation or identification of any reference in that application should not be construed as an admission that such reference is available prior to the technique of the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

权利要求:
Claims (8)
[0001]
1. Method of increasing seed production, biomass, growth rate, vigor, tolerance to nitrogen deficiency, efficiency in the use of nitrogen and / or reducing the time for flowering of a plant, characterized by overexpressing within the plant a polynucleotide comprising the nucleic acid sequence shown in: (i) SEQ ID NO: 34 or 317, which encodes the polypeptide shown in SEQ ID NO: 521, or degenerate sequences thereof encoding said polypeptide shown in SEQ ID NO: 521; (ii) SEQ ID NO: 907, which encodes the polypeptide shown in SEQ ID NO: 4938, or degenerate sequences thereof that encode said polypeptide shown in SEQ ID NO: 4938; (iii) SEQ ID NO: 908, which encodes the polypeptide shown in SEQ ID NO: 4939, or degenerate sequences thereof that encode said polypeptide shown in SEQ ID NO: 4939; (iv) SEQ ID NO: 909, which encodes the polypeptide shown in SEQ ID NO: 4940, or degenerate sequences thereof that encode said polypeptide shown in SEQ ID NO: 4940; (v) SEQ ID NO: 910, which encodes the polypeptide shown in SEQ ID NO: 4941, or degenerate sequences thereof that encode said polypeptide shown in SEQ ID NO: 4941; or (vi) SEQ ID NO: 911, which encodes the polypeptide shown in SEQ ID NO: 4942, or degenerate sequences thereof that encode said polypeptide shown in SEQ ID NO: 4942.
[0002]
2. Method according to claim 1, characterized in that said polynucleotide is shown in SEQ ID NO: 34 or 317, which encode the polypeptide shown in SEQ ID NO: 521, or degenerate sequences of the same that encode said polypeptide shown in SEQ
[0003]
Method according to claim 1, characterized in that said polynucleotide is shown in SEQ ID Nos: 34, 317, 907, 908, 909, 910 or 911.
[0004]
4. Method according to claim 1, characterized in that said polynucleotide is shown in SEQ ID NO: 34 or 317.
[0005]
5. Method for producing a transgenic plant, characterized in that it comprises transforming a plant with a nucleic acid construct comprising an isolated polynucleotide comprising a nucleic acid sequence as shown in: (i) SEQ ID NO: 34 or 317, which encodes the polypeptide shown in SEQ ID NO: 521, or degenerate sequences thereof that encode said polypeptide shown in SEQ ID NO: 521; (ii) SEQ ID NO: 907, which encodes the polypeptide shown in SEQ ID NO: 4938, or degenerate sequences thereof that encode said polypeptide shown in SEQ ID NO: 4938; (iii) SEQ ID NO: 908, which encodes the polypeptide shown in SEQ ID NO: 4939, or degenerate sequences thereof that encode said polypeptide shown in SEQ ID NO: 4939; (iv) SEQ ID NO: 909, which encodes the polypeptide shown in SEQ ID NO: 4940, or degenerate sequences thereof that encode said polypeptide shown in SEQ ID NO: 4940; (v) SEQ ID NO: 910, which encodes the polypeptide shown in SEQ ID NO: 4941, or degenerate sequences thereof that encode said polypeptide shown in SEQ ID NO: 4941; or (vi) SEQ ID NO: 911, which encodes the polypeptide shown in SEQ ID NO: 4942, or degenerate sequences thereof encoding said polypeptide shown in SEQ ID NO: 4942; and a heterologous promoter to direct the transcription of said
[0006]
6. Method according to claim 5, characterized in that said polynucleotide is shown in SEQ ID NO: 34 or 317, which encode the polypeptide shown in SEQ ID NO: 521, or degenerate sequences of the same that encode said polypeptide shown in SEQ ID NO: 521.
[0007]
Method according to claim 5, characterized in that said polynucleotide is shown in SEQ ID Nos: 34, 317, 907, 908, 909, 910 or 911.
[0008]
8. Method according to claim 5, characterized in that said polynucleotide is shown in SEQ ID NO: 34 or 317.

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法律状态:
2020-07-21| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-10-27| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-02-02| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-09| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 09/03/2021, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US28218309P| true| 2009-12-28|2009-12-28|

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US61/282,183|2009-12-28|

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US34520510P| true| 2010-05-17|2010-05-17|

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US61/345,205|2010-05-17|

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PCT/IB2010/056023|WO2011080674A2|2009-12-28|2010-12-22|Isolated polynucleotides and polypeptides and methods of using same for increasing plant yield, biomass, growth rate, vigor, oil content, abiotic stress tolerance of plants and nitrogen use efficiency|

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