Plants with Improved Nitrogen Utilization and Stress Tolerance

Abstract

The present invention relates to transgenic plants that have increased nitrogen use efficiency, stress tolerance, or both and that have been transformed using a novel vector construct including nucleic acid sequences that modulate nitrogen use in plants. In various embodiments, the vector construct comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 7, 9, 11, 13, 15, 17, 22, 24, 26, 28, 30, 32, 34, 36, or 38. The invention also relates to isolated vectors for transforming plants and to antibodies used for detecting transformed plants. The invention also relates to methods of expressing in plants the nucleic acid molecules corresponding to the nucleic acid sequences that modulate nitrogen use in plants or are modulated by nitrogen conditions.

Description

[0001] This application claims priority to U.S. application Ser. No. 11/977,768, filed Oct. 26, 2007, which claims priority to U.S. Application Ser. No. 60/854,927, filed Oct. 27, 2006, which is incorporated herein in its entirety by this reference.

BACKGROUND OF THE INVENTION

[0002] The invention relates generally to plants with improved nitrogen utilization and stress tolerance, more specifically, to corn plants transformed with a gene that improves stress tolerance and nitrogen uptake, metabolism or both.

[0003] Plants require nitrogen during their vegetative and reproductive growth phases. Nitrogen is made available to the plant through soil mineralization, the application of nitrogen fertilizer, or both. It has been estimated, however, that between 50 and 70 percent of nitrogen applied to crops is lost from the plant-soil system [Peoples, M. B. et al., "Minimizing Gaseous Losses of Nitrogen," In Nitrogen Fertilizer in the Environment (Bacon, P. E., ed.) Marcel Dekker, pp. 565-606 (1995)]. Nitrogen is one of the most expensive plant nutrients to supply, nitrogen fertilizer is not always available at a reasonable cost, and excessive application of nitrogen fertilizer can result in pollution problems in runoff. Corn is an example of an agronomically important plant that often requires nitrogen fertilizers to perform at its genetic potential.

[0004] The development of plant varieties that use nitrogen more efficiently will reduce the need for excessive inputs of nitrogen, save production costs for farmers, benefit farmers in developing countries who do not have access to fertilizer inputs, and reduce pollution associated with the application of excessive nitrogen fertilizers. One approach that has been used in the development of plant varieties with improved nitrogen utilization relies on conventional plant breeding techniques. An alternative approach that may be more efficient would use genetic engineering to attempt to transform plants by the introduction of genes that have been identified by various groups as having a potential impact on nitrogen uptake and use.

SUMMARY OF THE INVENTION

[0005] The present invention relates to transgenic plants that have increased nitrogen use efficiency, stress tolerance, or both, that have been transformed using a novel vector construct including nucleic acid sequences that modulate nitrogen use in plants. The invention also relates to isolated vectors for transforming plants and to antibodies for detecting expression of the nucleotide sequence of interest in the transformed plants. The invention also relates to methods of expressing in plants the nucleic acid molecules corresponding to the nucleic acid sequences that modulate nitrogen use in plants.

[0006] Specifically, vectors for transforming plants have been constructed using nucleotide sequences selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 38, as well as variants, fragments, and complements thereof. These vectors include a 5' DNA promoter sequence and a 3' terminator sequence, wherein the nucleic acid sequence, the DNA promoter sequence, and the terminator sequence are operatively coupled to permit transcription of the nucleotide sequence. The promoter sequence may be a constitutive plant promoter or a tissue specific promoter.

[0007] The invention also includes polyclonal antibodies, comprising polyclonal antibodies to a nucleotide sequence selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36; and SEQ ID NO: 38.

[0008] The invention also includes a novel methodology for assaying nitrogen use efficiency.

[0009] The invention also includes plants transformed with nucleotide sequences selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 38, as well as variants and fragments thereof. The plant is selected from the group consisting of rice, corn, soybean, canola, wheat, alfalfa, barley, rye, cotton, sunflower, peanut, sweet potato, bean, pea, potato, oilseed rape, sorghum, forage grass, pasture grass, turf grass, and sugarcane. The invention also includes a component part of such plants, plant seed produced from such plants, and a plant seed transformed with a vector construct of the present invention.

[0010] The invention also includes a host cell transformed with a nucleotide sequence selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36; and SEQ ID NO: 38. The host cell may be a bacterial cell or a plant cell.

[0011] The invention also includes a method of expressing a nucleic acid molecule modulated by nitrogen in a plant, said method comprising the steps of providing a transgenic plant or plant seed transformed with a vector construct according to the present invention, and growing the transgenic plant or a plant grown from the transgenic plant seed under conditions effective to express the nucleic acid molecule in said transgenic plant or said plant grown from the transgenic plant seed. Growing of the transgenic plant is effective in reducing nitrogen uptake of said transgenic plant or said plant grown from the transgenic plant seed, in increasing nitrogen uptake of said transgenic plant or said plant grown from the transgenic plant seed, and in increasing efficiency of nitrogen utilization of said transgenic plant or said plant grown from the transgenic plant seed. The invention also includes the foregoing methods wherein a transgenic plant is provided or a transgenic seed is provided. The invention also includes the foregoing method wherein the plant is selected from the group consisting of rice, corn, soybean, canola, wheat, alfalfa, barley, rye, cotton, sunflower, peanut, sweet potato, bean, pea, potato, oilseed rape, sorghum, forage grass, pasture grass, turf grass, sugarcane.

[0012] The invention also includes a method of improving the stress tolerance of a plant by expressing a nucleic acid molecule modulated by nitrogen in a plant, said method comprising the steps of providing a transgenic plant or plant seed transformed with a vector construct according to the present invention and growing the transgenic plant or a plant grown from the transgenic plant seed under conditions effective to express the nucleic acid molecule in said transgenic plant or said plant grown from the transgenic plant seed.

[0013] The invention also includes a method of altering the morphology of a plant by expressing a nucleic acid molecule modulated by nitrogen in a plant, said method comprising the steps of providing a transgenic plant or plant seed transformed with a vector construct according to the present invention and growing the transgenic plant or a plant grown from the transgenic plant seed under conditions effective to express the nucleic acid molecule in said transgenic plant or said plant grown from the transgenic plant seed.

[0014] The invention also includes a vector construct, comprising a nucleotide sequence encoding an amino acid sequence selected from the list consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 37, a 5' DNA promoter sequence, and a 3' terminator sequence, wherein the nucleotide sequence, the DNA promoter sequence, and the terminator sequence are operatively coupled to permit transcription of the nucleotide sequence.

[0015] The invention also includes a vector construct comprising a nucleotide sequence that is modulated by nitrogen in a plant, wherein said nucleotide sequence is selected from the group consisting of a nucleotide sequence selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36; and SEQ ID NO: 38, and a nucleotide sequence encoding an amino acid sequence selected from the list consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 39, wherein said construct further comprises a 5' DNA promoter sequence and a 3' terminator sequence, wherein the nucleotide sequence, the DNA promoter sequence, and the terminator sequence are operatively coupled to permit transcription of the nucleotide sequence.

[0016] The invention also includes a vector construct comprising a nucleotide sequence that is modulated by nitrogen in a plant, wherein said nucleotide sequence is selected from the group consisting of a nucleotide sequence selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 38, a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38, wherein said nucleotide sequence is modulated by nitrogen in a plant, and a nucleotide sequence encoding an amino acid sequence selected from the list consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39, and a nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39, wherein said nucleotide sequence is modulated by nitrogen in a plant, wherein said construct further comprises a 5' DNA promoter sequence and a 3' terminator sequence, wherein the nucleotide sequence, the DNA promoter sequence, and the terminator sequence are operatively coupled to permit transcription of the nucleotide sequence.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0017] There is a need to develop plant cultivars that absorb and use nitrogen more efficiently. Plant scientists have adopted the shorthand term nitrogen use efficiency (NUE), and a variety of methods of measuring and evaluating NUE have been developed [Craswell, E. T. and Godwin, D. C. (1984) The efficiency of nitrogen fertilizers applied to cereals grown in different climates. In Advances in Plant Nutrition (Vol. 1) (Tinker, P. B. and Lauchli, A., eds), pp. 1-55, Praeger Publishers; Steenbjerg, F. and Jakobsen, S. T. (1963) Plant nutrition and yield curves. Soil Sci. 95, 69-90; Siddiqi, M. Y. and Glass, D. M. (1981) Utilization index: a modified approach to the estimation and comparison of nutrient utilization efficiency in plants. J. Plant Nutr. 4, 289-302; Moll, R. H. et al. (1982) Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization. Agron. J. 74, 562-564]. There are differences in the definitions. Some definitions are based on total biomass while others are based on the weight of grain yielded. Another set of definitions uses the efficiency of extracting nitrogen from the soil. The efficiency with which applied nitrogen is used to improve grain yield may be measured by agronomic efficiency (AE), the product of physiological efficiency and utilization efficiency, or NUEg which is the product of uptake efficiency and utilization efficiency. Other definitions take physiological factors into account.

[0018] As used in this specification, the term nitrogen use efficiency, or NUE, is defined to include a measurable change in any of the main nitrogen metabolic pool sizes in the assimilation pathways (for example, may include a measurable change in one or more of the following: nitrate, nitrite, ammonia, glutamic acid, aspartic acid, glutamine, asparagine, lysine, leucine, threonine, methionine, glycine, tryptophan, tyrosine, total protein content of a plant part, total nitrogen content of a plant part, chlorophyll content), or where the plant is shown to provide the same or elevated yield at lower nitrogen fertilization levels, or where the plant is shown to provide elevated yields at the same nitrogen fertilization levels when compared to a plant that has not been transformed with an nitrogen-modulated nucleic acid construct of the invention. A "measurable change" can include an increase or a decrease in the amount of any component ("metabolic pool") of the nitrogen assimilation pathway. A change can include either a decrease or an increase in one or more metabolic pools in the pathway, or a decrease in one or more pools with a concomitant increase in one or more other pool(s). For example, the level of one pool (e.g., glutamate) can decrease while the level of another pool (e.g., glutamine) can increase.

[0019] An increase in nitrogen utilization efficiency can be associated with about a 5%, about a 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, about a 200% or greater measurable change in any of the main nitrogen metabolic pool sizes in the assimilation pathway. In one embodiment, the transgenic plants of the invention have an increased nitrogen uptake from the environment when compared to a plant that does not contain a nitrogen-modulated sequence of the invention. In another embodiment, the transgenic plants of the invention have a decreased nitrogen uptake from the environment when compared to a plant that does not contain a nitrogen-modulated sequence of the invention. By "nitrogen modulating sequence" is intended a nucleotide or amino acid sequence that is modulated (e.g., increased or decreased, or upregulated or downregulated) in response to exposure to nitrogen.

[0020] The present invention further provides a method of improving stress tolerance in a plant by expressing a nitrogen-modulated nucleotide sequence within the plant. In one embodiment, the nitrogen-modulated nucleotide sequence is SEQ ID NO: 2, 4, 7, 9, 11, 13, 15, 17, 22, 24, 26, 28, 30, 32, 34, 36, or 38, or variants and fragments thereof. In another embodiment, the nitrogen-modulated nucleotide sequence is a nucleotide sequence that encodes SEQ ID NO: 3, 5, 8, 10, 12, 14, 16, 18, 23, 25, 27, 29, 31, 33, 35, 37, or 39, or variants and fragments thereof.

[0021] As used herein, the term "stress" or "stress condition" refers to the exposure of a plant, plant cell, or the like, to a physical or chemical agent or condition that has an adverse effect on metabolism, growth, development, propagation and/or survival of the plant (collectively "growth"). A stress can be imposed on a plant due, for example, to an environmental factor such as water (e.g., flooding, drought, dehydration), anaerobic conditions (e.g., a low level of oxygen), abnormal osmotic conditions, salinity or temperature (e.g., hot/heat, cold, freezing, frost), a deficiency of nutrients such as nitrogen or exposure to pollutants, or by a hormone, second messenger or other molecule. Anaerobic stress, for example, is due to a reduction in oxygen levels (hypoxia or anoxia) sufficient to produce a stress response. A flooding stress can be due to prolonged or transient immersion of a plant, plant part, tissue or isolated cell in a liquid medium such as occurs during monsoon, wet season, flash flooding or excessive irrigation of plants, or the like. A cold stress or heat stress can occur due to a decrease or increase, respectively, in the temperature from the optimum range of growth temperatures for a particular plant species. Such optimum growth temperature ranges are readily determined or known to those skilled in the art. Dehydration stress can be induced by the loss of water, reduced turgor, or reduced water content of a cell, tissue, organ or whole plant. Drought stress can be induced by or associated with the deprivation of water or reduced supply of water to a cell, tissue, organ or organism. Saline stress (salt stress) can be associated with or induced by a perturbation in the osmotic potential of the intracellular or extracellular environment of a cell. Osmotic stress also can be associated with or induced by a change, for example, in the concentration of molecules in the intracellular or extracellular environment of a plant cell, particularly where the molecules cannot be partitioned across the plant cell membrane.

[0022] Transformation of Bacterial or Plant Cells

[0023] Provided herein are novel nucleotide sequences that modulate nitrogen utilization efficiency in plants. Also provided are amino acid sequences of the nitrogen-modulated proteins of the invention.

[0024] The nitrogen-modulated nucleotide sequences of the invention may be modified to obtain or enhance expression in plant cells. The nitrogen-modulated sequences of the invention may be provided in expression cassettes for expression in the plant of interest. "Plant expression cassette" includes DNA constructs that are capable of resulting in the expression of a protein from an open reading frame in a plant cell. The cassette will include in the 5'-3' direction of transcription, a transcriptional initiation region (i.e., promoter) operably-linked to a DNA sequence of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants. The cassette may additionally contain at least one additional gene to be cotransformed into the organism, such as a selectable marker gene. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites for insertion of the nitrogen-modulated sequence to be under the transcriptional regulation of the regulatory regions.

[0025] By "promoter" is intended a nucleic acid sequence that functions to direct transcription of a downstream coding sequence. The promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also termed as "control sequences"), are necessary for the expression of a DNA sequence of interest. Preferably, the promoter is one that is known to stimulate transcription in the organism into which the nucleotide sequence of the invention is being introduced.

[0026] The promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is "native" or "homologous" to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is "foreign" or "heterologous" to the DNA sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention. "Heterologous" generally refers to the nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like. By "operably linked" is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, "operably linked" means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

[0027] In one embodiment, the promoter is a constitutive promoter. Suitable constitutive promoters for use in plants include: the promoters from plant viruses, such as the peanut chlorotic streak caulimovirus (PC1SV) promoter (U.S. Pat. No. 5,850,019); the 35S promoter from cauliflower mosaic virus (CaMV) (Odell et al. (1985) Nature 313:810-812); promoters of Chlorella virus methyltransferase genes (U.S. Pat. No. 5,563,328) and the full-length transcript promoter from figwort mosaic virus (FMV) (U.S. Pat. No. 5,378,619); the promoters from such genes as rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689), including the TrpPro5 promoter (U.S. patent application Ser. No. 10/377,318; filed Mar. 16, 2005); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); maize H3 histone (Lepetit et al. (1992) Mol. Gen. Genet. 231:276-285 and Atanassova et al. (1992) Plant J. 2(3):291-300); Brassica napus ALS3 (PCT application WO 97/41228); and promoters of various Agrobacterium genes (see U.S. Pat. Nos. 4,771,002; 5,102,796; 5,182,200; and 5,428,147).

[0028] In another embodiment, the promoter is a tissue-specific promoter. A list of commonly-used tissue-specific promoters can be found in Reviewed in Moore et al. (2006) Plant J. 45(4):651-683, which is herein incorporated by reference in its entirety.

[0029] Often, such constructs will also contain 5' and 3' untranslated regions. Such constructs may contain a "signal sequence" or "leader sequence" to facilitate co-translational or post-translational transport of the peptide of interest to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus, or to be secreted. For example, the gene can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum. By "signal sequence" is intended a sequence that is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. By "leader sequence" is intended any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a sub-cellular organelle. Thus, this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. It may also be preferable to engineer the plant expression cassette to contain an intron, such that mRNA processing of the intron is required for expression.

[0030] By "3' untranslated region" is intended a nucleotide sequence located downstream of a coding sequence. Polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor are 3' untranslated regions. By "5' untranslated region" is intended a nucleotide sequence located upstream of a coding sequence.

[0031] Other upstream or downstream untranslated elements include enhancers. Enhancers are nucleotide sequences that act to increase the expression of a promoter region. Enhancers are well known in the art and include, but are not limited to, the SV40 enhancer region and the 35S enhancer element.

[0032] The termination region may be native with the transcriptional initiation region, may be native with the nitrogen-modulated sequence of the present invention, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions, or the potato proteinase inhibitor II sequence (PinII) as described in Liu et al. (2004) Acta Biochim Biophys Sin 36(8):553-558. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0033] Where appropriate, the gene(s) may be optimized for increased expression in the transformed host cell. That is, the genes can be synthesized using host cell-preferred codons for improved expression, or may be synthesized using codons at a host-preferred codon usage frequency. Generally, the GC content of the gene will be increased. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are known in the art for synthesizing host-preferred genes. See, for example, U.S. Pat. Nos. 6,320,100; 6,075,185; 5,380,831; and 5,436,391, U.S. Published Application Nos. 20040005600 and 20010003849, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

[0034] In one embodiment, the nucleic acids of interest are targeted to the chloroplast for expression. In this manner, where the nucleic acid of interest is not directly inserted into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the gene product of interest to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.

[0035] The nucleic acids of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.

[0036] Typically this "plant expression cassette" will be inserted into a "plant transformation vector." By "transformation vector" is intended a DNA molecule that is necessary for efficient transformation of a cell. Such a molecule may consist of one or more expression cassettes, and may be organized into more than one "vector" DNA molecule. For example, binary vectors are plant transformation vectors that utilize two non-contiguous DNA vectors to encode all requisite cis- and trans-acting functions for transformation of plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). "Vector" refers to a nucleic acid construct designed for transfer between different host cells. "Expression vector" refers to a vector that has the ability to incorporate, integrate and express heterologous DNA sequences or fragments in a foreign cell.

[0037] This plant transformation vector may be comprised of one or more DNA vectors needed for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that are comprised of more than one contiguous DNA segment. These vectors are often referred to in the art as "binary vectors." Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a "nucleotide sequence of interest" (a nucleotide sequence engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the gene of interest are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science, 5:446-451). Several types of Agrobacterium strains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.

[0038] Altered or Improved Variants Useful in the Constructs of the Invention

[0039] It is recognized that nucleotide and amino acid sequences useful in the present invention may be altered by various methods, and that these alterations may result in sequences encoding proteins with amino acid sequences different than that encoded by the nitrogen-modulated sequences disclosed herein.

[0040] Nucleotide sequences useful in the present invention include the sequences set forth in SEQ ID NO: 2, 4, 7, 9, 11, 13, 15, 17, 22, 24, 26, 28, 30, 32, 34, 36, and 38, and variants, fragments, and complements thereof. As used herein, the term "nucleotide sequence" or "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecules can be single-stranded or double-stranded, but preferably are double-stranded DNA. By "complement" is intended a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence such that it can hybridize to the given nucleotide sequence to thereby form a stable duplex. The corresponding amino acid sequences for the nitrogen-modulated proteins encoded by these nucleotide sequences are set forth in SEQ ID NO: 3, 5, 8, 10, 12, 14, 16, 18, 23, 25, 27, 29, 31, 33, 35, 37, and 39, as well as variants and fragments thereof. The invention also encompasses the use of nucleic acid molecules comprising nucleotide sequences encoding partial-length nitrogen-modulated proteins, and complements thereof.

[0041] Nucleic acid molecules that are fragments of these nitrogen-modulated nucleotide sequences are also useful in the present invention. By "fragment" is intended a portion of a nucleotide sequence encoding a nitrogen-modulated protein. A fragment of a nucleotide sequence may encode a biologically active portion of a nitrogen-modulated protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. Nucleic acid molecules that are fragments of a nitrogen-modulated nucleotide sequence comprise at least about 15, 20, 50, 75, 100, 200, 300, 350, or at least about 400 contiguous nucleotides, or up to the number of nucleotides present in a full-length nitrogen-modulated nucleotide sequence disclosed herein depending upon the intended use. By "contiguous" nucleotides is intended nucleotide residues that are immediately adjacent to one another.

[0042] Polypeptides that are fragments of these nitrogen-modulated polypeptides are also useful in the present invention. By "fragment" is intended a portion of an amino acid sequence encoding a nitrogen-modulated protein as set forth SEQ ID NO: 3, 5, 8, 10, 12, 14, 16, 18, 23, 25, 27, 29, 31, 33, 35, 37, or 39, and that retains nitrogen utilization efficiency. A biologically active portion of a nitrogen-modulated protein can be a polypeptide that is, for example, 10, 25, 50, 100, 125, 150, 175, 200, 250, 300, 350, 400 or more amino acids in length. Such biologically active portions can be prepared by recombinant techniques and evaluated for nitrogen utilization efficiency. As used here, a fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 3, 5, 8, 10, 12, 14, 16, 18, 23, 25, 27, 29, 31, 33, 35, 37, or 39. The invention encompasses other fragments, however, such as any fragment in the protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

[0043] The invention also encompasses the use of variant nucleic acid molecules, or variant amino acid sequences, in the methods and compositions of the inventions. "Variants" of the nitrogen-modulated nucleotide sequences include those sequences that encode a nitrogen-modulated protein disclosed herein but that differ conservatively because of the degeneracy of the genetic code, as well as those that are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the nitrogen-modulated proteins disclosed in the present invention as discussed below. Variant proteins useful in the present invention are biologically active, that is they retain the desired biological activity of the native protein, that is, nitrogen utilization efficiency.

[0044] By "variants" is intended proteins or polypeptides having an amino acid sequence that is at least about 60%, 65%, about 70%, 75%, 80%, 85%, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3, 5, 8, 10, 12, 14, 16, 18, 23, 25, 27, 29, 31, 33, 35, 37, or 39. Variants also include polypeptides encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule of SEQ ID NO: 2, 4, 7, 9, 11, 13, 15, 17, 22, 24, 26, 28, 30, 32, 34, 36, or 38, or a complement thereof, under stringent conditions. Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, retain nitrogen utilization efficiency.

[0045] Preferred nitrogen-modulated proteins useful in the present invention are encoded by a nucleotide sequence sufficiently identical to the nucleotide sequence of SEQ ID NO: 2, 4, 7, 9, 11, 13, 15, 17, 22, 24, 26, 28, 30, 32, 34, 36, or 38. The term "sufficiently identical" is intended an amino acid or nucleotide sequence that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80% or 85% sequence identity, or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0046] To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions).times.100). In one embodiment, the two sequences are the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

[0047] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to nitrogen-modulated nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to nitrogen-modulated protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. See www.ncbi.nlm.nih.gov. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, Calif.). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non-limiting example of a software program useful for analysis of ClustalW alignments is GENEDOC.TM.. GENEDOC.TM. (Karl Nicholas) allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package (available from Accelrys, Inc., 9865 Scranton Rd., San Diego, Calif., USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0048] A preferred program is GAP version 10, which used the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453. GAP Version 10 may be used with the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 Scoring Matrix. Equivalent programs may also be used. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0049] The skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded nitrogen-modulated protein, without altering the biological activity of the protein. Thus, variant isolated nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.

[0050] For example, conservative amino acid substitutions may be made at one or more predicted, preferably nonessential amino acid residues. A "nonessential" amino acid residue is a residue that can be altered from the wild-type sequence of a nitrogen-modulated protein without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. However, one of skill in the art would understand that functional variants may have minor conserved or nonconserved alterations in the conserved residues. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related sequences known to be involved in nitrogen assimilation. Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or related sequences known to be involved in nitrogen assimilation.

[0051] Alternatively, variant nucleotide sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ability to confer nitrogen utilization efficiency to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.

[0052] Using methods such as PCR, hybridization, and the like, corresponding nitrogen-modulated sequences can be identified, such sequences having substantial identity to the sequences of the invention. See, for example, Sambrook J., and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY). In a hybridization method, all or part of the nitrogen-modulated nucleotide sequence can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra.

[0053] Variants and fragments of the nucleotide or amino acid sequences of the present invention generally will encode protein fragments that retain the biological activity of the full-length nitrogen-modulated protein; i.e., retain nitrogen utilization efficiency. By "retains nitrogen utilization efficiency" is intended that the variant or fragment will have at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the nitrogen utilization efficiency of the full-length nitrogen-modulated protein disclosed herein as SEQ ID NO: 3, 5, 8, 10, 12, 14, 16, 18, 23, 25, 27, 29, 31, 33, 35, 37, or 39, or the full-length nitrogen-modulated nucleotide sequence disclosed herein as SEQ ID NO: 2, 4, 7, 9, 11, 13, 15, 17, 22, 24, 26, 28, 30, 32, 34, 36, or 38. Methods for monitoring nitrogen utilization efficiency include detecting a change in any of the main nitrogen metabolic pool sizes in the assimilation pathways (for example, a measurable change in nitrate, nitrite, ammonia, glutamic acid, aspartic acid, glutamine, asparagine, lysine, leucine, threonine, methionine, glycine, tryptophan, tyrosine, total protein content of a plant part, total nitrogen content of a plant part, and/or chlorophyll content) or detecting the ability of a plant to provide the same or elevated yield at lower nitrogen fertilization levels, or the ability of a plant to provide elevated yields at the same nitrogen fertilization levels when compared to a plant that does not contain or express a nitrogen-modulated sequence of the invention.

[0054] The polypeptide sequences useful in the present invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the nitrogen-modulated proteins disclosed herein can be prepared by mutations in the nucleotide sequences. This may also be accomplished by one of several forms of mutagenesis and/or in directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect function of the protein. Such variants will possess the desired nitrogen utilization efficiency. However, it is understood that the ability of the nitrogen-modulated sequences of the invention to alter or improve nitrogen utilization may be further improved by one use of such techniques upon the compositions of this invention. For example, one may express the nucleotide sequences disclosed herein in host cells that exhibit high rates of base misincorporation during DNA replication, such as XL-1 Red (Stratagene, La Jolla, Calif.). After propagation in such strains, one can isolate the DNA (for example by preparing plasmid DNA, or by amplifying by PCR and cloning the resulting PCR fragment into a vector), transform it into plants as described elsewhere herein, and measure nitrogen utilization efficiency.

[0055] Alternatively, alterations may be made to the protein sequence of many proteins at the amino or carboxy terminus without substantially affecting activity. This can include insertions, deletions, or alterations introduced by modern molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification. Alternatively, the protein sequences added can include entire protein-coding sequences, such as those used commonly in the art to generate protein fusions. Such fusion proteins are often used to (1) increase expression of a protein of interest, (2) introduce a binding domain, enzymatic activity, or epitope to facilitate either protein purification, protein detection, or other experimental uses known in the art, or, (3) target secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of gram-negative bacteria, or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in glycosylation of the protein.

[0056] Variant nucleotide and amino acid sequences of the present invention also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different nitrogen-modulated protein coding regions can be used to create a new nitrogen-modulated protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the nitrogen-modulated sequence useful in the present invention and other known nitrogen-modulated sequences to obtain a new sequence coding for a protein with an improved property of interest, such as improved nitrogen utilization. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0057] Plant Transformation

[0058] Methods of the invention involve introducing a nucleotide construct into a plant. By "introducing" is intended to present to the plant the nucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not require that a particular method for introducing a nucleotide construct to a plant is used, only that the nucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0059] In general, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g. immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass. Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent (i.e., antibiotics, such as spectinomycin and kanamycin). The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grow into mature plant and produce fertile seeds (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods for generating transgenic plants are found in Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar (1997) Maydica 42:107-120. Since the transformed material contains many cells, both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non-transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants. Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest in the genome of transgenic plant.

[0060] Generation of transgenic plants may be performed by one of several methods, including but not limited to introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, and various other non-particle direct-mediated methods (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750; Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239; Bommineni and Jauhar (1997) Maydica 42:107-120) to transfer DNA.

[0061] Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0062] Transformation of bacterial cells is accomplished by one of several techniques known in the art, including but not limited to electroporation or chemical transformation (see, for example, Ausubel, ed. (1994) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., Indianapolis, Ind.). Markers conferring resistance to toxic substances are useful in identifying transformed cells (having taken up and expressed the test DNA) from non-transformed cells (those not containing or not expressing the test DNA).

[0063] In one aspect of the invention, the nucleotide sequences of the invention are useful as markers to assess transformation of bacterial or plant cells. In this manner, transformation is assessed by monitoring nitrogen utilization efficiency as described above.

[0064] Transformation of plant cells can be accomplished in similar fashion. By "plant" is intended whole plants, or component parts including plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen). "Transgenic plants" or "transformed plants" or "stably transformed" plants or cells or tissues refer to plants that have incorporated or integrated exogenous nucleic acid sequences or DNA fragments into the plant cell. By "stable transformation" is intended that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof.

[0065] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

[0066] Methods to Increase Plant Yield by Modulating Nitrogen Utilization

[0067] Methods for increasing plant yield are provided. The methods comprise introducing into a plant or plant cell a nitrogen-modulated nucleotide sequence disclosed herein such that an increase in nitrogen utilization efficiency corresponds to an increase in plant yield. As defined herein, the "yield" of the plant refers to the quality and/or quantity of biomass produced by the plant. By "biomass" is intended any measured plant product. An increase in biomass production is any improvement in the yield of the measured plant product. Increasing plant yield has several commercial applications. For example, increasing plant leaf biomass may increase the yield of leafy vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products. An increase in yield can comprise any statistically significant increase including, but not limited to, at least a 1% increase, at least a 3% increase, at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater increase.

[0068] Plants

[0069] The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassaya, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, grasses (such as turf grasses, forage grasses, or pasture grasses), ornamentals, fruit trees, and conifers.

[0070] Vegetables include, but are not limited to, onions, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis such as cucumber, cantaloupe, and muskmelon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum. Preferably, plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

[0071] This invention is particularly suitable for any member of the monocot plant family including, but not limited to, maize, rice, barley, oats, wheat, sorghum, rye, sugarcane, pineapple, yams, onion, banana, coconut, and dates.

[0072] Evaluation of Plant Transformation

[0073] Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

[0074] PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated nucleotide sequences at the earlier stage before transplanting into the soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.

[0075] Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated in an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or "blot" is then probed with, for example, radiolabeled .sup.32P target DNA fragments to confirm the integration of the introduced gene in the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).

[0076] In Northern analysis, RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, 2001, supra). Expression of RNA encoded by the nucleotide sequence of the invention is then tested by hybridizing the filter to a radioactive probe derived from a polynucleotide of the invention, by methods known in the art (Sambrook and Russell, 2001, supra)

[0077] Western blot and biochemical assays and the like may be carried out on the transgenic plants to determine the presence of protein encoded by the nitrogen-modulated gene by standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present on the nitrogen-modulated protein. For example, the polyclonal antibodies generated by the methods of the present invention can be used to detect the presence of a nitrogen-modulated protein.

[0078] Antibodies

[0079] Antibodies to the polypeptides useful in the present invention, or to variants or fragments thereof, are also encompassed. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

EXPERIMENTAL

Materials and Methods

[0080] The majority of the starting genetic material for this project was provided in the form of maize expressed sequence tags, or "ESTs", derived from a microarray experiment to identify potential genes up- or down-regulated in response to nitrogen. The microarray experiment identified several hundred possible candidates for possible use in transformations. While these sequences were predictive of gene transcription as a response to nitrogen fluctuations, they did not provide a firm identification of genes that were regulated in response to nitrogen levels or genes that regulate nitrogen levels. The candidate ESTs from the microarray experiment were screened based on genomic selection criteria to analyze and determine a small number of priority candidates for subsequent use in transgenic expression as described in this specification. All EST sequences that were entered into the project (i.e., "project genes") were first examined to identify open reading frames that could encode a protein that was responsive to plant nitrogen levels. Multiple open reading frames were typically present within an EST. Ultimately, individual project genes were selected based on multiple criteria, including size of open reading frame wherein longer open reading frames were preferentially selected, and predicted function of translated genes, wherein individual open reading frames were translated and then subjected to a BLAST search to identify protein homologues. In cases where homologues were identified, we inferred that the gene was likely to encode a protein with a similar function. This information was used to assess if genes might encode protein functions with relevance to nitrogen assimilation in plants.

[0081] By this selection process, an individual gene target was selected from each EST. The complete gene sequence selected from each EST is disclosed in the following examples. One of the EST sequences (N-EST 77-A01) was used as a source for two different genes that were entered into the project (N-EST77A and N-EST77B) and for one other EST (EST N-EST76-H12). We discovered that the EST could be modified to generate an open reading frame that is longer than the reading frames present in the unmodified EST. In summary, three open reading frames were combined to create one longer gene ("N-EST76A").

[0082] In some cases, a DNA sample provided from the microarray experiment was used as the source material for all subsequent DNA cloning steps. In cases where the EST sample was not suitable, synthetic sequences were generated. The N-EST76b gene was ordered as a synthetic gene from the vendor Blue Heron Biotechnology, Inc. (Bothell, Wash.). The gene sequence for each EST and each synthetic gene was confirmed by DNA sequencing prior to subcloning each gene for protein overexpression.

[0083] Protein Overexpression and Purification

[0084] Each of the genes selected for the project were subcloned into an expression vector that facilitates protein overexpression in E. coli. The protein overexpression was carried out to allow individual proteins to be purified. The purified proteins can be used to generate polyclonal antibodies against each protein in a pair of rabbits. Finally, the polyclonal antibodies can be used to detect the presence of target proteins in transgenic plants.

[0085] Using methods known in the art, each of the project genes was subcloned into the E. coli expression vector pRSF1b (Invitrogen Corporation, Carlsbad, Calif.). Resulting clones were confirmed by DNA sequencing, and used to induce expression of each protein in E. coli. The expressed His-tagged protein was then purified as known in the art using a cobalt affinity resin (Clontech Laboratories, Inc., Mountain View, Calif.).

[0086] Plant Transformation

[0087] Representative project genes were subcloned into vectors to carry out Agrobacterium-mediated transformation of maize. Following vector construction and transformation of Agrobacterium, the vectors were confirmed by Southern blot by methods known in the art. Positive Agrobacterium strains that passed these tests were then grown on a solid medium to produce cell counts for large-scale transformation experiments.

[0088] The following examples describe the methods for plant vector construction and plant transformation.

[0089] Vector Construction for Plant Transformation

[0090] The open reading frame (ORF) for each project gene is amplified by PCR from the maize EST sequence or synthetic gene. Restriction sites (BamH I and Asc I, for example) are added to each end of the ORF during PCR. Additionally, the nucleotide sequence ACC is added immediately 5' to the start codon of the gene to increase translational efficiency (Kozak (1987) Nucleic Acids Research 15:8125-8148; Joshi (1987) Nucleic Acids Research 15:6643-6653). The PCR product is subcloned into an intermediate vector (for example, pRSF-1b) and sequenced, using techniques well known in the art, to ensure that no mutations are introduced during PCR. The plasmid containing the project gene is digested with, for example, BamH I and Pst I and a fragment containing the intact ORF is isolated and purified.

[0091] The purified DNA fragment containing the project ORF is then subcloned into a plasmid such as pSB11 (Japan Tobacco, Inc.), for example at a BamH I and Pst I site, to complete the plant expression vector. The plant expression vector contains, for example, a Tripsacum ubiquitin promoter, TripPro5 promoter (U.S. patent application Ser. No. 11/377,318 filed Mar. 16, 2006, incorporated herein by this reference) and the PinIl terminator (An et al. (1989) The Plant Cell 1:115-122) to form the final plasmid, referred to herein as pSB11-1A. pSB11-IA is organized such that the DNA fragment containing, for example, the promoter--NUE gene--terminator construct may be excised by appropriate restriction enzymes and also used for transformation into plants, for example, by aerosol beam injection. The structure of pSB11-1A is verified by restriction digest and gel electrophoresis, as well as by sequencing across the various cloning junctions.

[0092] The plasmid is mobilized into Agrobacterium tumefaciens strain LBA4404 which also harbors the plasmid pSB 1 (Japan Tobacco, Inc.), using triparental mating procedures well known in the art, and plated on media containing antibiotic. Plasmid pSB11-1A carries spectinomycin resistance but is a narrow host range plasmid and cannot replicate in Agrobacterium. Antibiotic resistant colonies arise when pSB11-1A integrates into the broad host range plasmid pSB1 through homologous recombination. The resulting cointegrate product is verified by Southern hybridization. The Agrobacterium strain harboring the cointegrate can be used to transform plants, for example, by the PureIntro method (Japan Tobacco, Inc.).

[0093] Transformation of Plant Cells by Agrobacterium-Mediated Transformation

[0094] Ears are collected 8-12 days after pollination. Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in size are used for transformation. Embryos are plated scutellum side-up on a suitable incubation media, and incubated overnight at 25.degree. C. in the dark. However, it is not necessary per se to incubate the embryos overnight. Embryos are contacted with an Agrobacterium strain containing the appropriate vectors for Ti plasmid mediated transfer for 5-10 min, and then plated onto co-cultivation media for 3 days (25.degree. C. in the dark). After co-cultivation, explants are transferred to recovery period media for five days (at 25.degree. C. in the dark). Explants are incubated in selection media for up to eight weeks, depending on the nature and characteristics of the particular selection utilized. After the selection period, the resulting callus is transferred to embryo maturation media, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light, and the process of regeneration is initiated as known in the art. The resulting shoots are allowed to root on rooting media, and the resulting plants are transferred to nursery pots and propagated as transgenic plants. At this time, leaf samples are isolated and the presence of the gene of interest is confirmed by PCR.

[0095] All plants generated in this manner were grown to seed set and crossed with pollen isolated from with Hi-II plants (Iowa State University, Ames, Iowa). The fertilized plants were grown until maturity. Mature seeds were harvested from individual plants and saved for future testing in the T1 generation, if necessary.

[0096] Protein Expression in Transgenic Plants

[0097] Protein expression in representative transgenic maize events was estimated by Western blot. Briefly, leaf samples were taken after 4 weeks of growth in the greenhouse and immediately frozen on dry ice. Total protein was extracted (P-PER plant protein extraction kit, Pierce) and the protein concentration determined by Bradford assay. Individual plant protein samples were separated by electrophoresis, transferred to nitrocellulose, and the immobilized proteins were contacted with rabbit polyclonal antiserum using methods known in the art. Bound antibody complexes were visualized with the ECL Plus Western Blotting detection system (GE Healthcare Bio-Sciences Corp., Piscataway, N.J.).

[0098] Nitrogen Assay Methods

[0099] In preparation for nitrogen assays, leaves were sliced from plants two or four weeks after transfer from tissue culture to the greenhouse (or four weeks from germination for T1 plants). The material was snap frozen on dry ice and stored at -80.degree. C. prior to processing.

[0100] Nitrate

[0101] Fifty milligrams of leaf material (fresh weight, no midrib) were freeze-dried for dry weight determination. The dehydrated leaf tissue was then ground in the presence of fresh Milli Q water using a MiniBeadbeater-96.TM. and 2.3 mm stainless-steel beads. The ground leaf tissue was filtered through a 0.45 .mu.m Polyvinylidene Fluoride (PVDF) filter and injected into an Agilent 1100 HPLC running a mobile phase of a mixture of 1.8 mM sodium carbonate and 1.7 mM sodium bicarbonate at 1.5 ml/min. Ions were separated using an IonPac AS9-SC ion chromatography column equipped with a guard column. Analysis was performed using anion auto-suppressed conductivity with a self-regenerating suppressor operating in recycle mode. Samples were compared to internal standards included in each sample run.

[0102] Ammonium

[0103] Fifty milligrams of leaf material (fresh weight, no midrib) were ground in the presence of 60% methanol using a MiniBeadbeater-96.TM. and 2.3 mm stainless-steel beads. The ground leaf tissue was filtered through a 0.45 .mu.m Polyvinylidene Fluoride (PVDF) filter and injected into an Agilent 1100 HPLC equipped with a 3.3 m, 63.degree. C. stainless steel coil and cooled autosampler. The mobile phase contained 3 mM o-phthalaldehyde (OPA), 10 mM .beta.-mercaptoethanol, and 100 mM phosphate buffer (pH6.8) running at 0.4 ml/min. Fluorescence (excitation 410 nm and emission 470 nm) and diode array detection (410 nm) were used for the quantification of ammonium in the leaf extracts. Internal ammonium standards were included in each run for comparison.

[0104] Amino Acids by HPLC

[0105] Fifty milligrams of leaf material (fresh weight, no midrib) were freeze-dried for dry weight determination. The dehydrated leaf tissue was then ground in the presence of fresh Milli Q water using a MiniBeadbeater-96.TM. and 2.3 mm stainless-steel beads. The ground leaf tissue was filtered through a 0.45 .mu.m Polyvinylidene Fluoride (PVDF) filter and injected into an Agilent 1100 HPLC using a Zorbax Eclipse AAA, 4.6.times.75 mm reverse phase column equipped with a guard column. A cooled autosampler was used to mix the leaf extract with 400 mM borate buffer (pH 10.2), 1% o-phthalaldehyde/1% 3-mercaptopropionic acid in methanol, which was then diluted with water prior to injection. The details of the injector program are as follows: 0.5 .mu.l sample are added to 2.5 .mu.l borate buffer and mixed at maximum speed two times. After a 0.5 minute hold, the needle is placed in water to remove residue from the tip and then 0.5 .mu.l OPA solution is added. The combined 3.5 .mu.l is mixed at maximum speed six times. The needle is again placed in water to rinse the tip and then placed into a vial containing fresh water. Next, 32 .mu.l Milli Q water are added to the sample mixture, and 18 .mu.l are mixed at maximum speed two times. The sample solution is then injected into the HPLC with the pump running a 2 ml/min mobile phase of 40 mM Na2HPO4 (pH 7.8) (A) with a gradient from 0 to 26% acetonitrile/methanol/water (45:45:10) (B) in five minutes followed by a 100% hold B for two minutes then 100% A for two minutes. Quantification of asparagine, glutamine, glutamic acid, and aspartic acid was performed by diode array detection (328 to 348 nm) and fluorescence detection (excitation 340 nm, emission 450 nm). Samples were compared to asparagine, glutamine, glutamic acid, and aspartic acid internal standards included in each sample run.

[0106] Total Amino Acids

[0107] Fifty milligrams of leaf material (fresh weight, no midrib) were freeze-dried for dry weight determination. The dehydrated leaf tissue was then ground in the presence of fresh Milli Q water using a MiniBeadbeater-96.TM. and 2.3 mm stainless-steel beads. The ground leaf tissue was filtered through a 0.45 .mu.m Polyvinylidene Fluoride (PVDF) filter. Dilutions of leaf extract were performed in water, and ninhydrin reagent solution (ninhydrin and hydrindantin in DMSO and lithium acetate buffer, pH 5.2) was added. The samples were then sealed with a thick foil tape, heated for ten minutes at 90.degree. C., cooled for exactly two minutes, and read in a spectrophotometer at 590 nm. Values were compared with internal standards included during each sample analysis.

[0108] Total Protein

[0109] Fifty milligrams of leaf material (fresh weight, no midrib) were freeze-dried for dry weight determination. The dehydrated leaf tissue was then ground in the presence of fresh Milli Q water using a MiniBeadbeater-96.TM. and 2.3 mm stainless-steel beads. The ground leaf tissue was filtered through a 0.45 .mu.m Polyvinylidene Fluoride (PVDF) filter. Bio-Rad Protein Dye was added to leaf samples diluted in water, and a Bradford protein assay was performed and read in the spectrophotometer at 595 nm vs. internal protein standards included in the assay.

[0110] Chlorophyll

[0111] Fifty milligrams of leaf material (fresh weight, no midrib) were ground in the presence of 60% methanol using a MiniBeadbeater-96.TM. and 2.3 mm stainless-steel beads. The ground leaf tissue was filtered through a 1.0 .mu.m A/B glass fiber filter, and 100 .mu.l extract was placed in a Corning 3370 flat bottom microplate and read in spectrophotometer with wells blanked with an equivalent volume of 60% methanol. SoftMax Pro software was used to convert the light pathlength to 1 cm. Calculations of chlorophyll content were performed using equations from Porra, R. J. Photosynthesis Research, 73: 149-156, 2002.

Example 1

Identification of Candidate ESTs

[0112] The nucleotide sequence information for each of the candidate nitrogen-modulated genes was generated in a differential nitrogen microarray experiment conducted at the direction of applicant by Dr. Pat Schnable at Iowa State University. This microarray experiment was used as an initial screen to select a sub-set of ESTs that may be related to nitrogen conditions.

[0113] From the large number of EST sequences showing some difference in the microarray (136 with both 3' and 5' data), further selections were made following a bioinformatics analysis. This analysis included checking for nucleotide sequence similarities in the International Nucleotide Sequence Database (housed at NCBI), checking for predicted protein similarities in the protein databases, such as NCBI and Swisspro, exploring information concerning known or predicted function, and checking the nucleotide and protein databases at the patent office. Using the results of these analyses, as well as supporting key information, a subset of ESTs was selected for transgenic overexpression in corn in relation to nitrogen use efficiency. For each of the EST sequences, an open reading frame was identified and translated into an amino acid sequence. A list of the candidate nitrogen-modulated sequences is provided in Table 1.

[0114] Vector Construction for Overexpression of Nitrogen-Modulated Sequences in Plants

[0115] An open reading frame for each of the candidate nitrogen-modulated ESTs and subsequently introduced into vectors for plant expression. Using an approach well-known in the art, two different selectable marker systems which allow selection of transformed plants in the presence of a selection agent were employed.

[0116] Maize Transformation with Nitrogen-Modulated Genes

[0117] The plant vectors described are useful for plant transformation experiments to introduce the nitrogen-modulated genes into the maize genome using the methods described above.

TABLE-US-00001 TABLE 1 Nitrogen-modulated sequences EST Open reading Protein Sequence frame sequence (SEQ ID (SEQ ID (SEQ ID pAX EST Name NO:) NO:) NO:) number N-EST213 1 2 3 pAX2411 pAX2410 N-EST45-C08 4 5 pAX3404 N-EST77-A.sup.1 6 7 8 pAX3405 N-EST77-B.sup.1 6 9 10 pAX3406 N-EST61-A10 11 12 pAX2422 N-EST88-H03 13 14 pAX2425 N-EST15 15 16 pAX2437 N-EST42-B12 17 18 pAX2435 N-EST76a.sup.2 19 22 23 pAX2433 N-EST76b.sup.2 19 24 25 pAX2431 N-EST31-A10 26 27 pAX2441 N-EST43 28 29 pAX2443 N-EST264 30 31 pAX2437 N-EST28 32 33 pAX2439 N-EST13A-A08 34 35 pAX2454 N-EST13E-E07 36 37 pAX2457 N-EST55C-C10 38 39 pAX2460 .sup.1See Example 2 .sup.2See Example 3

Example 2

Two Maize Proteins N-EST 77A, N-EST 77B

[0118] This invention describes the use of a maize gene sequences (from EST N-EST77-A01) to confer enhanced nitrogen utilization in transgenic maize (Zea mays). Two open reading frames are joined to a highly active plant promoter and a terminus to express each protein following integration into the maize genome. The ectopically expressed proteins will enhance the maize plant's ability to utilize available nitrogen.

[0119] Bioinformatics analysis revealed that there was no significant sequence homology with other sequences in the NCBI database. One portion showed some homology to a CCAAT-binding transcription factor in other species but not in maize. When the nucleotide sequence was received from the microarray experiment, there was also a predicted protein sequence. The predicted protein is referred to herein as N-EST77A. Examination of the nucleotide sequence indicated that the nucleotide could code for another protein (subsequently confirmed), and that protein sequence is referred to as N-EST77B. This second protein was not predicted in any information received from the microarray experiment.

[0120] For expression of N-EST 77B, the first amino acid was changed from a leucine to a methionine to improve protein expression.

Example 3

Maize Protein N-EST76

[0121] This Example describes the use of a maize gene sequence (from EST N-EST76-H12) to confer enhanced nitrogen utilization in transgenic maize (Zea mays). This particular EST possesses part of the nucleotide sequence that is homologous to the so-called "bZIP" class of transcription factors. For this invention, two separate gene constructs are overexpressed in plants. One construct ("N-EST76a") contains the modified version of the N-EST76-H12 EST to allow a longer open reading frame to be expressed in maize. This modified gene contains 3 substitutions when compared to the gene sequence in the native EST. A second gene is also created which adds a basic region leucine zipper sequence to the 3' end of the gene. The resulting gene is referred to as "N-EST76b"

[0122] The full-length clone sequence appeared to contain two different regions that code for proteins, protein I of 108 amino acids and protein II of 122 amino acids. It was recognized, however, that if the full-length clone had not been sequenced accurately and a mistake had been made in the sequencing in the middle of the clone, a frameshift may have artificially generated a new start codon when it should not be there, thus suggesting two regions when there is only one longer region. To accommodate this possibility, the sequence analysis was done assuming that both the two shorter regions and the one longer region existed. Briefly, the nucleotide sequence searches returned results that indicated that the "I" sequence had some homology with a hypothetical protein from rice (genomic DNA from the rice genome program), and minor homology with some putative bZIP TFs. The nucleotide patent database search showed that sequence I had some homology (E=2e-06) with sequences that were noted to be transcription factors (e.g. WO03007699). A predicted amino acid sequence for 1 from the microarray assay was used to search against the databases and no significant hits were found. However, when the nucleotide sequence I was re-translated using GenBank tools, or the ExPasy tool, the predicted protein sequences were found to have: (1) Hits against the GenBank protein dbase (e.g. E=9e-09) with suggested function being a bZIP transcription factor; and (2) hits against the patent protein database (e.g. E=7e-05) with function being associated with a bZIP transcription factor (especially from rice), or an ABA-responsive element-binding protein (mostly from Arabidopsis, e.g. U.S. Pat. No. 6,245,905).

[0123] Confirmation of DNA Sequence

[0124] The DNA construct that contained N-EST76-H12 was sequenced to confirm the sequence provided from the microarray assay. This sequencing effort revealed a single nucleotide substitution at position 1121 of SEQ ID NO: 19, in which a "G" is present in place of a "C". This substitution is located in an open reading frame described for N-EST76, and leads to the substitution of a glutamine for a glutamic acid in the protein sequence. The correct DNA sequence for the full N-EST76-H12 EST is represented in SEQ ID NO: 19.

[0125] Cloning Strategy to Generate N-EST76a and N-EST76b

[0126] The DNA sequence in N-EST76-H12 contains 3 open reading frames that are separated by two stop codons and one frameshift. The cloning strategy employed was to eliminate both stop codons and the frameshift to produce a continuous open reading frame that is more similar to known bZIP proteins and is thus more likely to function properly when expressed. Additionally, bZIP proteins typically contain a basic region leucine zipper at the C-terminal end of the protein. N-EST76-H12 does not contain such a domain. Thus, a second protein was created which adds a basic region leucine zipper domain to the end of the N-EST76 protein.

[0127] Elimination of Stop Codons and Frameshift in N-EST76-H12

[0128] For this Example, the maize sequence described in the EST N-EST76-H12 (SEQ ID NO:19) was modified to produce a longer open reading frame that is more homologous to full-length bZIP proteins. This required 3 modifications to the N-EST76 sequence:

[0129] Substitution of cytosine in place of thymine at nucleotide position 444

[0130] Substitution of guanine in place of adenine at nucleotide position 673

[0131] Addition of guanine after nucleotide position 722

[0132] The first two substitutions served to remove a pair of stop codons that are present in the N-EST76 EST in reading frame 3. The last change (addition after nucleotide position 722) introduced a frameshift to connect reading frame 3 to reading frame 2 to generate a reading frame that is more homologous to full-length bZIP proteins. The DNA sequence is presented in SEQ ID NO: 22 and the protein that is expressed from the resulting construct is referred to as "N-EST76a" (SEQ ID NO:23).

[0133] Addition of Basic Region Leucine Zipper to N-EST76a

[0134] Additionally, we create a second gene in which a DNA fragment encoding a basic region leucine zipper was added to the 3' end of N-EST76a. This zipper domain is lacking in the EST for N-EST76, and is added here to create a N-EST76-derived protein that is more similar to the bZIP proteins described in the literature. Thus, a protein which is identical to N-EST76a is created except that it possesses an added zipper domain at the C-terminus. This new DNA sequence is represented in SEQ ID NO: 24 and the protein is referred to as "N-EST76b" (SEQ ID NO: 25).

[0135] These cloning strategies are summarized below.

[0136] Selection of bZIP Domain for Project

[0137] The selection of a bZIP domain for this project was carried out by selecting proteins with high homology to the translated N-EST76a sequence using the blastx search algorithm. This approach led to the identification of a rice bZIP protein with significant homology to the N-EST76a protein. The protein sequence of this rice bZIP protein (accession number BAD17130) is presented herein as SEQ ID NO: 20, with the bZIP domain represented by amino acid positions 275-357 of SEQ ID NO: 20.

[0138] The DNA sequence encoding the complete rice bZIP protein is presented in SEQ ID NO: 21, with the DNA fragment coding for the basic region leucine zipper represented by nucleotide positions 826-1074 of SEQ ID NO:21. This bZIP DNA sequence was optimized for maize codon usage and then added to the 3' end of the N-EST76a gene sequence (nucleotide position 1130 in N-EST76-H12) to create the N-EST76b gene sequence (SEQ ID NO: 24).

Example 4

Generation of Transgenic Maize Events and Nitrogen Assimilation in Maize Plants Expressing N-EST76a and N-EST76b

[0139] As described in a previous Example, the plant transformation vectors pAX2433 and pAX2431 were constructed to direct overexpression of the N-EST76a and N-EST76b proteins in maize.

[0140] Each vector was introduced into an Agrobacterium tumefaciens strain by electroporation. This strain also contained the vector pSB 1, which allows pSB 1 and pAX2433 or pAX2431 to recombine in vivo to create a vector that can direct insertion of the N-EST76a or N-EST76b cassette into the maize genome. The formation of each recombinant vector (pAG2433, pAG2431) was confirmed by Southern blot hybridization of the Agrobacterium strain.

[0141] The Agrobacterium strains containing pAG2433 or pAG2431 were co-cultivated with maize embryos using methods known in the art. Following co-cultivation, the embryos were grown on selection medium. Individual events that survived selective growth in the presence of the selection agent were then moved to regeneration medium and grown to the plantlet stage using methods known in the art.

[0142] Nitrogen Assimilation in Maize Plants Expressing N-EST76a, N-EST76b

[0143] Nitrogen Assays, T0 Events

[0144] A series of assays that quantify nitrogen intermediates in plants have been developed. These assays were utilized here to analyze a total of 24 transgenic plants containing the N-EST76a gene and 6 plants containing the N-EST76b gene. Each of the plants was sampled following 4 weeks of growth in soil in a greenhouse. These leaf samples were processed to determine their nitrate, asparagine, glutamine, aspartic acid, glutamic acid, ammonium, total amino acid, chlorophyll and total protein levels. Included alongside in the analysis were plants that were transformed with a construct containing only the selectable marker (no N-EST76a or N-EST76b). These plants were likewise sampled at 4 weeks and are referred to as "non GOT" plants. The results of the nitrogen assays carried out on both types of plants are shown below in Table 2.

TABLE-US-00002 TABLE 2 Nitrogen levels, N-EST76a and N-EST76b vs. non GOI maize events, 4 weeks following transfer to soil Total Total Aspartic Glutamic Amino Total Chlorophyll Nitrate Asparagine Glutamine Acid Acid Ammonium Acids protein (a + b) Plant # GOI (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (mg/g) (mg/g) (mg/g) 6164 N-EST76a 259 181 159 798 2596 112 120 9.18 0.048 6165 N-EST76a 557 288 204 546 3156 179 164 18.43 0.057 6166 N-EST76a 170 132 180 377 2921 122 163 15.97 0.040 6167 N-EST76a 394 346 178 459 2430 122 132 12.70 0.056 6170 N-EST76a 292 113 172 449 2857 126 147 10.09 0.028 6172 N-EST76a 259 160 198 326 2856 130 156 17.13 0.040 6173 N-EST76a 300 211 210 140 2024 179 152 17.52 0.069 6174 N-EST76a 15572 1604 208 470 2287 433 161 17.83 0.143 6175 N-EST76a 448 287 247 574 2542 247 166 15.73 0.078 6176 N-EST76a 272 231 207 306 3146 172 161 14.20 0.049 6178 N-EST76a 380 816 387 503 3152 170 169 6.71 0.044 6287 N-EST76a 772 126 372 290 2821 188 133 9.08 0.050 6288 N-EST76a 418 222 367 214 3010 114 153 10.02 0.036 6289 N-EST76a 153 90 288 109 2529 168 136 8.46 0.073 6290 N-EST76a 388 758 428 490 2584 196 169 9.78 0.074 6291 N-EST76a 241 112 277 697 1905 170 126 7.31 0.082 6292 N-EST76a 186 136 426 561 2259 158 144 14.21 0.066 6293 N-EST76a 628 277 712 491 2582 490 185 10.01 0.070 6294 N-EST76a 470 271 413 744 2794 182 121 10.96 0.073 6295 N-EST76a 197 169 484 263 2395 160 146 19.75 0.063 6296 N-EST76a 291 528 391 314 2537 153 130 13.80 0.046 6297 N-EST76a 173 217 406 358 2886 167 160 15.94 0.132 6298 N-EST76a 383 149 570 684 2277 629 121 8.41 0.046 6299 N-EST76a 524 364 439 191 1723 404 166 13.55 0.061 6155 N-EST76b 426 378 338 564 3587 142 180 14.57 0.045 6156 N-EST76b 114 190 208 987 3209 122 171 12.04 0.041 6158 N-EST76b 1449 690 258 298 2986 127 171 19.55 0.042 6160 N-EST76b 629 541 290 272 3279 162 181 23.69 0.094 6161 N-EST76b 347 352 198 704 3214 145 154 16.00 0.048 6162 N-EST76b 483 226 183 581 2912 148 146 10.35 0.086 5986 non-GOI 148 73 285 364 3107 98 85 6.29 0.045 5987 non-GOI 652 32 280 544 2111 124 75 7.62 0.040 5988 non-GOI 232 22 186 199 1420 124 95 8.48 0.036 5989 non-GOI 123 55 256 354 2904 107 108 9.20 0.045 Avg N-EST76a 355 269 336 430 2608 215 149 12.56 0.060 (excl. 6174) Avg N-EST76b 575 396 246 568 3198 141 167 16.03 0.059 Avg non-GOI 289 46 252 365 2386 114 91 7.90 0.041

Example 5

Generation of N-EST213 Antibodies

[0145] Synthetic peptides were generated to match the N-terminal fragment of N-EST213 (1.sup.st 20 amino acids of SEQ ID NO.3) and the C-terminal fragment of N-EST213 (last 20 amino acids of SEQ ID NO.3). These peptides are used to immunize rabbits using methods known in the art for the purpose of generating polyclonal antibodies against N-EST213 peptide.

Example 6

Generation of Transgenic Maize Events Using the N-EST213 Gene and Nitrogen Assimilation in Maize Plants Expressing N-EST213 (T0 Plants)

[0146] Generation of Transgenic Maize Plants that Overexpress the N-EST213 Protein

[0147] The plant transformation vector pAX2411 was constructed to direct overexpression of the N-EST213 protein in maize as described in a previous Example. The vector pAX2411 was introduced into an Agrobacterium tumefaciens strain by electroporation. This strain also contained the vector pSB 1, which allows pSB 1 and pAX2411 to recombine in vivo to create a vector that can direct insertion of the N-EST213 cassette into the maize genome. The formation of this recombinant vector (pAG2411) was confirmed by Southern blot hybridization of this Agrobacterium strain.

[0148] The Agrobacterium strain containing pAG2411 was co-cultivated with maize embryos using methods known in the art. Individual events that survived selective growth in the presence of the selection agent were then moved to regeneration medium and grown to the plantlet stage using methods known in the art.

[0149] Surprisingly, some of the plants transformed with the N-EST213 DNA were found to display an unusual phenotype. These plants were significantly shorter than non-transformed plants, with "nodal compression" present along the stalk. Seven of the 20 plants in this study exhibited this "short" phenotype. An additional 8 plants were scored as "medium" height, and an additional 4 plants were scored as "tall" height. The shorter plants developed a tassel and an ear, but both organs were sometimes undersized, and the husks were sometimes discolored or not completely formed.

[0150] Nitrogen Assimilation in Maize Plants Expressing N-EST213

[0151] A series of assays that quantify nitrogen intermediates in plants have been developed. These assays were utilized here to analyze a total of 23 transgenic plants containing the N-EST213 gene. Each of the plants was sampled following 4 weeks of growth in soil in a greenhouse. These leaf samples were processed to determine their nitrate, asparagine, glutamine, aspartic acid, glutamic acid, ammonium, total amino acid, chlorophyll and total protein levels. The results of these nitrogen assays are shown below in Table 3.

TABLE-US-00003 TABLE 3 Nitrogen levels, N-EST213 maize events, 4 weeks following transfer to soil Total Total Aspartic Glutamic Amino Total Chlorophyll Nitrate Asparagine Glutamine Acid Acid Ammonium Acids protein (a + b) Plant # GOI (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (mg/g) (mg/g) (mg/g) 2811 N-EST213 242 644 697 72 3.10 0.101 2812 N-EST213 158 618 541 30 1.99 0.078 2813 N-EST213 245 268 15 798 416 63 9.10 0.045 2814 N-EST213 296 49 77 1124 484 124 2.40 0.081 2815 N-EST213 202 62 1133 546 127 4.69 0.097 2816 N-EST213 616 27 66 1977 720 179 17.35 0.052 2817 N-EST213 6915 119 216 125 1464 509 230 14.48 0.057 2818 N-EST213 9380 221 81 2413 829 228 14.08 0.119 2822 N-EST213 3483 109 50 1458 401 150 13.42 0.074 2823 N-EST213 839 79 229 2510 671 173 8.83 0.160 2824 N-EST213 328 527 421 67 1.63 0.051 2825 N-EST213 162 566 382 75 2.62 0.084 2826 N-EST213 272 394 367 50 1.27 0.119 2827 N-EST213 181 351 384 72 2.45 0.109 2828 N-EST213 163 256 416 49 1.78 0.014 2829 N-EST213 171 274 358 71 5.25 0.098 2830 N-EST213 185 15 217 368 54 2.20 0.063 2832 N-EST213 205 742 375 53 4.01 0.102 2833 N-EST213 152 354 383 53 1.89 0.057 2835 N-EST213 232 15 100 447 363 43 2.75 0.123 2837 N-EST213 249 139 666 390 43 0.14 0.061 2838 N-EST213 2997 547 418 67 0.113 2841 N-EST213 188 300 355 70 3.03 0.071 Average 214 194 83 103 860 469 93 5 0.084 Std Dev 52 83 56 676 136 68 5.69 0.033 CV 0.24 0.00 1.01 0.54 0.79 0.29 0.73 1.06 0.39 # plants 23 23 2 9 9 23 23 23 22 23 with positive values 2816 to 2823, 2838 excluded

[0152] Control samples were also generated from transgenic maize plants that contained the selectable marker cassette only (no N-EST213). These samples were likewise sampled at 4 weeks, and the nitrogen levels were determined. These data are shown in Table 4.

TABLE-US-00004 TABLE 4 Nitrogen levels, non-GOI plants, 4 weeks following planting Total Total Aspartic Glutamic Amino Total Chlorophyll Nitrate Asparagine Glutamine Acid Acid Ammonium Acids protein (a + b) Plant # GOI (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (mg/g) (mg/g) (mg/g) 2760 non GOI 24939 257 92 1423 551 151 16.77 0.022 2761 non GOI 7159 196 74 1151 607 146 17.59 0.038 2762 non GOI 1625 146 225 58 1177 445 141 13.33 0.036 2763 non GOI 4421 197 119 1172 487 111 11.08 0.038 2765 non GOI 1901 131 69 874 366 92 9.10 0.032 2766 non-GOI 233 14 184 352 56 5.33 0.085 2768 non-GOI 210 9 256 346 64 5.54 0.062 2769 non-GOI 245 17 249 481 56 4.08 0.055 Average 229 146 131 83 811 454 102 10 0.046 Std Dev 18 104 24 503 96 41 5 0.020 CV 0.08 0.79 0.29 0.62 0.21 0.40 0.51 0.44 # plants with 8 3 1 8 5 8 8 8 8 8 positive values

Example 7

Generation of Transgenic Maize Events and Nitrogen Assimilation in Maize Plants Expressing N-EST45 (T0 and T1 Plants)

[0153] Generation of Transgenic Maize Plants that Overexpress the N-EST45 Protein

[0154] As described in the previous Example, the plant transformation vector pAX3404 was constructed to direct overexpression of the N-EST45 protein in maize.

[0155] The vector pAX3404 was introduced into an Agrobacterium tumefaciens strain by electroporation. This strain also contained the vector pSB 1, which allows pSB 1 and pAX3404 to recombine in vivo to create a vector that can direct insertion of the N-EST45 cassette into the maize genome. The formation of this recombinant vector (pAG3404) was confirmed by Southern blot hybridization of this Agrobacterium strain.

[0156] The Agrobacterium strain containing pAG3404 was co-cultivated with maize embryos using methods known in the art. Following co-cultivation, the embryos were grown on selection medium. Individual events that survived selective growth in the presence of the selection agent were then moved to regeneration medium and grown to the plantlet stage using methods known in the art.

[0157] Nitrogen Assimilation in Maize Plants Expressing N-EST45

[0158] Nitrogen Assays, T0 Events

[0159] A series of assays that quantify nitrogen intermediates in plants have been developed. These assays were utilized here to analyze a total of 16 transgenic plants containing the N-EST45 gene. Each of the plants was sampled following 4 weeks of growth in soil in a greenhouse. These leaf samples were processed to determine their nitrate, asparagine, glutamine, aspartic acid, glutamic acid, ammonium, total amino acid, chlorophyll and total protein levels. Included alongside in the analysis were plants that were transformed with a construct containing only the selectable marker (no N-EST45). These plants were likewise sampled at 4 weeks and are referred to as "non GOT" plants. The results of the nitrogen assays carried out on both types of plants are shown below in Table 5.

TABLE-US-00005 TABLE 5 Nitrogen levels, N-EST45 vs. non GOI maize events, 4 weeks following transfer to soil Total Total Aspartic Glutamic Amino Total Chlorophyll Nitrate Asparagine Glutamine Acid Acid Ammonium Acids protein (a + b) Plant # GOI (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (mg/g) (mg/g) (mg/g) 3751 N-EST45 216 178 155 102 1383 403 84 1.30 0.074 3752 N-EST45 251 129 317 1485 407 104 1.34 0.092 3754 N-EST45 457 446 402 557 3378 768 240 2.59 0.104 3755 N-EST45 293 416 321 645 2143 484 144 1.81 0.065 3759 N-EST45 656 211 379 2068 413 135 1.73 0.051 3760 N-EST45 7172 221 238 661 2421 627 184 1.88 0.092 3762 N-EST45 809 150 273 1752 369 128 1.24 0.090 3764 N-EST45 233 108 203 1919 301 106 0.86 0.073 3765 N-EST45 598 121 284 1409 437 112 1.47 0.137 3768 N-EST45 271 118 321 1430 321 116 1.14 0.052 3771 N-EST45 480 117 189 462 1221 525 129 1.89 0.183 3772 N-EST45 565 231 167 395 2083 410 111 1.05 0.076 3773 N-EST45 659 105 263 1634 445 101 1.63 0.088 3779 N-EST45 533 96 138 354 1745 397 126 0.99 0.095 3781 N-EST45 500 109 144 286 1477 490 137 1.28 0.094 3784 N-EST45 1209 90 144 228 1446 424 135 1.11 0.089 2771 (non-GOI) non-GOI 354 224 132 1065 506 117 0.88 0.073 2773 (non-GOI) non-GOI 183 188 209 1040 365 109 1.27 0.044 2774 (non-GOI) non-GOI 135 223 158 629 470 102 1.38 0.045 Average (GOI) 466 212 177 358 1812 451 131 1.46 0.091 Std Dev 187 135 82 156 537 115 37 0.44 0.032 CV 0.40 0.64 0.46 0.43 0.30 0.25 0.28 0.30 0.35 3760, 3784 excluded

[0160] Nitrogen Assays, T1 Events

[0161] The nitrogen levels present in the T0 N-EST45 maize events were examined and several plants were selected for characterization as T1 plants. Events ("plant #") 3755, 3759, 3760, 3765, 3773 and 3781 were chosen. Non-GOI events 3822 and 3828 were selected as negative controls. To generate T1 plants, pollen was collected from each of the T0 events and used to pollinate ears on Hi-II (A188.times.B73) plants. Following seed set and seed harvest, dried seeds from these crosses were germinated in soil. Approximately 2 weeks after planting, segregants containing the N-EST45 gene (or selectable marker gene in non-GOI plants) were identified and grown until 4 weeks of age. Leaf samples were taken from these events at 4 weeks and entered into the same nitrogen testing scheme utilized for the T0 plants (nitrate, asparagine, glutamine, aspartic acid, glutamic acid, ammonium, total amino acid, chlorophyll and total protein). The results of these nitrogen assays are shown in Table 6.

TABLE-US-00006 TABLE 6 Nitrogen levels, T1 plants, N-EST45 vs. non-GOI events Total Total Aspartic Glutamic Amino Total Chlorophyll Nitrate Asparagine Glutamine Acid Acid Ammonium Acids protein (a + b) Plant # GOI (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (mg/g) (mg/g) (mg/g) 3751 N-EST45 216 178 155 102 1383 403 84 1.30 0.074 3752 N-EST45 251 129 317 1485 407 104 1.34 0.092 3754 N-EST45 457 446 402 557 3378 768 240 2.59 0.104 3755 N-EST45 293 416 321 645 2143 484 144 1.81 0.065 3759 N-EST45 656 211 379 2068 413 135 1.73 0.051 3760 N-EST45 7172 221 238 661 2421 627 184 1.88 0.092 3762 N-EST45 809 150 273 1752 369 128 1.24 0.090 3764 N-EST45 233 108 203 1919 301 106 0.86 0.073 3765 N-EST45 598 121 284 1409 437 112 1.47 0.137 3768 N-EST45 271 118 321 1430 321 116 1.14 0.052 3771 N-EST45 480 117 189 462 1221 525 129 1.89 0.183 3772 N-EST45 565 231 167 395 2083 410 111 1.05 0.076 3773 N-EST45 659 105 263 1634 445 101 1.63 0.088 3779 N-EST45 533 96 138 354 1745 397 126 0.99 0.095 3781 N-EST45 500 109 144 286 1477 490 137 1.28 0.094 3784 N-EST45 1209 90 144 228 1446 424 135 1.11 0.089 2771 (non-GOI) non-GOI 354 224 132 1065 506 117 0.88 0.073 2773 (non-GOI) non-GOI 183 188 209 1040 365 109 1.27 0.044 2774 (non-GOI) non-GOI 135 223 158 629 470 102 1.38 0.045 Average (GOI) 466* 212 177 358 1812 451 131 1.46 0.091 Std Dev 187* 135 82 156 537 115 37 0.44 0.032 CV 0.40* 0.64 0.46 0.43 0.30 0.25 0.28 0.30 0.35 Average (non GOI) 224 undet 211 166 911 447 110 1.17 0.054 Std Dev 115 20 39 245 73 8 0.26 0.017 CV 0.52 0.10 0.23 0.27 0.16 0.07 0.22 0.31 *3760, 3784 excluded

Example 8

Generation of Transgenic Maize Events and Nitrogen Assimilation in Maize Plants Expressing N-EST61

[0162] As described in the previous Example, the plant transformation vector pAX2422 was constructed to direct overexpression of the N-EST61 protein in maize.

[0163] The vector pAX2422 was introduced into an Agrobacterium tumefaciens strain by electroporation. This strain also contained the vector pSB 1, which allows pSB 1 and pAX2422 to recombine in vivo to create a vector that can direct insertion of the N-EST61 cassette into the maize genome. The formation of this recombinant vector (pAG2422) was confirmed by Southern blot hybridization of this Agrobacterium strain.

[0164] The Agrobacterium strain containing pAG2422 was co-cultivated with maize embryos using methods known in the art. Following co-cultivation, the embryos were grown on selection medium. Individual events that survived selective growth in the presence of the selection agent were then moved to regeneration medium and grown to the plantlet stage using methods known in the art.

[0165] Nitrogen Assimilation in Maize Plants Expressing N-EST61

[0166] Nitrogen Assays, T0 Events

[0167] A series of assays that quantify nitrogen intermediates in plants have been developed. These assays were utilized here to analyze a total of 8 transgenic plants containing the N-EST61 gene. Each of the plants was sampled following 4 weeks of growth in soil in a greenhouse. These leaf samples were processed to determine their nitrate, asparagine, glutamine, aspartic acid, glutamic acid, ammonium, total amino acid, chlorophyll and total protein levels. Included alongside in the analysis were plants that were transformed with a construct containing only the selectable marker (no N-EST61). These plants were likewise sampled at 4 weeks and are referred to as "non GOT" plants. The results of the nitrogen assays carried out on both types of plants are shown below in Table 7.

TABLE-US-00007 TABLE 7 Nitrogen levels, N-EST61 vs. non GOI maize events, 4 weeks following transfer to soil Total Total Aspartic Glutamic Amino Total Chlorophyll Nitrate Asparagine Glutamine Acid Acid Ammonium Acids protein (a + b) Plant # GOI (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (mg/g) (mg/g) (mg/g) 5629 N-EST61 205 79 267 287 1954 204 71 25.29 0.095 5630 N-EST61 790 59 223 976 2371 202 98 18.91 0.092 5632 N-EST61 222 144 266 651 2630 292 100 12.41 0.076 5633 N-EST61 193 64 314 1132 1738 387 80 8.88 0.067 5635 N-EST61 383 54 202 574 1431 248 72 8.37 0.080 5636 N-EST61 449 67 349 594 1545 402 87 9.93 0.042 5637 N-EST61 354 292 368 389 2519 244 116 20.12 0.127 5638 N-EST61 1477 37 292 835 1360 224 84 8.51 0.051 5983 non-GOI 345 61 264 71 1435 215 296 7.98 0.107 5984 non-GOI 213 155 850 398 3670 117 355 14.11 0.081 5985 non-GOI 212 73 199 566 2039 182 294 2.67 0.058 Average (N-EST61) 509 100 285 680 1943 275 89 14.05 0.079 Average (non-GOI) 256 96 438 345 2381 171 315 8.25 0.082

Example 9

Generation of Transgenic Maize Events and Nitrogen Assimilation in Maize Plants Expressing N-EST15

[0168] As described in the previous Example, the plant transformation vector pAX2437 was constructed to direct overexpression of the N-EST15 protein in maize.

[0169] The vector pAX2437 was introduced into an Agrobacterium tumefaciens strain by electroporation. This strain also contained the vector pSB 1, which allows pSB 1 and pAX2437 to recombine in vivo to create a vector that can direct insertion of the N-EST15 cassette into the maize genome. The formation of this recombinant vector (pAG2437) was confirmed by Southern blot hybridization of this Agrobacterium strain.

[0170] The Agrobacterium strain containing pAG2437 was co-cultivated with maize embryos using methods known in the art. Following co-cultivation, the embryos were grown on selection medium. Individual events that survived selective growth in the presence of the selection agent were then moved to regeneration medium and grown to the plantlet stage using methods known in the art.

[0171] Nitrogen Assimilation in Maize Plants Expressing N-EST15

[0172] Nitrogen Assays, T0 Events

[0173] A series of assays that quantify nitrogen intermediates in plants have been developed. These assays were utilized here to analyze a total of 8 transgenic plants containing the N-EST15 gene. Each of the plants was sampled following 4 weeks of growth in soil in a greenhouse. These leaf samples were processed to determine their nitrate, asparagine, glutamine, aspartic acid, glutamic acid, ammonium, total amino acid, chlorophyll and total protein levels. Included alongside in the analysis were plants that were transformed with a construct containing only the selectable marker (no N-EST15). These plants were likewise sampled at 4 weeks and are referred to as "non GOT" plants. The results of the nitrogen assays carried out on both types of plants are shown below in Table 8.

TABLE-US-00008 TABLE 8 Nitrogen levels, N-EST15 vs. non GOI maize events, 4 weeks following transfer to soil Total Total Aspartic Glutamic Amino Total Chlorophyll Nitrate Asparagine Glutamine Acid Acid Ammonium Acids protein (a + b) Plant # GOI (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (mg/g) (mg/g) (mg/g) 5923 N-EST15 111 245 267 1036 3244 142 321 10.41 0.074 5924 N-EST15 401 470 330 685 3271 142 349 8.65 0.056 5926 N-EST15 554 65 202 308 2376 183 327 9.22 0.072 5929 N-EST15 591 256 238 1551 2562 168 338 9.98 0.053 5930 N-EST15 1873 909 477 3059 2495 174 458 12.98 0.065 5931 N-EST15 2275 382 268 1023 3590 196 357 6.42 0.073 5932* N-EST15 414 1107 739 1751 4049 430 683 16.79 0.125 5934 N-EST15 272 290 293 863 2306 163 312 7.37 0.047 5983 non-GOI 345 61 264 71 1435 215 296 7.98 0.107 5984 non-GOI 213 155 850 398 3670 117 355 14.11 0.081 5985 non-GOI 212 73 199 566 2039 182 294 2.67 0.058 Average (N-EST15) 811 465 352 1285 2987 200 393 10.23 0.071 Average (non-GOI) 256 96 438 345 2381 171 315 8 0.082 *5932 water content measured higher than others (93% vs. avg of 85%); also extremely fibrous and easily shredded

Example 9

Generation of Transgenic Maize Events and Nitrogen Assimilation in Maize Plants Expressing N-EST28

[0174] As described in the previous Example, the plant transformation vector pAX2439 was constructed to direct overexpression of the N-EST28 protein in maize.

[0175] The vector pAX2439 was introduced into an Agrobacterium tumefaciens strain by electroporation. This strain also contained the vector pSB 1, which allows pSB 1 and pAX2439 to recombine in vivo to create a vector that can direct insertion of the N-EST28 cassette into the maize genome. The formation of this recombinant vector (pAG2439) was confirmed by Southern blot hybridization of this Agrobacterium strain.

[0176] The Agrobacterium strain containing pAG2439 was co-cultivated with maize embryos using methods known in the art. Following co-cultivation, the embryos were grown on selection medium. Individual events that survived selective growth in the presence of the selection agent were then moved to regeneration medium and grown to the plantlet stage using methods known in the art.

[0177] Nitrogen Assimilation in Maize Plants Expressing N-EST28

[0178] Nitrogen Assays, T0 Events

[0179] A series of assays that quantify nitrogen intermediates in plants have been developed. These assays were utilized here to analyze a total of 5 transgenic plants containing the N-EST28 gene. Each of the plants was sampled following 4 weeks of growth in soil in a greenhouse. These leaf samples were processed to determine their nitrate, asparagine, glutamine, aspartic acid, glutamic acid, ammonium, total amino acid, chlorophyll and total protein levels. Included alongside in the analysis were plants that were transformed with a construct containing only the selectable marker (no N-EST28). These plants were likewise sampled at 4 weeks and are referred to as "non GOT" plants. The results of the nitrogen assays carried out on both types of plants are shown below in Table 9.

TABLE-US-00009 TABLE 9 Nitrogen levels, N-EST28 vs. non GOI maize events, 4 weeks following transfer to soil Total Total Aspartic Glutamic Amino Total Chlorophyll Nitrate Asparagine Glutamine Acid Acid Ammonium Acids protein (a + b) Plant # GOI (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (.mu.g/g) (mg/g) (mg/g) (mg/g) 6179 N-EST28 230 393 385 458 3209 116 137 15.22 0.026 6180 N-EST28 282 392 415 151 3613 126 175 22.09 0.074 6182 N-EST28 271 132 248 340 2722 133 103 8.77 0.050 6183 N-EST28 244 177 291 1098 2976 122 113 9.59 0.088 6184 N-EST28 183 253 325 496 3143 123 119 11.40 0.050 5986 non-GOI 148 73 285 364 3107 98 85 6.29 0.045 5987 non-GOI 652 32 280 544 2111 124 75 7.62 0.040 5988 non-GOI 232 22 186 199 1420 124 95 8.48 0.036 5989 non-GOI 123 55 256 354 2904 107 108 9.20 0.045 Avg N-EST28 242 270 333 509 3133 124 129 13.41 0.057 Avg non-GOI 289 46 252 365 2386 114 91 7.90 0.041

Example 11

Generation of Transgenic Maize Events and Nitrogen Assimilation in Maize Plants Expressing N-EST88, N-EST42, N-EST31, N-EST264

[0180] As described in the previous Example, the plant transformation vectors pAX2424 (N-EST88), pAX2435 (N-EST42), pAX2441 (N-EST31) and pAX2437 (N-EST264) were constructed to direct overexpression of the N-EST88, N-EST42, N-EST31 and N-EST264 proteins in maize.

[0181] Each vector was introduced into an Agrobacterium tumefaciens strain by electroporation. This strain also contained the vector pSB1, which allows pSB1 and pAX2424, pAX2435, pAX2441 or pAX2437 to recombine in vivo to create a vector that can direct insertion of the N-EST28 cassette into the maize genome. The formation of these recombinant vectors (pAG2424, pAG2435, pAG2441 or pAG2437) was confirmed by Southern blot hybridization of this Agrobacterium strain.

[0182] The Agrobacterium strains containing pAG2424, pAG2435, pAG2441 or pAG2437 were co-cultivated with maize embryos using methods known in the art. Following co-cultivation, the embryos were grown on selection medium. Individual events that survived selective growth in the presence of the selection agent were then moved to regeneration medium and grown to the plantlet stage using methods known in the art.

Example 12

Generation of Plasmids to Direct Overexpression of the N-EST43, N-EST13A, N-EST13E or N-EST55C Proteins in Transgenic Maize Events

[0183] As described in the previous Example, the plant transformation vectors pAX2443 (N-EST43), pAX2454 (N-EST13A), pAX2457 (N-EST13E) and pAX2460 (N-EST55C) were constructed to direct overexpression of the N-EST43, N-EST13A, N-EST13E or N-EST55C proteins in maize.

[0184] Each vector was introduced into an Agrobacterium tumefaciens strain by electroporation. This strain also contained the vector pSB1, which allows pSB1 and pAX2443, pAX2454, pAX2457 or pAX2460 to recombine in vivo to create a vector that can direct insertion of the N-EST43, N-EST13A, N-EST13E or N-EST55C cassette into the maize genome. The formation of these recombinant vectors (pAG2443, pAG2454, pAG2457 or pAG2460) was confirmed by Southern blot hybridization of this Agrobacterium strain.

[0185] The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto. Those skilled in the art who have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention.

TABLE-US-00010 SEQUENCE LISTING SEQ ID NO: 1 TCGACTGGAGCACGAGGACACTGACATGGACTGAAGGAGTAGAAAATCACCTAGCTAGAAAGGAGAGCAC CGAGCGCTGCACCACTACTGCTGATATGAGCACCTGAACCTTCTGGGCAACCACATCGTCCCTGCCCCTG ATCATCCGCAGCAGCCATGGCGCAGCAGCAGGAGAAGAAGCAGCAGCAGAGGGGGAAGCTGCAGAGGGTG CTAAGGGAGCAGAAGGCTCGGCTCTACATCATCCGCCGATGCGCGTCATGCTCCTCTGCTGGAGTGACTG ATCCATCTCAAGCATGCATGATAAACCTGTGCTCTTTTTTTTTCCTTCTGTTTTTTCCCCTCTTTTTCCC ATCCTTTTCACCTTGCCACTTTGGTGGGCG SEQ ID NO: 2 ATGGCGCAGCAGCAGGAGAAGAAGCAGCAGCAGAGGGGGAAGCTGCAGAGGGTGCTAAGGGAGCAGAAGG CTCGGCTCTACATCATCCGCCGATGCGCGTCATGCTCCTCTGCTGGAGTGACTGA SEQ ID NO: 3 MAQQQEKKQQQRGKLQRVLREQKARLYIIRRCVVMLLCWSD SEQ ID NO.: 4 ATGTGCATTGCTGCATGGATTTGGCAGGCTCACCCTGTGCACCAACTCCTCCTGCTTCTCAACAGAGATG AGTTCCACAGCAGGCCTACAAAAGCAGTAGGATGGTGGGGTGAAGGCTCAAAGAAGATCCTTGGTGGCAG GGATGTGCTTGGTGGAGGAACATGGATGGGGTGCACCAAGGATGGAAGGCTTGCCTTCCTGACCAATGTG CTTGAACCAGATGCCATGCCCGGTGCACGGACTAGGGGAGATCTGCCTCTCAAATTCCTGCAGAGCAACA AGAGCCCACTCGAAGTTGCAACTGAAGTGGCAGAAGAAGCTGATGAATACAATGGCTTCAACCTCATACT AGCTGATCTAACAACAAATATCATGGTTTATGTGTCAAACCGGCCTAAGGGTCAGCCTGCAACAATTCAA CTCGTGTCACCAGGACTCCATGTGCTGTCCAATGCAAGGCTAGATAGCCCTTGGCAGAAGGCAATTCTCC TCGGTAAAAACTTCAGGGAGCTTCTTAGGGAGCATGGTGCTGATGAGGTTGAAGTGAAGGATATAGTTGA GAGGCTAATGACTGACACCACAAAGGCTGACAAAGATAGACTGCCAAACACTGGTTGTGATCCCAACTGG GAGCATGGTCTGAGCTCCATCTTCATTGAGGTGCAAACTGACCAAGGGCCCTATGGGACACGGAGCACAG CCGTTTTATCAGTGAACTATGATGGCGAAGCTAGCTTGTACGAGAAGTATCTTGAGAGTGGTATATGGAA GGATCACACAGTGAGTTACCAGATAGAGTAG SEQ ID NO: 5 MCIAAWIWQA HPVHQLLLLL NRDEFHSRPT KAVGWWGEGS KKILGGRDVL GGGTWMGCTK DGRLAFLTNV LEPDAMPGAR TRGDLPLKFL QSNKSPLEVA TEVAEEADEY NGFNLILADL TTNIMVYVSN RPKGQPATIQ LVSPGLHVLS NARLDSPWQK AILLGKNFRE LLREHGADEV EVKDIVERLM TDTTKADKDR LPNTGCDPNW EHGLSSIFIE VQTDQGPYGT RSTAVLSVNY DGEASLYEKY LESGIWKDHT VSYQIE SEQ ID NO: 6 ATTCCCGTCTTACCTAGCGCTAGGGTTAGTACGCGTCCACGGCGACGACCTCTGCGCGGAGTGTGCTCCG ATTGGCTGGCCTCCTCGATCCTCCTTCCCGCGAACGCACGCGCGCGCGAGGGAGAGGTTGAGACTTGAGA GATAGACGAAAGACGAAACAAGGGAAGGAGACGCCGTGCTCGCCTATTGGCCGCCGCCTCCGCTCCTTCG CGCCCAATGGCTTCTGCAGCATATCAATATCATGCAGCATAGCAGTACTCAGACCCTTACTACGCAGGCG TTGTTGCTCCCTATGGAAGTCAAGATGTGTGTCCGAGGAGCCTGTCTATGTGAACGCCAAGCAGTACCGC GGCATTCTAAGACGGCGGCAGTCACGTGCCAAGGCCGAGCTTGAGAGAAAGCGCTGGTCAAAGCAAGAAA GCCGTATCTTCACGAGTCCCCGTCATCAGCACGCGATGACGAGGAGGGCGAGAGGGAACGGTGGACGCTT CCTAAACACGAAGAAGAGTGACCGTGTCCCTCCTGATGACTTGATACAGCTACGACGACACAACGAGGCT TGAAGAGGTAGCGGTCTGGCTGGCATCCTAGAGCAGCGGTTTCTGTCCACAGGCACGTGCATCTGAGACC GGATCCGTAGCTCCACTCCACAGCATATGCGCAGCCCATCCATCTCGTGCACACTTG SEQ ID NO: 7 ATGCAGCATAGCAGTACTCAGACCCTTACTACGCAGGCGTTGTTGCTCCCTATGGAAGTCAAGATGTGTG TCCGAGGAGCCTGTCTATGTGAACGCCAAGCAGTACCGCGGCATTCTAAGACGGCGGCAGTCACGTGCCA AGGCCGAGCTTGA SEQ ID NO: 8 MQHSSTQTLT TQALLLPMEV KMCVRGACLC ERQAVPRHSK TAAVTCQGRA SEQ ID NO: 9 TTGAGAGATAGACGAAAGACGAAACAAGGGAAGGAGACGCCGTGCTCGCCTATTGGCCGCCGCCTCCGCT CCTTCGCGCCCAATGGCTTCTGCAGCATATCAATATCATGCAGCATAGCAGTACTCAGACCCTTACTACG CAGGCGTTGTTGCTCCCTATGGAAGTCAAGATGTGTGTCCGAGGAGCCTGTCTATGTGAACGCCAAGCAG TACCGCGGCATTCTAAGACGGCGGCAGTCACGTGCCAAGGCCGAGCTTGAGAGAAAGCGCTGGTCAAAGC AAGAAAGCCGTATCTTCACGAGTCCCCGTCATCAGCACGCGATGACGAGGAGGGCGAGAGGGAACGGTGG ACGCTTCCTAAACACGAAGAAGAGTGACCGTGTCCCTCCTGATGACTTGATACAGCTACGACGACACAAC GAGGCTTGA SEQ ID NO: 10 LRDRRKTKQG KETPCSPIGR RLRSFAPNGF CSISISCSIA VLRPLLRRRC CSLWKSRCVS EEPVYVNAKQ YRGILRRRQS RAKAELERKR WSKQESRIFT SPRHQHAMTR RARGNGGRFL NTKKSDRVPP DDLIQLRRHN EA SEQ ID NO: 11 ATGACTGCTCACCAGACTTGCTGCGATGATGCCGTTGCCGCCGGCACTGCACCGGCTGCC AGGAGGAGGCGCCTCAAATTGACGAGGCCGTCGGCCTCGCTCTTGATGGCGAGGAAGCTA AGGAAGAAGGCTGCCGGCAGCAAACGCCCAAGGGCGGCAGCGTCGAGGAAGCGCGCGATG GCGATCAGGAGGAAGATGGAAGCGCTGAGGCTGCTCGTGCCACTCTGCGGCCGAGACAAC GGCTCGGTGACCGGTGGGGCGGTCGAACGACTGGACGAGCTCCTCATGCACGCCGCCGGG TACATCCTGCGCCTCCAGATGCAGGTCAGAGTGATGCAGCTTATGGTCCATGCACTAAAT GACCGGCCCGAGGATTAA SEQ ID NO: 12 MTAHQTCCDDAVAAGTAPAARRRRLKLTRPSASLLMARKLRKKAAGSKRPRAAASRKRAMAIRRKMEALR LLVPLCGRDNGSVTGGAVERLDELLMHAAGYILRLQMQVRVMQLMVHALNDRPED SEQ ID NO: 13 ATGTCGGCGGCGCTCGCGGTGACGGACGAGGTGGCCCTGCCGATCCGGGCGGTGGGGGAT CTAGCGGCCGCCGCCGAGGTCTCGCGGGAGGAGGTCGCCGTCATCACCCAGTGCGCGGCG CTCGGTGGGAAGTTGCCTTTTGAAGATGCATCAGTTGGTGCGGTTCTTGCAGTCATTAAA AACGTGGAAAGCTTGAGGGAGCAATTGGTTGCTGAAATCAGGCGGGTGCTGAAAGCTGGT GGAAGAGTATTGGTGCAGAGCCCTGCACCCTCATCCAGTCAGAAGCCGAACACTGATATT GAGCGCAAGTTACTGATGGGTGGATTTGCTGAAGTGCAATCTTCTGCTGCAAGCTCGCAG GATAGCGTGCAATCTGTTACAGTTAAGGCAAAGAAGGCTAGCTGGAGCATGGGCTCTTCT TTTCCCCTTAAGAAAACAACAAAAGCCCTTCCCAAGATTCAAATTGACGACGACTCTGAT CTGATTGATGAAGACAGTCTCTTGACTGAGGAGGACCTGAAGAAACCACAACTTCCAGTT GTTGGGGACTGTGAGGTGGGGGCAGCAAAGAAAGCATGCAAGAACTGTACTTGTGGCAGG GCTGAGGCCGAGGAGAAGGTTGGGAAGCTGGAGCTCACTGCGGAGCAGATCAATAACCCT CAGTCAGCTTGTGGCAGTTGTGGGTTGGGTGATGCCTTCCGCTGTGGAACCTGTCCCTAC AGAGGTCTTCCACCATTCAAGCCTGGCGAGAAGGTTTCCTTGTCTGGCAACTTCCTTGCT GCTGACATATGA SEQ ID NO: 14 MSAALAVTDEVALPIRAVGDLAAAAEVSREEVAVITQCAALGGKLPFEDASVGAVLAVIKNVESLREQLV AEIRRVLKAGGRVLVQSPAPSSSQKPNTDIERKLLMGGFAEVQSSAASSQDSVQSVTVKAKKASWSMGSS FPLKKTTKALPKIQIDDDSDLIDEDSLLTEEDLKKPQLPVVGDCEVGAAKKACKNCTCGRAEAEEKVGKL ELTAEQINNPQSACGSCGLGDAFRCGTCPYRGLPPFKPGEKVSLSGNFLAADI SEQ ID NO: 15 ATGGCGATGCAGACGGGGGTCGCGACCTCCAAGGTCCTCATCCTCGTCGGTGCAGGGATGACGGGCTCGA TCCTGCTGCGGAATGGCCGCTTATCTGATGTGTTGGGAGAACTCCAGGAGATTATGAAGGGTGTAAATCA AGGAACTTCTTCGGGTCCCTATGACATTGCACTTATTCAAGCTCAGATTCGGAATTTAGCGCAAGAAGTC AGAGATTTGACATTGTCAAAGCCCATTACCATACTGAATGGCAAATCTGACTCGGGAGGCAGTTTATCAT CCTACATACTGCCAGCAGCAGCAGTTGGAGCAATGGGTTATTGCTACATGTGGTGGAAGGGGTTGTCTCT CTCAGATGTCATGTTTGTCACAAAACACAACATGGCAAATGCTGTTCAGAGCATGTCAAAGCAGTTGGAG CAAGTTTCATCAGCACTAGCTGCAACAAAAAGACATCTAACTCAACGGCTTGAGAATTTGGATGGCAAAA TGGATGAACAAGTAGAGGTCTCCAAAGCTATTAGAAATGAGGTCAATGATGTTAAAGATGACCTGTCTCA AATTGGATTTGATGTCGAATCAATTCAGAAAATGGTTGCTGGATTGGAGGGAAAGATCGAGTTACTTGAG AACAAACAGGACGTGGCTAATACTGGTATCTGGTATCTCTGCCAAGTAGCAGGCGGTTTAAAAGATGGAA TAAACACCAGGTTTTTCCAGGAAACCAGTGAGAAGCTGAAGCTCTCACATTCAGCTCAACCTGAAAACAA GCCAGTGAAGGGGCTTGAATTTTTTTCGGAAAGCACCATGGAACAGAAAGTAGCTGACTCCAAACCAATT GCGGTGACAGTCGACGCTGAGAAGCCTGAGAAAACCGCTGCTGTAATGGGCACCACAGTGCACAGGTCTA TCAGGTTCTCATATCGGAAGGCAGGCCTTGCTTTGTGA SEQ ID NO: 16 MAMQTGVATS KVLILVGAGM TGSILLRNGR LSDVLGELQE IMKGVNQGTS SGPYDIALIQ AQIRNLAQEV RDLTLSKPIT ILNGKSDSGG SLSSYILPAA AVGAMGYCYM WWKGLSLSDV MFVTKHNMAN AVQSMSKQLE QVSSALAATK RHLTQRLENL DGKMDEQVEV SKAIRNEVND VKDDLSQIGF DVESIQKMVA GLEGKIELLE NKQDVANTGI WYLCQVAGGL KDGINTRFFQ ETSEKLKLSH SAQPENKPVK GLEFFSESTM EQKVADSKPI AVTVDAEKPE KTAAVMGTTV HRSIRFSYRK AGLAL SEQ ID NO: 17 ATGTGCTCGGTAGCGAGGCTGGCGTTTGTGCTTGCACTGGCCATAGCCGCCTCGTCAATTGAGGTTGCGG AGAGCAGAGATTTTAATATCTTTGCTCAGGGCAGCTTGCCTGATGCAACCAAGGGATCGTCTGGTCTAGC TGCAACCAGTGGAAAGTTGTGTCAGTTATGCGAGCAGTACTCATCCGAGGCGCTCCTCTATCTCACACAA AACGAGACCCAGACTGAGATTCTTAGCATTCTACACCATGAATGTGCCAGCCTTGCCCCTCTCAAACAGC AGTGCATCACGCTGGTTGACTACTACGTACCCCTTTTCTTCTTGGAGGTCTCCATGGTTACCCCTGAGAA GTTCTGCGAGTCGATGCATCTCTGCAAGAAGGGGATGAAGATTAGCCTACCCACCCGGGAGGGTACTTGT GGTTTGTGCCACCATGTTGTTGTTGAAATTCTTATCATGCTTAAAGACCCCAACATGCAGCTGGAAGTAA TCGACCTACTCACCAAAACATGCAGCAAGGCGCAGAACTATGAACAGTAG SEQ ID NO: 18 MCSVARLAFV LALAIAASSI EVAESRDFNI FAQGSLPDAT KGSSGLAATS GKLCQLCEQY SSEALLYLTQ NETQTEILSI LHHECASLAP LKQQCITLVD YYVPLFFLEV SMVTPEKFCE SMHLCKKGMK ISLPTREGTC GLCHHVVVEI LIMLKDPNMQ LEVIDLLTKT CSKAQNYEQ SEQ ID NO: 19 GGACACTGACATGGACTGAAGGAGTAGAAAATCCATCCATTCCCCTCGCCAAGCCGCCACGGCCTGACTT TCCCTCCCGCACACCCGCGACCATACAGGCAAGTCAGGCATACACCAACAACGCTCGTCGTGCACCTCGC GCCTCAGGTCACCCCACCAAATTCCTCTTGATACGCCGAATTTCTTTTGCTAATTCTGCTACCTCCTGTC GCTAAGCCACCATATTCAGTCTAACCCCTGCTCTGAGCTCACCTGATTGGCGGCTCCGTTCGGCCTCTGG GCCTGGGTGTACCGACTACCGAGGGCTCTTTCGAAATGTCAATTGGGTCGAGTTTGGTGGGCTACGTGAA GCATGGATGAATTTCCCGGCTGGAAGCGGGAGGCGGCAGCAGCATCCGGGGCCGGAGCACCTGTCGCCGA TGACGCCGCTCCCGCTGGCGCGGTAGGGGTCGGTCTACTCGCTCACGTTCGACGAGTTCCAGAGCTCGCT

CGGTGGGGCCACCAAGGACTTCGGATCCATGAACATGGACGAGCTCCTCCGCAACATCTGGTCGGCGGAG GAGACACACAGCGTCACAGCTGCGGACCATGCCGCGCGGGCGCCGTACGTCCAGTGCCAGGGCTCGCTCA CCCTCCCCTGCACGCTCAGCCAGAAGACCGTCGACGAGGTCTAGCGTGACCTCGTGTGCAACGGTGGAGG ACCCTCCGACGAGGCTGTGGCGCCGCCCCACCGGCCCAACGGCAGCCGACGCTCGGGGAGATCATGCTGG AGGAGTTCCTCGTCCGCGCCGGCGTGGTGAGGGAGGACATGATGGCGGCGGCGCCCGTACCACCAGCGCC GGGTTGCCCACCACCTCATCTGCAACCGCCAATGCTGTTTCCACATGGCAATGTGTTTGCTCCCTTAGTG CCTCCGCTCCAATTCGGGAATGGGTTTGTGTCGGGGGCTCTCAGTCAGCAGCAGGGAGGTGTTCTTGAGG CCCCGGCGGTATCGCCGCGGCCGGTGACGGCAAGCGGGTTCGGGAAGATGGAAGGAGACGACTTGTCGCA TCTGTCGCCATCACCGGTGTCGTACGTTTTTTTGTGCTGGTTTGAGGGGAAGGAAGCCACCAGCTGTGGA GAAGGTGGTTGAGAGGAGGCAACGCC SEQ ID No: 20 mdfpggsgrq qqlppmtplp larqgsvysl tfdefqstlg gvgkdfgsmn mdellrsiwt aeeshavgaa ttttattasv aaaehaavga ppvqrqgslt 1prtlsqktv devwrdmmcf ggggastapa aaeppppahr qqtlgeitle eflvragvvr edmsvppvpp aptptaaavp pppppqqqtp mlfgqsnvfp pmvpplslgn glvsgavghg gggaaslvsp vrpvssngfg kmeggdlssl spspvpyvfk gglrgrkapg iekvverrqr rmiknresaa rsrqrkqaym meleaevakl kelndelqkk qdemleqqkn evlermsrqv gptakriclr rtltgpw SEQ ID NO: 21 atg gattttccgg gagggagcgg gaggcagcag cagctgccgc cgatgacgcc gctgccgttg gcgaggcagg ggtcggtgta ctcgctcacg ttcgacgagt tccagagcac gctgggcggg gtcgggaagg acttcgggtc gatgaacatg gacgagctcc tccgcagcat ctggacggcc gaggagtcgc acgccgtcgg cgccgccacg acgacgacgg cgacgacggc gtccgtggcg gcggcggagc acgcggcggt gggggcgccg cccgttcaga ggcaggggtc gctgaccctc ccccgcacgc tcagccagaa gaccgtcgac gaggtctggc gcgacatgat gtgcttcggt ggcggcggcg cctccaccgc gccggccgcc gcggagcccc cgccgccggc gcaccggcag cagacgctcg gggagatcac gctggaggag ttcctcgtgc gggccggcgt ggtgagggag gacatgtcgg tcccgcccgt cccgccggcg ccgactccta cggcggctgc tgtacctccc ccgccgccgc cgcagcagca gacgccgatg ttgttcggtc agagcaatgt gttccctccg atggtgcctc cgctctcgct gggaaatggg ctggtctcgg gagctgtcgg acacggcggt ggtggtgccg cgtcgttggt ttcgccggtg aggccggtct cgtccaatgg cttcggcaag atggaaggcg gggacctgtc gtcgctgtcg ccatcgccgg tgccgtacgt tttcaaaggt gggctgaggg gaaggaaggc accgggcatc gagaaggttg tcgagagaag acagcggcgg atgatcaaga acagggagtc tgccgcgagg tcgcgccaga ggaaacaggc atatatgatg gaattggaag ctgaggtagc aaaacttaag gagctgaacg atgaactcca gaaaaagcag gatgaaatgt tggagcagca aaagaatgag gttctagaga gaatgagccg acaagttgga ccgacagcaa agagaatttg ccttcggagg actctgacgg gtccatggtg a SEQ ID NO: 22 ATGAATTTCCCGGCTGGAAGCGGGAGGCGGCAGCAGCATCCGGGGCCGGAGCACCTGTCGCCGATGACGC CGCTCCCGCTGGCGCGGCAGGGGTCGGTCTACTCGCTCACGTTCGACGAGTTCCAGAGCTCGCTCGGTGG GGCCACCAAGGACTTCGGATCCATGAACATGGACGAGCTCCTCCGCAACATCTGGTCGGCGGAGGAGACA CACAGCGTCACAGCTGCGGACCATGCCGCGCGGGCGCCGTACGTCCAGTGCCAGGGCTCGCTCACCCTCC CCTGCACGCTCAGCCAGAAGACCGTCGACGAGGTCTGGCGTGACCTCGTGTGCAACGGTGGAGGACCCTC CGACGAGGCTGTGGCGGCCGCCCCACCGGCCCAACGGCAGCCGACGCTCGGGGAGATCATGCTGGAGGAG TTCCTCGTCCGCGCCGGCGTGGTGAGGGAGGACATGATGGCGGCGGCGCCCGTACCACCAGCGCCGGGTT GCCCACCACCTCATCTGCAACCGCCAATGCTGTTTCCACATGGCAATGTGTTTGCTCCCTTAGTGCCTCC GCTCCAATTCGGGAATGGGTTTGTGTCGGGGGCTCTCAGTCAGCAGCAGGGAGGTGTTCTTGAGGCCCCG GCGGTATCGCCGCGGCCGGTGACGGCAAGCGGGTTCGGGAAGATGGAAGGAGACGACTTGTCGCATCTGT CGCCATCACCGGTGTCGTACGTTTTTTTGTGCTGGTTTGAGGGGAAGGAAGCCACCAGCTGTGGAGAAGG TGGTTGA SEQ ID NO: 23 MNFPAGSGRR QQHPGPEHLS PMTPLPLARQ GSVYSLTFDE FQSSLGGATK DFGSMNMDEL LRNIWSAEET HSVTAADHAA RAPYVQCQGS LTLPCTLSQK TVDEVWRDLV CNGGGPSDEA VAAAPPAQRQ PTLGEIMLEE FLVRAGVVRE DMMAAAPVPP APGCPPPHLQ PPMLFPHGNV FAPLVPPLQF GNGFVSGALS QQQGGVLEAP AVSPRPVTAS GFGKMEGDDL SHLSPSPVSY VFLCWFEGKE ATSCGEGG SEQ ID NO: 24 ATGAATTTCCCGGCTGGAAGCGGGAGGCGGCAGCAGCATCCGGGGCCGGAGCACCTGTCGCCGATGACGC CGCTCCCGCTGGCGCGGCAGGGGTCGGTCTACTCGCTCACGTTCGACGAGTTCCAGAGCTCGCTCGGTGG GGCCACCAAGGACTTCGGATCTATGAACATGGACGAGCTCCTCCGCAACATCTGGTCGGCGGAGGAGACA CACAGCGTCACAGCTGCGGACCATGCCGCGCGGGCGCCGTACGTCCAGTGCCAGGGCTCGCTCACCCTCC CCTGCACGCTCAGCCAGAAGACCGTCGACGAGGTCTGGCGTGACCTCGTGTGCAACGGTGGAGGACCCTC CGACGAGGCTGTGGCGGCCGCCCCACCGGCCCAACGGCAGCCGACGCTCGGGGAGATCATGCTGGAGGAG TTCCTCGTCCGCGCCGGCGTGGTGAGGGAGGACATGATGGCGGCGGCGCCCGTACCACCAGCGCCGGGTT GCCCACCACCTCATCTGCAACCGCCAATGCTGTTTCCACATGGCAATGTGTTTGCTCCCTTAGTGCCTCC GCTCCAATTCGGGAATGGGTTTGTGTCGGGGGCTCTCAGTCAGCAGCAGGGAGGTGTTCTTGAGGCCCCG GCGGTATCGCCGCGGCCGGTGACGGCAAGCGGGTTCGGGAAGATGGAAGGAGACGACTTGTCGCATCTGT CGCCATCACCGGTGTCGTACGTTTTTTTGTGCTGGTTTGAGGGGAAGGAAGCCACCAGCTGTGGACAAGG TGGTGAGAGAAGACAGAGGAGGATGATCAAGAACAGGGAGTCTGCCGCGAGGTCGAGGCAGAGGAAACAG GCATATATGATGGAATTGGAAGCTGAGGTAGCAAAGCTCAAGGAGCTGAACGACGAGCTCCAGAAGAAGC AGGACGAGATGCTGGAGCAGCAGAAGAACGAGGTGCTGGAGAGGATGTCC AGGCAGGTGGGCCCAACCGCCAAGAGGATTTGCCTGAGGAGGACCCTGACCGGCCCATGGTGA SEQ ID NO: 25 MNFPAGSGRR QQHPGPEHLS PMTPLPLARQ GSVYSLTFDE FQSSLGGATK DFGSMNMDEL LRNIWSAEET HSVTAADHAA RAPYVQCQGS LTLPCTLSQK TVDEVWRDLV CNGGGPSDEA VAAAPPAQRQ PTLGEIMLEE FLVRAGVVRE DMMAAAPVPP APGCPPPHLQ PPMLFPHGNV FAPLVPPLQF GNGFVSGALS QQQGGVLEAP AVSPRPVTAS GFGKMEGDDL SHLSPSPVSY VFLCWFEGKE ATSCGQGGER RQRRMIKNRE SAARSRQRKQ AYMMELEAEV AKLKELNDEL QKKQDEMLEQ QKNEVLERMS RQVGPTAKRI CLRRTLTGPW SEQ ID NO: 26 ATGATGTTCTCCTCCTCCCTCTCTGTGGTGGAGTTTTACTTCCTGCACAGATTCCCCCTG CCTTTTGCTGGCTACCTCATCTTCATTTCCATATTGGCTGGATTCTGGGGCCAGTGTTTG GTTAGGAAGATCGTGCATGTGCTCAAGAGAGCATCGCTTATTGTCTTCATCCTCTCCTCT GTTATCTTCGTCAGTGCTCTTACGATGGGTGTCGTTGGAACCCAGAAGAGCATTTCGATG ATCAACAATCACGAATATATGGGGTTCCTCAACTTCTGCGAGTAA SEQ ID NO: 27 MMFSSSLSVV EFYFLHRFPL PFAGYLIFIS ILAGFWGQCL VRKIVHVLKR ASLIVFILSS VIFVSALTMG VVGTQKSISM INNHEYMGFL NFCE SEQ ID NO: 28 ATGGCGTCTGCAGTGACCAGCAGCGACAAGGAGCAGGCCGTCCCTACCATCGACGCTGAC GAAGCGCACGCGCTGCTGAGCTCCGGCCATGGCTACGTGGATGTCAGGATGCGGGGGGAC TTCCACAAGGCGCATGCGCCCGGTGCTCGGAACGTTCCCTACTACCTGTCCGTCACGCCG CAAGGGAAGGAGAAGAACCCACACTTTGTAGAGGAAGTGGCTGCCTTCTGTGGGAAGGAT GATGTCTTCATTGTGGGTTGCAACACGGGGAACAGATCCAGGTTCGCGACGGCAGACCTT CTGAACGCGGGGTTCAAGAACGTGAGGAACCTGCAAGGTGGTTACCGCTCCTTTCAGCAG CGAGCTCAACAGCAGTA SEQ ID NO: 29 MASAVTSSDK EQAVPTIDAD EAHALLSSGH GYVDVRMRGD FHKAHAPGAR NVPYYLSVTP QGKEKNPHFV EEVAAFCGKD DVFIVGCNTG NRSRFATADL LNAGFKNVRN LQGGYRSFQQ RAQQQ SEQ ID NO: 30 ATGGAGGCGAAGAAGAAGCCGTCGGCCCCCGCCGCCGTCGGAGCCGCGCCGCCGCCGCCG GGTAACGGGTACTTCAGCACCGTCTTCTCCGCGCCGACTGCGGGAAGCGCAAGTGACGCA AAGCATGCGGACTTGTACACGATGCTGAACAAGCAGAGCTCCAGAGGGCAGAATGGCAGA GATGGCAAATCCCACAGCCGCCCTACTTACAAGGATGGCAAACATGCTCATCCAAATGAG CCATCAGAATCTCCTTACTTTGGCTCATCCGTGCATTACGGTGGTCGGGAGTTCTACAGC AGCGTTTTACGGAAGCAACCAGCCAATGAACCCCATACGGATTACAAGGGGGACAACCCG GATGGCTCTGCTACCAGAGGTGATTGGTGGCAAGGTTCACTTTATTACTGA SEQ ID NO: 31 MEAKKKPSAP AAVGAAPPPP GNGYFSTVFS APTAGSASDA KHADLYTMLN KQSSRGQNGR DGKSHSRPTY KDGKHAHPNE PSESPYFGSS VHYGGREFYS SVLRKQPANE PHTDYKGDNP DGSATRGDWW QGSLYY SEQ ID NO: 32 ATGGACCGGAACCTGAGCGGGTTTCTGATCGGGTGCCTGGGCGCCGCCGTGACGCTGCTG GCGTACCAGCAGACGGTGGTGACCAGCACGCAGAGCGTCGCGGCGGGCTTCGTCGTCATC CTCTTCGCCCTCTTCGTCAAGGAAGGATTCATTTCCCTCTGA SEQ ID NO: 33 MDRNLSGFLI GCLGAAVTLL AYQQTVVTST QSVAAGFVVI LFALFVKEGF ISL SEQ ID NO: 34 ATGGCATGCGTCAGCACCTTCCAGAGCTGCCCCATTGCCAGAAGAGCAAAGATCAACACCAGGTCCAGGG GCAGCAGCAGTAGCGTGGCGAAGGGGTCACCACCACCAGCCTTCCAGTTCCAGTGCAGGGCGTCGACTTT CGCGGCGGACACCAGCCTCCGGCTCGAGCTGGACGAGAACCCCGAGGCGATCATCTCGGGGGCGTGGCCC GGGAACTGCTCCCTCCTCAGCTACGACGACCTCCGCGCCTACCTCGAGTCGCAGGAGACGGCGGCCCAGG CAGACGATCAGCGCGGCGTGGCGCTCCTGAGCGAGACCATGTCCACACCCGTGCTGGTGGCCACAGCAGA CCAGACCCTGGAGGACGTCGAGTGCCACTTCGAGGCCGTGTCGGGGCTTCCGGTCGTCGACAGCGGCCTC AGATGCGTCGGGGTGATCGTCAAGAACGACCGGGCAAGAGCCTCTCATGGGTCCAAGACGAAGATATCGG AAGTGATGACATCTCCAGCTATCACACTATCGTCTGACAAAACCGTGATGGATGCTGCTGTTCTCATGCT CAAGAAGAAGATCCACAGATTACCAGTTGTAAACCAGGACGAAAAAGTAATAGGTATAGTTACCCGCGCT GATGTTCTTCGCGTGTTGGAAGGCATGTTGAAGATTTAG SEQ ID NO: 35 MACVSTFQSC PIARRAKINT RSRGSSSSVA KGSPPPAFQF QCRASTFAAD TSLRLELDEN PEAIISGAWP GNCSLLSYDD LRAYLESQET AAQADDQRGV ALLSETMSTP VLVATADQTL EDVECHFEAV SGLPVVDSGL RCVGVIVKND RARASHGSKT KISEVMTSPA ITLSSDKTVM DAAVLMLKKK IHRLPVVNQD EKVIGIVTRA DVLRVLEGML KI

SEQ ID NO: 36 ATGGGCGACCTCTCTGTCGGCCACAGCCGCCGCTGGTGCGGCCGTTTCGCGGCCGTCCTTTGCCTGTGCG CGGCCTTCTGCAAGCCAGATGAACTCCCGATGGATCCACTGCCGAACTTGCCGCCGACGAGGTCGCTGCA GTGCTTCGAGGACGAACAGGTGTACAGCTGCTGCGAGGGCGCGTACAGGCTAAACCCATCGGGAATCATC GCCGTTCCCGTCGGCGCGGTGGACTACTACTGCGGCGGCGCGTGCGTGGTGGAGACGGAGGACGTGCTCA ACTGCGTGGCCAGCGCCCTGGACGGCTTCGCCTTCTACAACGGGGCCTCCGTGGAGGACGTGCGCTACGC ACTCAGGCGGGGCTGCAGCCACACCGCCAGAAGAGGCGACTTCAACGATTTGGAGCCGCATCTGGGCGAC TACCCTGACATCTATGGCGACGATGATGAGCACAGCTTTGGCAGCAAGGTTGTTGCAGCTCCTCTGAGGT TGCTCGCGTTTCTTGGCGGTGCGGGGCTGTTCTTCCTGGGCCCTTGA SEQ ID NO: 37 MGDLSVGHSR RWCGRFAAVL CLCAAFCKPD ELPMDPLPNL PPTRSLQCFE DEQVYSCCEG AYRLNPSGII AVPVGAVDYY CGGACVVETE DVLNCVASAL DGFAFYNGAS VEDVRYALRR GCSHTARRGD FNDLEPHLGD YPDIYGDDDE HSFGSKVVAA PLRLLAFLGG AGLFFLGP SEQ ID NO: 38 ATGGATTCGGAGGCGGTGCAGCACGGCCTTCTCCCTCTGTCTGCCTGTCCTCCTACCGCCAACAGCTGCG CGCATTACAGCCGTGGGTGCAGCGTCGTGGCGCCCTGCTGCGGCCAGGCCTTCGGCTGCCGCCATTGCCA CAACGACGCCAAGAACTCGCTGGAGGTCGACCCGCGCGACCGGCACGAGATCCCCCGCCACGAAATAAAG AAGGTGATCTGTTCTCTCTGCTCCAAGGAACAGGACGTGCAACAGAACTGCTCCAGCTGTGGGGCCTGCA TGGGCAAGTACTTCTGTAAAGTATGCAAGTTCTTCGATGATGATGCCTCAAAGGGCCAGTACCACTGTGA CGGATGTGGAATATGTAGAACCGGCGGCGTGGAGAACTTTTTCCACTGTGATAAATGTGGGTGTTGCTAC AGCAATGTCTTGAAGGATTCCCACCACTGCGTCGAAAGAGCAATGCATCACAACTGCCCCGTCTGCTTTG AGTATCTGTTCGACTCCACGAAGGACATCAGCGTGCTGCAATGTGGGCATACCATCCATTTGGAGTGCAT GAACGAGATGAGAGCACACCATCACTTCTCATGCCCAGTGTGCTCGAGGTCCGCCTGCGACATGTCGGCC ACATGGCGGAAGCTCGACGAGGAGGTCGCGGCCACGCCGATGCCTGACATCTACCAGAAGCACATGGTGT GGATCCTGTGCAACGACTGCAGCGCGACCTCGAGCGTGCGGTTCCACGTGCTGGGGCACAAGTGCCCCGC GTGCAGCTCGTACAACACCCGGGAGACGAGGGCTGCGTGCCCCAGGATCTGA SEQ ID NO: 39 MDSEAVQHGL LPLSACPPTA NSCAHYSRGC SVVAPCCGQA FGCRHCHNDA KNSLEVDPRD RHEIPRHEIK KVICSLCSKE QDVQQNCSSC GACMGKYFCK VCKFFDDDAS KGQYHCDGCG ICRTGGVENF FHCDKCGCCY SNVLKDSHHC VERAMHHNCP VCFEYLFDST KDISVLQCGH TIHLECMNEM RAHHHFSCPV CSRSACDMSA TWRKLDEEVA ATPMPDIYQK HMVWILCNDC SATSSVRFHV LGHKCPACSS YNTRETRAAC PRI

Sequence CWU 1

1

391380DNAZea mays 1tcgactggag cacgaggaca ctgacatgga ctgaaggagt agaaaatcac ctagctagaa 60aggagagcac cgagcgctgc accactactg ctgatatgag cacctgaacc ttctgggcaa 120ccacatcgtc cctgcccctg atcatccgca gcagccatgg cgcagcagca ggagaagaag 180cagcagcaga gggggaagct gcagagggtg ctaagggagc agaaggctcg gctctacatc 240atccgccgat gcgcgtcatg ctcctctgct ggagtgactg atccatctca agcatgcatg 300ataaacctgt gctctttttt tttccttctg ttttttcccc tctttttccc atccttttca 360ccttgccact ttggtgggcg 3802125DNAZea mays 2atggcgcagc agcaggagaa gaagcagcag cagaggggga agctgcagag ggtgctaagg 60gagcagaagg ctcggctcta catcatccgc cgatgcgcgt catgctcctc tgctggagtg 120actga 125341PRTZea mays 3Met Ala Gln Gln Gln Glu Lys Lys Gln Gln Gln Arg Gly Lys Leu Gln1 5 10 15Arg Val Leu Arg Glu Gln Lys Ala Arg Leu Tyr Ile Ile Arg Arg Cys 20 25 30Val Val Met Leu Leu Cys Trp Ser Asp 35 404801DNAZea mays 4atgtgcattg ctgcatggat ttggcaggct caccctgtgc accaactcct cctgcttctc 60aacagagatg agttccacag caggcctaca aaagcagtag gatggtgggg tgaaggctca 120aagaagatcc ttggtggcag ggatgtgctt ggtggaggaa catggatggg gtgcaccaag 180gatggaaggc ttgccttcct gaccaatgtg cttgaaccag atgccatgcc cggtgcacgg 240actaggggag atctgcctct caaattcctg cagagcaaca agagcccact cgaagttgca 300actgaagtgg cagaagaagc tgatgaatac aatggcttca acctcatact agctgatcta 360acaacaaata tcatggttta tgtgtcaaac cggcctaagg gtcagcctgc aacaattcaa 420ctcgtgtcac caggactcca tgtgctgtcc aatgcaaggc tagatagccc ttggcagaag 480gcaattctcc tcggtaaaaa cttcagggag cttcttaggg agcatggtgc tgatgaggtt 540gaagtgaagg atatagttga gaggctaatg actgacacca caaaggctga caaagataga 600ctgccaaaca ctggttgtga tcccaactgg gagcatggtc tgagctccat cttcattgag 660gtgcaaactg accaagggcc ctatgggaca cggagcacag ccgttttatc agtgaactat 720gatggcgaag ctagcttgta cgagaagtat cttgagagtg gtatatggaa ggatcacaca 780gtgagttacc agatagagta g 8015266PRTZea mays 5Met Cys Ile Ala Ala Trp Ile Trp Gln Ala His Pro Val His Gln Leu1 5 10 15Leu Leu Leu Leu Asn Arg Asp Glu Phe His Ser Arg Pro Thr Lys Ala 20 25 30Val Gly Trp Trp Gly Glu Gly Ser Lys Lys Ile Leu Gly Gly Arg Asp 35 40 45Val Leu Gly Gly Gly Thr Trp Met Gly Cys Thr Lys Asp Gly Arg Leu 50 55 60Ala Phe Leu Thr Asn Val Leu Glu Pro Asp Ala Met Pro Gly Ala Arg65 70 75 80Thr Arg Gly Asp Leu Pro Leu Lys Phe Leu Gln Ser Asn Lys Ser Pro 85 90 95Leu Glu Val Ala Thr Glu Val Ala Glu Glu Ala Asp Glu Tyr Asn Gly 100 105 110Phe Asn Leu Ile Leu Ala Asp Leu Thr Thr Asn Ile Met Val Tyr Val 115 120 125Ser Asn Arg Pro Lys Gly Gln Pro Ala Thr Ile Gln Leu Val Ser Pro 130 135 140Gly Leu His Val Leu Ser Asn Ala Arg Leu Asp Ser Pro Trp Gln Lys145 150 155 160Ala Ile Leu Leu Gly Lys Asn Phe Arg Glu Leu Leu Arg Glu His Gly 165 170 175Ala Asp Glu Val Glu Val Lys Asp Ile Val Glu Arg Leu Met Thr Asp 180 185 190Thr Thr Lys Ala Asp Lys Asp Arg Leu Pro Asn Thr Gly Cys Asp Pro 195 200 205Asn Trp Glu His Gly Leu Ser Ser Ile Phe Ile Glu Val Gln Thr Asp 210 215 220Gln Gly Pro Tyr Gly Thr Arg Ser Thr Ala Val Leu Ser Val Asn Tyr225 230 235 240Asp Gly Glu Ala Ser Leu Tyr Glu Lys Tyr Leu Glu Ser Gly Ile Trp 245 250 255Lys Asp His Thr Val Ser Tyr Gln Ile Glu 260 2656687DNAZea mays 6attcccgtct tacctagcgc tagggttagt acgcgtccac ggcgacgacc tctgcgcgga 60gtgtgctccg attggctggc ctcctcgatc ctccttcccg cgaacgcacg cgcgcgcgag 120ggagaggttg agacttgaga gatagacgaa agacgaaaca agggaaggag acgccgtgct 180cgcctattgg ccgccgcctc cgctccttcg cgcccaatgg cttctgcagc atatcaatat 240catgcagcat agcagtactc agacccttac tacgcaggcg ttgttgctcc ctatggaagt 300caagatgtgt gtccgaggag cctgtctatg tgaacgccaa gcagtaccgc ggcattctaa 360gacggcggca gtcacgtgcc aaggccgagc ttgagagaaa gcgctggtca aagcaagaaa 420gccgtatctt cacgagtccc cgtcatcagc acgcgatgac gaggagggcg agagggaacg 480gtggacgctt cctaaacacg aagaagagtg accgtgtccc tcctgatgac ttgatacagc 540tacgacgaca caacgaggct tgaagaggta gcggtctggc tggcatccta gagcagcggt 600ttctgtccac aggcacgtgc atctgagacc ggatccgtag ctccactcca cagcatatgc 660gcagcccatc catctcgtgc acacttg 6877153DNAZea mays 7atgcagcata gcagtactca gacccttact acgcaggcgt tgttgctccc tatggaagtc 60aagatgtgtg tccgaggagc ctgtctatgt gaacgccaag cagtaccgcg gcattctaag 120acggcggcag tcacgtgcca aggccgagct tga 153850PRTZea mays 8Met Gln His Ser Ser Thr Gln Thr Leu Thr Thr Gln Ala Leu Leu Leu1 5 10 15Pro Met Glu Val Lys Met Cys Val Arg Gly Ala Cys Leu Cys Glu Arg 20 25 30Gln Ala Val Pro Arg His Ser Lys Thr Ala Ala Val Thr Cys Gln Gly 35 40 45Arg Ala 509429DNAZea mays 9ttgagagata gacgaaagac gaaacaaggg aaggagacgc cgtgctcgcc tattggccgc 60cgcctccgct ccttcgcgcc caatggcttc tgcagcatat caatatcatg cagcatagca 120gtactcagac ccttactacg caggcgttgt tgctccctat ggaagtcaag atgtgtgtcc 180gaggagcctg tctatgtgaa cgccaagcag taccgcggca ttctaagacg gcggcagtca 240cgtgccaagg ccgagcttga gagaaagcgc tggtcaaagc aagaaagccg tatcttcacg 300agtccccgtc atcagcacgc gatgacgagg agggcgagag ggaacggtgg acgcttccta 360aacacgaaga agagtgaccg tgtccctcct gatgacttga tacagctacg acgacacaac 420gaggcttga 42910142PRTZea mays 10Leu Arg Asp Arg Arg Lys Thr Lys Gln Gly Lys Glu Thr Pro Cys Ser1 5 10 15Pro Ile Gly Arg Arg Leu Arg Ser Phe Ala Pro Asn Gly Phe Cys Ser 20 25 30Ile Ser Ile Ser Cys Ser Ile Ala Val Leu Arg Pro Leu Leu Arg Arg 35 40 45Arg Cys Cys Ser Leu Trp Lys Ser Arg Cys Val Ser Glu Glu Pro Val 50 55 60Tyr Val Asn Ala Lys Gln Tyr Arg Gly Ile Leu Arg Arg Arg Gln Ser65 70 75 80Arg Ala Lys Ala Glu Leu Glu Arg Lys Arg Trp Ser Lys Gln Glu Ser 85 90 95Arg Ile Phe Thr Ser Pro Arg His Gln His Ala Met Thr Arg Arg Ala 100 105 110Arg Gly Asn Gly Gly Arg Phe Leu Asn Thr Lys Lys Ser Asp Arg Val 115 120 125Pro Pro Asp Asp Leu Ile Gln Leu Arg Arg His Asn Glu Ala 130 135 14011378DNAZea mays 11atgactgctc accagacttg ctgcgatgat gccgttgccg ccggcactgc accggctgcc 60aggaggaggc gcctcaaatt gacgaggccg tcggcctcgc tcttgatggc gaggaagcta 120aggaagaagg ctgccggcag caaacgccca agggcggcag cgtcgaggaa gcgcgcgatg 180gcgatcagga ggaagatgga agcgctgagg ctgctcgtgc cactctgcgg ccgagacaac 240ggctcggtga ccggtggggc ggtcgaacga ctggacgagc tcctcatgca cgccgccggg 300tacatcctgc gcctccagat gcaggtcaga gtgatgcagc ttatggtcca tgcactaaat 360gaccggcccg aggattaa 37812125PRTZea mays 12Met Thr Ala His Gln Thr Cys Cys Asp Asp Ala Val Ala Ala Gly Thr1 5 10 15Ala Pro Ala Ala Arg Arg Arg Arg Leu Lys Leu Thr Arg Pro Ser Ala 20 25 30Ser Leu Leu Met Ala Arg Lys Leu Arg Lys Lys Ala Ala Gly Ser Lys 35 40 45Arg Pro Arg Ala Ala Ala Ser Arg Lys Arg Ala Met Ala Ile Arg Arg 50 55 60Lys Met Glu Ala Leu Arg Leu Leu Val Pro Leu Cys Gly Arg Asp Asn65 70 75 80Gly Ser Val Thr Gly Gly Ala Val Glu Arg Leu Asp Glu Leu Leu Met 85 90 95His Ala Ala Gly Tyr Ile Leu Arg Leu Gln Met Gln Val Arg Val Met 100 105 110Gln Leu Met Val His Ala Leu Asn Asp Arg Pro Glu Asp 115 120 12513792DNAZea mays 13atgtcggcgg cgctcgcggt gacggacgag gtggccctgc cgatccgggc ggtgggggat 60ctagcggccg ccgccgaggt ctcgcgggag gaggtcgccg tcatcaccca gtgcgcggcg 120ctcggtggga agttgccttt tgaagatgca tcagttggtg cggttcttgc agtcattaaa 180aacgtggaaa gcttgaggga gcaattggtt gctgaaatca ggcgggtgct gaaagctggt 240ggaagagtat tggtgcagag ccctgcaccc tcatccagtc agaagccgaa cactgatatt 300gagcgcaagt tactgatggg tggatttgct gaagtgcaat cttctgctgc aagctcgcag 360gatagcgtgc aatctgttac agttaaggca aagaaggcta gctggagcat gggctcttct 420tttcccctta agaaaacaac aaaagccctt cccaagattc aaattgacga cgactctgat 480ctgattgatg aagacagtct cttgactgag gaggacctga agaaaccaca acttccagtt 540gttggggact gtgaggtggg ggcagcaaag aaagcatgca agaactgtac ttgtggcagg 600gctgaggccg aggagaaggt tgggaagctg gagctcactg cggagcagat caataaccct 660cagtcagctt gtggcagttg tgggttgggt gatgccttcc gctgtggaac ctgtccctac 720agaggtcttc caccattcaa gcctggcgag aaggtttcct tgtctggcaa cttccttgct 780gctgacatat ga 79214263PRTZea mays 14Met Ser Ala Ala Leu Ala Val Thr Asp Glu Val Ala Leu Pro Ile Arg1 5 10 15Ala Val Gly Asp Leu Ala Ala Ala Ala Glu Val Ser Arg Glu Glu Val 20 25 30Ala Val Ile Thr Gln Cys Ala Ala Leu Gly Gly Lys Leu Pro Phe Glu 35 40 45Asp Ala Ser Val Gly Ala Val Leu Ala Val Ile Lys Asn Val Glu Ser 50 55 60Leu Arg Glu Gln Leu Val Ala Glu Ile Arg Arg Val Leu Lys Ala Gly65 70 75 80Gly Arg Val Leu Val Gln Ser Pro Ala Pro Ser Ser Ser Gln Lys Pro 85 90 95Asn Thr Asp Ile Glu Arg Lys Leu Leu Met Gly Gly Phe Ala Glu Val 100 105 110Gln Ser Ser Ala Ala Ser Ser Gln Asp Ser Val Gln Ser Val Thr Val 115 120 125Lys Ala Lys Lys Ala Ser Trp Ser Met Gly Ser Ser Phe Pro Leu Lys 130 135 140Lys Thr Thr Lys Ala Leu Pro Lys Ile Gln Ile Asp Asp Asp Ser Asp145 150 155 160Leu Ile Asp Glu Asp Ser Leu Leu Thr Glu Glu Asp Leu Lys Lys Pro 165 170 175Gln Leu Pro Val Val Gly Asp Cys Glu Val Gly Ala Ala Lys Lys Ala 180 185 190Cys Lys Asn Cys Thr Cys Gly Arg Ala Glu Ala Glu Glu Lys Val Gly 195 200 205Lys Leu Glu Leu Thr Ala Glu Gln Ile Asn Asn Pro Gln Ser Ala Cys 210 215 220Gly Ser Cys Gly Leu Gly Asp Ala Phe Arg Cys Gly Thr Cys Pro Tyr225 230 235 240Arg Gly Leu Pro Pro Phe Lys Pro Gly Glu Lys Val Ser Leu Ser Gly 245 250 255Asn Phe Leu Ala Ala Asp Ile 26015948DNAZea mays 15atggcgatgc agacgggggt cgcgacctcc aaggtcctca tcctcgtcgg tgcagggatg 60acgggctcga tcctgctgcg gaatggccgc ttatctgatg tgttgggaga actccaggag 120attatgaagg gtgtaaatca aggaacttct tcgggtccct atgacattgc acttattcaa 180gctcagattc ggaatttagc gcaagaagtc agagatttga cattgtcaaa gcccattacc 240atactgaatg gcaaatctga ctcgggaggc agtttatcat cctacatact gccagcagca 300gcagttggag caatgggtta ttgctacatg tggtggaagg ggttgtctct ctcagatgtc 360atgtttgtca caaaacacaa catggcaaat gctgttcaga gcatgtcaaa gcagttggag 420caagtttcat cagcactagc tgcaacaaaa agacatctaa ctcaacggct tgagaatttg 480gatggcaaaa tggatgaaca agtagaggtc tccaaagcta ttagaaatga ggtcaatgat 540gttaaagatg acctgtctca aattggattt gatgtcgaat caattcagaa aatggttgct 600ggattggagg gaaagatcga gttacttgag aacaaacagg acgtggctaa tactggtatc 660tggtatctct gccaagtagc aggcggttta aaagatggaa taaacaccag gtttttccag 720gaaaccagtg agaagctgaa gctctcacat tcagctcaac ctgaaaacaa gccagtgaag 780gggcttgaat ttttttcgga aagcaccatg gaacagaaag tagctgactc caaaccaatt 840gcggtgacag tcgacgctga gaagcctgag aaaaccgctg ctgtaatggg caccacagtg 900cacaggtcta tcaggttctc atatcggaag gcaggccttg ctttgtga 94816315PRTZea mays 16Met Ala Met Gln Thr Gly Val Ala Thr Ser Lys Val Leu Ile Leu Val1 5 10 15Gly Ala Gly Met Thr Gly Ser Ile Leu Leu Arg Asn Gly Arg Leu Ser 20 25 30Asp Val Leu Gly Glu Leu Gln Glu Ile Met Lys Gly Val Asn Gln Gly 35 40 45Thr Ser Ser Gly Pro Tyr Asp Ile Ala Leu Ile Gln Ala Gln Ile Arg 50 55 60Asn Leu Ala Gln Glu Val Arg Asp Leu Thr Leu Ser Lys Pro Ile Thr65 70 75 80Ile Leu Asn Gly Lys Ser Asp Ser Gly Gly Ser Leu Ser Ser Tyr Ile 85 90 95Leu Pro Ala Ala Ala Val Gly Ala Met Gly Tyr Cys Tyr Met Trp Trp 100 105 110Lys Gly Leu Ser Leu Ser Asp Val Met Phe Val Thr Lys His Asn Met 115 120 125Ala Asn Ala Val Gln Ser Met Ser Lys Gln Leu Glu Gln Val Ser Ser 130 135 140Ala Leu Ala Ala Thr Lys Arg His Leu Thr Gln Arg Leu Glu Asn Leu145 150 155 160Asp Gly Lys Met Asp Glu Gln Val Glu Val Ser Lys Ala Ile Arg Asn 165 170 175Glu Val Asn Asp Val Lys Asp Asp Leu Ser Gln Ile Gly Phe Asp Val 180 185 190Glu Ser Ile Gln Lys Met Val Ala Gly Leu Glu Gly Lys Ile Glu Leu 195 200 205Leu Glu Asn Lys Gln Asp Val Ala Asn Thr Gly Ile Trp Tyr Leu Cys 210 215 220Gln Val Ala Gly Gly Leu Lys Asp Gly Ile Asn Thr Arg Phe Phe Gln225 230 235 240Glu Thr Ser Glu Lys Leu Lys Leu Ser His Ser Ala Gln Pro Glu Asn 245 250 255Lys Pro Val Lys Gly Leu Glu Phe Phe Ser Glu Ser Thr Met Glu Gln 260 265 270Lys Val Ala Asp Ser Lys Pro Ile Ala Val Thr Val Asp Ala Glu Lys 275 280 285Pro Glu Lys Thr Ala Ala Val Met Gly Thr Thr Val His Arg Ser Ile 290 295 300Arg Phe Ser Tyr Arg Lys Ala Gly Leu Ala Leu305 310 31517540DNAZea mays 17atgtgctcgg tagcgaggct ggcgtttgtg cttgcactgg ccatagccgc ctcgtcaatt 60gaggttgcgg agagcagaga ttttaatatc tttgctcagg gcagcttgcc tgatgcaacc 120aagggatcgt ctggtctagc tgcaaccagt ggaaagttgt gtcagttatg cgagcagtac 180tcatccgagg cgctcctcta tctcacacaa aacgagaccc agactgagat tcttagcatt 240ctacaccatg aatgtgccag ccttgcccct ctcaaacagc agtgcatcac gctggttgac 300tactacgtac cccttttctt cttggaggtc tccatggtta cccctgagaa gttctgcgag 360tcgatgcatc tctgcaagaa ggggatgaag attagcctac ccacccggga gggtacttgt 420ggtttgtgcc accatgttgt tgttgaaatt cttatcatgc ttaaagaccc caacatgcag 480ctggaagtaa tcgacctact caccaaaaca tgcagcaagg cgcagaacta tgaacagtag 54018179PRTZea mays 18Met Cys Ser Val Ala Arg Leu Ala Phe Val Leu Ala Leu Ala Ile Ala1 5 10 15Ala Ser Ser Ile Glu Val Ala Glu Ser Arg Asp Phe Asn Ile Phe Ala 20 25 30Gln Gly Ser Leu Pro Asp Ala Thr Lys Gly Ser Ser Gly Leu Ala Ala 35 40 45Thr Ser Gly Lys Leu Cys Gln Leu Cys Glu Gln Tyr Ser Ser Glu Ala 50 55 60Leu Leu Tyr Leu Thr Gln Asn Glu Thr Gln Thr Glu Ile Leu Ser Ile65 70 75 80Leu His His Glu Cys Ala Ser Leu Ala Pro Leu Lys Gln Gln Cys Ile 85 90 95Thr Leu Val Asp Tyr Tyr Val Pro Leu Phe Phe Leu Glu Val Ser Met 100 105 110Val Thr Pro Glu Lys Phe Cys Glu Ser Met His Leu Cys Lys Lys Gly 115 120 125Met Lys Ile Ser Leu Pro Thr Arg Glu Gly Thr Cys Gly Leu Cys His 130 135 140His Val Val Val Glu Ile Leu Ile Met Leu Lys Asp Pro Asn Met Gln145 150 155 160Leu Glu Val Ile Asp Leu Leu Thr Lys Thr Cys Ser Lys Ala Gln Asn 165 170 175Tyr Glu Gln191146DNAZea mays 19ggacactgac atggactgaa ggagtagaaa atccatccat tcccctcgcc aagccgccac 60ggcctgactt tccctcccgc acacccgcga ccatacaggc aagtcaggca tacaccaaca 120acgctcgtcg tgcacctcgc gcctcaggtc accccaccaa attcctcttg atacgccgaa 180tttcttttgc taattctgct acctcctgtc gctaagccac catattcagt ctaacccctg 240ctctgagctc acctgattgg cggctccgtt cggcctctgg gcctgggtgt accgactacc 300gagggctctt tcgaaatgtc aattgggtcg agtttggtgg gctacgtgaa gcatggatga 360atttcccggc tggaagcggg aggcggcagc agcatccggg gccggagcac ctgtcgccga 420tgacgccgct cccgctggcg cggtaggggt cggtctactc gctcacgttc gacgagttcc 480agagctcgct cggtggggcc accaaggact tcggatccat gaacatggac gagctcctcc 540gcaacatctg gtcggcggag gagacacaca gcgtcacagc tgcggaccat gccgcgcggg 600cgccgtacgt ccagtgccag ggctcgctca ccctcccctg cacgctcagc cagaagaccg 660tcgacgaggt ctagcgtgac ctcgtgtgca acggtggagg accctccgac gaggctgtgg 720cgccgcccca ccggcccaac ggcagccgac gctcggggag atcatgctgg aggagttcct 780cgtccgcgcc ggcgtggtga gggaggacat

gatggcggcg gcgcccgtac caccagcgcc 840gggttgccca ccacctcatc tgcaaccgcc aatgctgttt ccacatggca atgtgtttgc 900tcccttagtg cctccgctcc aattcgggaa tgggtttgtg tcgggggctc tcagtcagca 960gcagggaggt gttcttgagg ccccggcggt atcgccgcgg ccggtgacgg caagcgggtt 1020cgggaagatg gaaggagacg acttgtcgca tctgtcgcca tcaccggtgt cgtacgtttt 1080tttgtgctgg tttgagggga aggaagccac cagctgtgga gaaggtggtt gagaggaggc 1140aacgcc 114620357PRTOryza glaberrima 20Met Asp Phe Pro Gly Gly Ser Gly Arg Gln Gln Gln Leu Pro Pro Met1 5 10 15Thr Pro Leu Pro Leu Ala Arg Gln Gly Ser Val Tyr Ser Leu Thr Phe 20 25 30Asp Glu Phe Gln Ser Thr Leu Gly Gly Val Gly Lys Asp Phe Gly Ser 35 40 45Met Asn Met Asp Glu Leu Leu Arg Ser Ile Trp Thr Ala Glu Glu Ser 50 55 60His Ala Val Gly Ala Ala Thr Thr Thr Thr Ala Thr Thr Ala Ser Val65 70 75 80Ala Ala Ala Glu His Ala Ala Val Gly Ala Pro Pro Val Gln Arg Gln 85 90 95Gly Ser Leu Thr Leu Pro Arg Thr Leu Ser Gln Lys Thr Val Asp Glu 100 105 110Val Trp Arg Asp Met Met Cys Phe Gly Gly Gly Gly Ala Ser Thr Ala 115 120 125Pro Ala Ala Ala Glu Pro Pro Pro Pro Ala His Arg Gln Gln Thr Leu 130 135 140Gly Glu Ile Thr Leu Glu Glu Phe Leu Val Arg Ala Gly Val Val Arg145 150 155 160Glu Asp Met Ser Val Pro Pro Val Pro Pro Ala Pro Thr Pro Thr Ala 165 170 175Ala Ala Val Pro Pro Pro Pro Pro Pro Gln Gln Gln Thr Pro Met Leu 180 185 190Phe Gly Gln Ser Asn Val Phe Pro Pro Met Val Pro Pro Leu Ser Leu 195 200 205Gly Asn Gly Leu Val Ser Gly Ala Val Gly His Gly Gly Gly Gly Ala 210 215 220Ala Ser Leu Val Ser Pro Val Arg Pro Val Ser Ser Asn Gly Phe Gly225 230 235 240Lys Met Glu Gly Gly Asp Leu Ser Ser Leu Ser Pro Ser Pro Val Pro 245 250 255Tyr Val Phe Lys Gly Gly Leu Arg Gly Arg Lys Ala Pro Gly Ile Glu 260 265 270Lys Val Val Glu Arg Arg Gln Arg Arg Met Ile Lys Asn Arg Glu Ser 275 280 285Ala Ala Arg Ser Arg Gln Arg Lys Gln Ala Tyr Met Met Glu Leu Glu 290 295 300Ala Glu Val Ala Lys Leu Lys Glu Leu Asn Asp Glu Leu Gln Lys Lys305 310 315 320Gln Asp Glu Met Leu Glu Gln Gln Lys Asn Glu Val Leu Glu Arg Met 325 330 335Ser Arg Gln Val Gly Pro Thr Ala Lys Arg Ile Cys Leu Arg Arg Thr 340 345 350Leu Thr Gly Pro Trp 355211074DNAOryza glaberrima 21atggattttc cgggagggag cgggaggcag cagcagctgc cgccgatgac gccgctgccg 60ttggcgaggc aggggtcggt gtactcgctc acgttcgacg agttccagag cacgctgggc 120ggggtcggga aggacttcgg gtcgatgaac atggacgagc tcctccgcag catctggacg 180gccgaggagt cgcacgccgt cggcgccgcc acgacgacga cggcgacgac ggcgtccgtg 240gcggcggcgg agcacgcggc ggtgggggcg ccgcccgttc agaggcaggg gtcgctgacc 300ctcccccgca cgctcagcca gaagaccgtc gacgaggtct ggcgcgacat gatgtgcttc 360ggtggcggcg gcgcctccac cgcgccggcc gccgcggagc ccccgccgcc ggcgcaccgg 420cagcagacgc tcggggagat cacgctggag gagttcctcg tgcgggccgg cgtggtgagg 480gaggacatgt cggtcccgcc cgtcccgccg gcgccgactc ctacggcggc tgctgtacct 540cccccgccgc cgccgcagca gcagacgccg atgttgttcg gtcagagcaa tgtgttccct 600ccgatggtgc ctccgctctc gctgggaaat gggctggtct cgggagctgt cggacacggc 660ggtggtggtg ccgcgtcgtt ggtttcgccg gtgaggccgg tctcgtccaa tggcttcggc 720aagatggaag gcggggacct gtcgtcgctg tcgccatcgc cggtgccgta cgttttcaaa 780ggtgggctga ggggaaggaa ggcaccgggc atcgagaagg ttgtcgagag aagacagcgg 840cggatgatca agaacaggga gtctgccgcg aggtcgcgcc agaggaaaca ggcatatatg 900atggaattgg aagctgaggt agcaaaactt aaggagctga acgatgaact ccagaaaaag 960caggatgaaa tgttggagca gcaaaagaat gaggttctag agagaatgag ccgacaagtt 1020ggaccgacag caaagagaat ttgccttcgg aggactctga cgggtccatg gtga 107422777DNAZea mays 22atgaatttcc cggctggaag cgggaggcgg cagcagcatc cggggccgga gcacctgtcg 60ccgatgacgc cgctcccgct ggcgcggcag gggtcggtct actcgctcac gttcgacgag 120ttccagagct cgctcggtgg ggccaccaag gacttcggat ccatgaacat ggacgagctc 180ctccgcaaca tctggtcggc ggaggagaca cacagcgtca cagctgcgga ccatgccgcg 240cgggcgccgt acgtccagtg ccagggctcg ctcaccctcc cctgcacgct cagccagaag 300accgtcgacg aggtctggcg tgacctcgtg tgcaacggtg gaggaccctc cgacgaggct 360gtggcggccg ccccaccggc ccaacggcag ccgacgctcg gggagatcat gctggaggag 420ttcctcgtcc gcgccggcgt ggtgagggag gacatgatgg cggcggcgcc cgtaccacca 480gcgccgggtt gcccaccacc tcatctgcaa ccgccaatgc tgtttccaca tggcaatgtg 540tttgctccct tagtgcctcc gctccaattc gggaatgggt ttgtgtcggg ggctctcagt 600cagcagcagg gaggtgttct tgaggccccg gcggtatcgc cgcggccggt gacggcaagc 660gggttcggga agatggaagg agacgacttg tcgcatctgt cgccatcacc ggtgtcgtac 720gtttttttgt gctggtttga ggggaaggaa gccaccagct gtggagaagg tggttga 77723258PRTZea mays 23Met Asn Phe Pro Ala Gly Ser Gly Arg Arg Gln Gln His Pro Gly Pro1 5 10 15Glu His Leu Ser Pro Met Thr Pro Leu Pro Leu Ala Arg Gln Gly Ser 20 25 30Val Tyr Ser Leu Thr Phe Asp Glu Phe Gln Ser Ser Leu Gly Gly Ala 35 40 45Thr Lys Asp Phe Gly Ser Met Asn Met Asp Glu Leu Leu Arg Asn Ile 50 55 60Trp Ser Ala Glu Glu Thr His Ser Val Thr Ala Ala Asp His Ala Ala65 70 75 80Arg Ala Pro Tyr Val Gln Cys Gln Gly Ser Leu Thr Leu Pro Cys Thr 85 90 95Leu Ser Gln Lys Thr Val Asp Glu Val Trp Arg Asp Leu Val Cys Asn 100 105 110Gly Gly Gly Pro Ser Asp Glu Ala Val Ala Ala Ala Pro Pro Ala Gln 115 120 125Arg Gln Pro Thr Leu Gly Glu Ile Met Leu Glu Glu Phe Leu Val Arg 130 135 140Ala Gly Val Val Arg Glu Asp Met Met Ala Ala Ala Pro Val Pro Pro145 150 155 160Ala Pro Gly Cys Pro Pro Pro His Leu Gln Pro Pro Met Leu Phe Pro 165 170 175His Gly Asn Val Phe Ala Pro Leu Val Pro Pro Leu Gln Phe Gly Asn 180 185 190Gly Phe Val Ser Gly Ala Leu Ser Gln Gln Gln Gly Gly Val Leu Glu 195 200 205Ala Pro Ala Val Ser Pro Arg Pro Val Thr Ala Ser Gly Phe Gly Lys 210 215 220Met Glu Gly Asp Asp Leu Ser His Leu Ser Pro Ser Pro Val Ser Tyr225 230 235 240Val Phe Leu Cys Trp Phe Glu Gly Lys Glu Ala Thr Ser Cys Gly Glu 245 250 255Gly Gly241023DNAZea mays 24atgaatttcc cggctggaag cgggaggcgg cagcagcatc cggggccgga gcacctgtcg 60ccgatgacgc cgctcccgct ggcgcggcag gggtcggtct actcgctcac gttcgacgag 120ttccagagct cgctcggtgg ggccaccaag gacttcggat ctatgaacat ggacgagctc 180ctccgcaaca tctggtcggc ggaggagaca cacagcgtca cagctgcgga ccatgccgcg 240cgggcgccgt acgtccagtg ccagggctcg ctcaccctcc cctgcacgct cagccagaag 300accgtcgacg aggtctggcg tgacctcgtg tgcaacggtg gaggaccctc cgacgaggct 360gtggcggccg ccccaccggc ccaacggcag ccgacgctcg gggagatcat gctggaggag 420ttcctcgtcc gcgccggcgt ggtgagggag gacatgatgg cggcggcgcc cgtaccacca 480gcgccgggtt gcccaccacc tcatctgcaa ccgccaatgc tgtttccaca tggcaatgtg 540tttgctccct tagtgcctcc gctccaattc gggaatgggt ttgtgtcggg ggctctcagt 600cagcagcagg gaggtgttct tgaggccccg gcggtatcgc cgcggccggt gacggcaagc 660gggttcggga agatggaagg agacgacttg tcgcatctgt cgccatcacc ggtgtcgtac 720gtttttttgt gctggtttga ggggaaggaa gccaccagct gtggacaagg tggtgagaga 780agacagagga ggatgatcaa gaacagggag tctgccgcga ggtcgaggca gaggaaacag 840gcatatatga tggaattgga agctgaggta gcaaagctca aggagctgaa cgacgagctc 900cagaagaagc aggacgagat gctggagcag cagaagaacg aggtgctgga gaggatgtcc 960aggcaggtgg gcccaaccgc caagaggatt tgcctgagga ggaccctgac cggcccatgg 1020tga 102325340PRTZea mays 25Met Asn Phe Pro Ala Gly Ser Gly Arg Arg Gln Gln His Pro Gly Pro1 5 10 15Glu His Leu Ser Pro Met Thr Pro Leu Pro Leu Ala Arg Gln Gly Ser 20 25 30Val Tyr Ser Leu Thr Phe Asp Glu Phe Gln Ser Ser Leu Gly Gly Ala 35 40 45Thr Lys Asp Phe Gly Ser Met Asn Met Asp Glu Leu Leu Arg Asn Ile 50 55 60Trp Ser Ala Glu Glu Thr His Ser Val Thr Ala Ala Asp His Ala Ala65 70 75 80Arg Ala Pro Tyr Val Gln Cys Gln Gly Ser Leu Thr Leu Pro Cys Thr 85 90 95Leu Ser Gln Lys Thr Val Asp Glu Val Trp Arg Asp Leu Val Cys Asn 100 105 110Gly Gly Gly Pro Ser Asp Glu Ala Val Ala Ala Ala Pro Pro Ala Gln 115 120 125Arg Gln Pro Thr Leu Gly Glu Ile Met Leu Glu Glu Phe Leu Val Arg 130 135 140Ala Gly Val Val Arg Glu Asp Met Met Ala Ala Ala Pro Val Pro Pro145 150 155 160Ala Pro Gly Cys Pro Pro Pro His Leu Gln Pro Pro Met Leu Phe Pro 165 170 175His Gly Asn Val Phe Ala Pro Leu Val Pro Pro Leu Gln Phe Gly Asn 180 185 190Gly Phe Val Ser Gly Ala Leu Ser Gln Gln Gln Gly Gly Val Leu Glu 195 200 205Ala Pro Ala Val Ser Pro Arg Pro Val Thr Ala Ser Gly Phe Gly Lys 210 215 220Met Glu Gly Asp Asp Leu Ser His Leu Ser Pro Ser Pro Val Ser Tyr225 230 235 240Val Phe Leu Cys Trp Phe Glu Gly Lys Glu Ala Thr Ser Cys Gly Gln 245 250 255Gly Gly Glu Arg Arg Gln Arg Arg Met Ile Lys Asn Arg Glu Ser Ala 260 265 270Ala Arg Ser Arg Gln Arg Lys Gln Ala Tyr Met Met Glu Leu Glu Ala 275 280 285Glu Val Ala Lys Leu Lys Glu Leu Asn Asp Glu Leu Gln Lys Lys Gln 290 295 300Asp Glu Met Leu Glu Gln Gln Lys Asn Glu Val Leu Glu Arg Met Ser305 310 315 320Arg Gln Val Gly Pro Thr Ala Lys Arg Ile Cys Leu Arg Arg Thr Leu 325 330 335Thr Gly Pro Trp 34026285DNAZea mays 26atgatgttct cctcctccct ctctgtggtg gagttttact tcctgcacag attccccctg 60ccttttgctg gctacctcat cttcatttcc atattggctg gattctgggg ccagtgtttg 120gttaggaaga tcgtgcatgt gctcaagaga gcatcgctta ttgtcttcat cctctcctct 180gttatcttcg tcagtgctct tacgatgggt gtcgttggaa cccagaagag catttcgatg 240atcaacaatc acgaatatat ggggttcctc aacttctgcg agtaa 2852794PRTZea mays 27Met Met Phe Ser Ser Ser Leu Ser Val Val Glu Phe Tyr Phe Leu His1 5 10 15Arg Phe Pro Leu Pro Phe Ala Gly Tyr Leu Ile Phe Ile Ser Ile Leu 20 25 30Ala Gly Phe Trp Gly Gln Cys Leu Val Arg Lys Ile Val His Val Leu 35 40 45Lys Arg Ala Ser Leu Ile Val Phe Ile Leu Ser Ser Val Ile Phe Val 50 55 60Ser Ala Leu Thr Met Gly Val Val Gly Thr Gln Lys Ser Ile Ser Met65 70 75 80Ile Asn Asn His Glu Tyr Met Gly Phe Leu Asn Phe Cys Glu 85 9028377DNAZea mays 28atggcgtctg cagtgaccag cagcgacaag gagcaggccg tccctaccat cgacgctgac 60gaagcgcacg cgctgctgag ctccggccat ggctacgtgg atgtcaggat gcggggggac 120ttccacaagg cgcatgcgcc cggtgctcgg aacgttccct actacctgtc cgtcacgccg 180caagggaagg agaagaaccc acactttgta gaggaagtgg ctgccttctg tgggaaggat 240gatgtcttca ttgtgggttg caacacgggg aacagatcca ggttcgcgac ggcagacctt 300ctgaacgcgg ggttcaagaa cgtgaggaac ctgcaaggtg gttaccgctc ctttcagcag 360cgagctcaac agcagta 37729125PRTZea mays 29Met Ala Ser Ala Val Thr Ser Ser Asp Lys Glu Gln Ala Val Pro Thr1 5 10 15Ile Asp Ala Asp Glu Ala His Ala Leu Leu Ser Ser Gly His Gly Tyr 20 25 30Val Asp Val Arg Met Arg Gly Asp Phe His Lys Ala His Ala Pro Gly 35 40 45Ala Arg Asn Val Pro Tyr Tyr Leu Ser Val Thr Pro Gln Gly Lys Glu 50 55 60Lys Asn Pro His Phe Val Glu Glu Val Ala Ala Phe Cys Gly Lys Asp65 70 75 80Asp Val Phe Ile Val Gly Cys Asn Thr Gly Asn Arg Ser Arg Phe Ala 85 90 95Thr Ala Asp Leu Leu Asn Ala Gly Phe Lys Asn Val Arg Asn Leu Gln 100 105 110Gly Gly Tyr Arg Ser Phe Gln Gln Arg Ala Gln Gln Gln 115 120 12530411DNAZea mays 30atggaggcga agaagaagcc gtcggccccc gccgccgtcg gagccgcgcc gccgccgccg 60ggtaacgggt acttcagcac cgtcttctcc gcgccgactg cgggaagcgc aagtgacgca 120aagcatgcgg acttgtacac gatgctgaac aagcagagct ccagagggca gaatggcaga 180gatggcaaat cccacagccg ccctacttac aaggatggca aacatgctca tccaaatgag 240ccatcagaat ctccttactt tggctcatcc gtgcattacg gtggtcggga gttctacagc 300agcgttttac ggaagcaacc agccaatgaa ccccatacgg attacaaggg ggacaacccg 360gatggctctg ctaccagagg tgattggtgg caaggttcac tttattactg a 41131136PRTZea mays 31Met Glu Ala Lys Lys Lys Pro Ser Ala Pro Ala Ala Val Gly Ala Ala1 5 10 15Pro Pro Pro Pro Gly Asn Gly Tyr Phe Ser Thr Val Phe Ser Ala Pro 20 25 30Thr Ala Gly Ser Ala Ser Asp Ala Lys His Ala Asp Leu Tyr Thr Met 35 40 45Leu Asn Lys Gln Ser Ser Arg Gly Gln Asn Gly Arg Asp Gly Lys Ser 50 55 60His Ser Arg Pro Thr Tyr Lys Asp Gly Lys His Ala His Pro Asn Glu65 70 75 80Pro Ser Glu Ser Pro Tyr Phe Gly Ser Ser Val His Tyr Gly Gly Arg 85 90 95Glu Phe Tyr Ser Ser Val Leu Arg Lys Gln Pro Ala Asn Glu Pro His 100 105 110Thr Asp Tyr Lys Gly Asp Asn Pro Asp Gly Ser Ala Thr Arg Gly Asp 115 120 125Trp Trp Gln Gly Ser Leu Tyr Tyr 130 13532162DNAZea mays 32atggaccgga acctgagcgg gtttctgatc gggtgcctgg gcgccgccgt gacgctgctg 60gcgtaccagc agacggtggt gaccagcacg cagagcgtcg cggcgggctt cgtcgtcatc 120ctcttcgccc tcttcgtcaa ggaaggattc atttccctct ga 1623353PRTZea mays 33Met Asp Arg Asn Leu Ser Gly Phe Leu Ile Gly Cys Leu Gly Ala Ala1 5 10 15Val Thr Leu Leu Ala Tyr Gln Gln Thr Val Val Thr Ser Thr Gln Ser 20 25 30Val Ala Ala Gly Phe Val Val Ile Leu Phe Ala Leu Phe Val Lys Glu 35 40 45Gly Phe Ile Ser Leu 5034669DNAZea mays 34atggcatgcg tcagcacctt ccagagctgc cccattgcca gaagagcaaa gatcaacacc 60aggtccaggg gcagcagcag tagcgtggcg aaggggtcac caccaccagc cttccagttc 120cagtgcaggg cgtcgacttt cgcggcggac accagcctcc ggctcgagct ggacgagaac 180cccgaggcga tcatctcggg ggcgtggccc gggaactgct ccctcctcag ctacgacgac 240ctccgcgcct acctcgagtc gcaggagacg gcggcccagg cagacgatca gcgcggcgtg 300gcgctcctga gcgagaccat gtccacaccc gtgctggtgg ccacagcaga ccagaccctg 360gaggacgtcg agtgccactt cgaggccgtg tcggggcttc cggtcgtcga cagcggcctc 420agatgcgtcg gggtgatcgt caagaacgac cgggcaagag cctctcatgg gtccaagacg 480aagatatcgg aagtgatgac atctccagct atcacactat cgtctgacaa aaccgtgatg 540gatgctgctg ttctcatgct caagaagaag atccacagat taccagttgt aaaccaggac 600gaaaaagtaa taggtatagt tacccgcgct gatgttcttc gcgtgttgga aggcatgttg 660aagatttag 66935222PRTZea mays 35Met Ala Cys Val Ser Thr Phe Gln Ser Cys Pro Ile Ala Arg Arg Ala1 5 10 15Lys Ile Asn Thr Arg Ser Arg Gly Ser Ser Ser Ser Val Ala Lys Gly 20 25 30Ser Pro Pro Pro Ala Phe Gln Phe Gln Cys Arg Ala Ser Thr Phe Ala 35 40 45Ala Asp Thr Ser Leu Arg Leu Glu Leu Asp Glu Asn Pro Glu Ala Ile 50 55 60Ile Ser Gly Ala Trp Pro Gly Asn Cys Ser Leu Leu Ser Tyr Asp Asp65 70 75 80Leu Arg Ala Tyr Leu Glu Ser Gln Glu Thr Ala Ala Gln Ala Asp Asp 85 90 95Gln Arg Gly Val Ala Leu Leu Ser Glu Thr Met Ser Thr Pro Val Leu 100 105 110Val Ala Thr Ala Asp Gln Thr Leu Glu Asp Val Glu Cys His Phe Glu 115 120 125Ala Val Ser Gly Leu Pro Val Val Asp Ser Gly Leu Arg Cys Val Gly 130 135 140Val Ile Val Lys Asn Asp Arg Ala Arg Ala Ser His Gly Ser Lys Thr145 150 155 160Lys Ile Ser Glu Val Met Thr Ser Pro Ala Ile Thr Leu Ser Ser Asp 165 170 175Lys Thr Val Met Asp Ala Ala Val Leu Met Leu Lys Lys Lys Ile His 180

185 190Arg Leu Pro Val Val Asn Gln Asp Glu Lys Val Ile Gly Ile Val Thr 195 200 205Arg Ala Asp Val Leu Arg Val Leu Glu Gly Met Leu Lys Ile 210 215 22036537DNAZea mays 36atgggcgacc tctctgtcgg ccacagccgc cgctggtgcg gccgtttcgc ggccgtcctt 60tgcctgtgcg cggccttctg caagccagat gaactcccga tggatccact gccgaacttg 120ccgccgacga ggtcgctgca gtgcttcgag gacgaacagg tgtacagctg ctgcgagggc 180gcgtacaggc taaacccatc gggaatcatc gccgttcccg tcggcgcggt ggactactac 240tgcggcggcg cgtgcgtggt ggagacggag gacgtgctca actgcgtggc cagcgccctg 300gacggcttcg ccttctacaa cggggcctcc gtggaggacg tgcgctacgc actcaggcgg 360ggctgcagcc acaccgccag aagaggcgac ttcaacgatt tggagccgca tctgggcgac 420taccctgaca tctatggcga cgatgatgag cacagctttg gcagcaaggt tgttgcagct 480cctctgaggt tgctcgcgtt tcttggcggt gcggggctgt tcttcctggg cccttga 53737178PRTZea mays 37Met Gly Asp Leu Ser Val Gly His Ser Arg Arg Trp Cys Gly Arg Phe1 5 10 15Ala Ala Val Leu Cys Leu Cys Ala Ala Phe Cys Lys Pro Asp Glu Leu 20 25 30Pro Met Asp Pro Leu Pro Asn Leu Pro Pro Thr Arg Ser Leu Gln Cys 35 40 45Phe Glu Asp Glu Gln Val Tyr Ser Cys Cys Glu Gly Ala Tyr Arg Leu 50 55 60Asn Pro Ser Gly Ile Ile Ala Val Pro Val Gly Ala Val Asp Tyr Tyr65 70 75 80Cys Gly Gly Ala Cys Val Val Glu Thr Glu Asp Val Leu Asn Cys Val 85 90 95Ala Ser Ala Leu Asp Gly Phe Ala Phe Tyr Asn Gly Ala Ser Val Glu 100 105 110Asp Val Arg Tyr Ala Leu Arg Arg Gly Cys Ser His Thr Ala Arg Arg 115 120 125Gly Asp Phe Asn Asp Leu Glu Pro His Leu Gly Asp Tyr Pro Asp Ile 130 135 140Tyr Gly Asp Asp Asp Glu His Ser Phe Gly Ser Lys Val Val Ala Ala145 150 155 160Pro Leu Arg Leu Leu Ala Phe Leu Gly Gly Ala Gly Leu Phe Phe Leu 165 170 175Gly Pro38822DNAZea mays 38atggattcgg aggcggtgca gcacggcctt ctccctctgt ctgcctgtcc tcctaccgcc 60aacagctgcg cgcattacag ccgtgggtgc agcgtcgtgg cgccctgctg cggccaggcc 120ttcggctgcc gccattgcca caacgacgcc aagaactcgc tggaggtcga cccgcgcgac 180cggcacgaga tcccccgcca cgaaataaag aaggtgatct gttctctctg ctccaaggaa 240caggacgtgc aacagaactg ctccagctgt ggggcctgca tgggcaagta cttctgtaaa 300gtatgcaagt tcttcgatga tgatgcctca aagggccagt accactgtga cggatgtgga 360atatgtagaa ccggcggcgt ggagaacttt ttccactgtg ataaatgtgg gtgttgctac 420agcaatgtct tgaaggattc ccaccactgc gtcgaaagag caatgcatca caactgcccc 480gtctgctttg agtatctgtt cgactccacg aaggacatca gcgtgctgca atgtgggcat 540accatccatt tggagtgcat gaacgagatg agagcacacc atcacttctc atgcccagtg 600tgctcgaggt ccgcctgcga catgtcggcc acatggcgga agctcgacga ggaggtcgcg 660gccacgccga tgcctgacat ctaccagaag cacatggtgt ggatcctgtg caacgactgc 720agcgcgacct cgagcgtgcg gttccacgtg ctggggcaca agtgccccgc gtgcagctcg 780tacaacaccc gggagacgag ggctgcgtgc cccaggatct ga 82239273PRTZea mays 39Met Asp Ser Glu Ala Val Gln His Gly Leu Leu Pro Leu Ser Ala Cys1 5 10 15Pro Pro Thr Ala Asn Ser Cys Ala His Tyr Ser Arg Gly Cys Ser Val 20 25 30Val Ala Pro Cys Cys Gly Gln Ala Phe Gly Cys Arg His Cys His Asn 35 40 45Asp Ala Lys Asn Ser Leu Glu Val Asp Pro Arg Asp Arg His Glu Ile 50 55 60Pro Arg His Glu Ile Lys Lys Val Ile Cys Ser Leu Cys Ser Lys Glu65 70 75 80Gln Asp Val Gln Gln Asn Cys Ser Ser Cys Gly Ala Cys Met Gly Lys 85 90 95Tyr Phe Cys Lys Val Cys Lys Phe Phe Asp Asp Asp Ala Ser Lys Gly 100 105 110Gln Tyr His Cys Asp Gly Cys Gly Ile Cys Arg Thr Gly Gly Val Glu 115 120 125Asn Phe Phe His Cys Asp Lys Cys Gly Cys Cys Tyr Ser Asn Val Leu 130 135 140Lys Asp Ser His His Cys Val Glu Arg Ala Met His His Asn Cys Pro145 150 155 160Val Cys Phe Glu Tyr Leu Phe Asp Ser Thr Lys Asp Ile Ser Val Leu 165 170 175Gln Cys Gly His Thr Ile His Leu Glu Cys Met Asn Glu Met Arg Ala 180 185 190His His His Phe Ser Cys Pro Val Cys Ser Arg Ser Ala Cys Asp Met 195 200 205Ser Ala Thr Trp Arg Lys Leu Asp Glu Glu Val Ala Ala Thr Pro Met 210 215 220Pro Asp Ile Tyr Gln Lys His Met Val Trp Ile Leu Cys Asn Asp Cys225 230 235 240Ser Ala Thr Ser Ser Val Arg Phe His Val Leu Gly His Lys Cys Pro 245 250 255Ala Cys Ser Ser Tyr Asn Thr Arg Glu Thr Arg Ala Ala Cys Pro Arg 260 265 270Ile

Claims

1. A method for increasing nitrogen utilization efficiency in a plant comprising: (a) transforming a plant cell with an expression vector comprising an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 5, (b) expressing said nucleic acid molecule in said plant cell; (c) generating from said plant cell a transformed plant in which said nucleotide sequence is overexpressed; and (d) selecting from a plurality of said transformed plants a plant having increased nitrogen use efficiency as compared to a control plant grown under the same conditions.

2. The method according to claim 1, said expression vector further comprising a 5' DNA promoter sequence and a 3' terminator sequence, wherein the nucleotide sequence, the DNA promoter sequence, and the terminator sequence are operatively coupled to permit transcription of the nucleotide sequence.

3. The method according to claim 2, wherein the promoter sequence is selected from the group consisting of constitutive plant promoters and tissue specific promoters.

4. A plant, comprising a plant produced by the method of claim 1.

5. A plant according to claim 4, wherein the plant is selected from the group consisting of corn (maize); sorghum; wheat; sunflower; crucifers; peppers; potato; cotton; rice; soybean; sugarbeet; sugarcane; tobacco; barley; oilseed rape; Brassica sp.; alfalfa; rye; millet; safflower; peanuts; sweet potato; cassaya; coffee; coconut; pineapple; citrus trees; cocoa; tea; banana; avocado; fig; guava; mango; olive; papaya; cashew; macadamia; almond; oats; vegetables; grasses; azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum; and conifers.

6. Transgenic plant tissue of a plant of claim 5.

7. Transgenic plant seed produced from a plant of claim 5.

8. A host cell, comprising a host cell transformed with at least a first nucleotide sequence using the method of claim 1.

9. The host cell according to claim 8, wherein the host cell is selected from the group consisting of bacterial cells and plant cells.

10. A method of expressing a nucleic acid molecule modulated by nitrogen in a plant, said method comprising the steps of providing a transgenic plant or plant seed transformed with a vector construct according to claim 1, and growing the transgenic plant or a plant grown from the transgenic plant seed under conditions effective to express the nucleic acid molecule in said transgenic plant or said plant grown from the transgenic plant seed.

11. A method according to claim 10, wherein expression of the nucleic acid molecule is effective in increasing nitrogen uptake of said transgenic plant or said plant grown from the transgenic plant seed.

12. A method according to claim 10, wherein expression of the nucleic acid molecule is effective in increasing efficiency of nitrogen utilization of said transgenic plant or said plant grown from the transgenic plant seed.

13. A method according to claim 10, wherein the plant is selected from the group consisting of rice, corn, soybean, canola, wheat, alfalfa, barley, rye, cotton, sunflower, peanut, sweet potato, bean, pea, potato, oilseed rape, sorghum, forage grass, pasture grass, turf grass, sugarcane.

14. A method of according to claim 10, wherein expression of the nucleic acid molecule is effective in improving the stress tolerance of said transgenic plant or said plant grown from the transgenic plant seed.

15. A method according to claim 10, wherein expression of the nucleic acid molecule is effective in altering the morphology of said transgenic plant or said plant grown from the transgenic plant seed.

16. A method for increasing nitrogen utilization efficiency in a plant comprising: (a) transforming a plant cell with an expression vector comprising an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes an amino acid sequence set forth in SEQ ID NO: 5, (b) expressing said nucleic acid molecule in said plant cell; (c) generating from said plant cell a transformed plant in which said nucleotide sequence is overexpressed; and (d) selecting from a plurality of said transformed plants a plant having increased nitrogen use efficiency as compared to a control plant grown under the same conditions.