BRPI0810658B1 - DNA CONSTRUCTION, METHOD OF PRODUCING A TRANSGENIC CORN PLANT WITH HIGH ASPARAGINE CONTENT, FOOD OR FEED AND METHOD OF PRODUCING THE SAME - Google Patents

Abstract

CORN PLANTS AND SEEDS WITH INCREASED ASPARAGINE AND PROTEIN CONTENT. The present invention relates to a corn seed with increased levels of protein and amino acids. The invention also relates to DNA constructs that provide expression in transgenic maize cells of an asparagine synthetase enzyme. DNA constructs are used in a method to produce transgenic corn plants and seeds and select plants and seeds with increased levels of protein and amino acids.

Description

BACKGROUND OF THE INVENTION 1. Field of Invention

[0001] The present invention relates generally to the field of plant biotechnology and more specifically to increasing asparagine and protein in plants and seeds.

2. Description of Related Art

[0002] Farmers and consumers desire crop plants with improved agronomic characteristics such as increased yield, increased seed protein production and improved nutritional composition. Desired nutritional components of crop plants include, among others, fiber, antioxidants such as Vitamin E, selenium, iron, magnesium, zinc, B vitamins, lignans, phenolic acids, essential amino acids and phytoestrogens. Although considerable efforts in plant breeding have provided some gains in these desirable traits, the ability to introduce specific non-host DNA into a plant genome provides more opportunities for generating plants with these traits. In particular, although the yield of conventional corn has increased uniformly over the years, there has not been a similar increase in the ability of corn plants to assimilate nitrogen more efficiently or increase seed protein content.

[0003] Nitrogen availability has a significant positive impact on plant productivity, biomass and crop yield including seed protein production. In plants, inorganic nitrogen is assimilated from the soil, reduced to ammonia, and incorporated into organic nitrogen in the form of the nitrogen-carrying amino acids asparagine, glutamine, aspartic acid, and glutamic acid. Asparagine (Asn) is the preferred amide transport molecule because of its high nitrogen to carbon ratio (2N:4C versus 2N:5C) and because it is relatively inert. Asn and other amino acids are also used as building blocks for protein synthesis.

[0004] In plants, Asn is synthesized from glutamine, aspartate and ATP, in a reaction catalyzed by the enzyme asparagine synthetase (AsnS). Glutamate, AMP and pyrophosphate are formed as byproducts. Two forms of AsnS have been described: a glutamine-dependent form and an ammonia-dependent form. Glutamine-dependent AsnS can catalyze both glutamine-dependent and ammonia-dependent reactions although glutamine is the preferred nitrogen source.

[0005] High protein concentration is considered an important quality characteristic for most major crops, including soybeans, corn and wheat. Varieties of corn, wheat and soybeans with a lot of protein, for example, were identified through traditional generation. However, most high-protein lines developed in this way have yield obstacles or other agronomic disadvantages. It would be desirable if the protein content of crops, especially corn, could be increased above currently available levels, both for human consumption and for use in animal feed. This would offer the benefit of greatly increased nutrient value when the crop is used as food and feed for humans and animals.

SUMMARY OF THE INVENTION

[0006] The invention provides methods and compositions for developing crops and plant products to increase protein and amino acid content. The methods and compositions increase the level of free amino acids and protein in plant tissues, particularly in seeds. More specifically, transgenic plants and seeds are provided, which contain heterologous DNA compositions that express a gene product involved in increased asparagine and increased protein biosynthesis. Product expression increases the nutritional value of food and feed corn sources and processed products derived from transgenic corn seed or parts thereof.

[0007] In one aspect, the invention provides methods for increasing the protein content in a corn plant. DNA constructs comprising a polynucleotide sequence encoding polypeptides with asparagine synthetase activity are also provided.

[0008] In another embodiment, the present invention comprises a corn plant cell transformed with the heterologous DNA composition encoding an asparagine synthetase identified here. In specific embodiments, heterologous expression of a corn AsnS4 (asparagine synthetase isozyme 4) polynucleotide molecule is provided, for example, to result in elevated asparagine and/or protein in a transgenic plant, including seeds, relative to plant of the same variety not expressing the heterologous maize AsnS2 polynucleotide molecule.

[0009] In particular embodiments, a nucleic acid sequence is provided, as well as methods of using the same, where the sequence is selected from the group consisting of: (a) a nucleic acid sequence comprising the sequence of SEQ ID NO: 50 ; (b) a nucleic acid sequence with at least about 90% identity to the sequence of SEQ ID NO: 50, wherein the nucleic acid sequence encodes a polypeptide comprising asparagine synthetase activity; (c) a nucleic acid sequence encoding the polypeptide sequence of SEQ ID NO: 51; (d) a nucleic acid sequence encoding a polypeptide sequence with at least about 90% identity to the sequence of SEQ ID NO: 51, wherein the polypeptide comprises asparagine synthetase activity; and (e) a nucleic acid sequence that hybridizes to a sequence of (a)-(d) under stringency conditions of about 0.2 x SSC and 65°C, wherein the nucleic acid sequence encodes a polypeptide comprising activity of asparagine synthetase. In particular embodiments, nucleic acids are provided having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence of SEQ ID NO: 50. In additional embodiments, there are provided nucleic acids encoding a polypeptide sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence of SEQ ID NO: 51. The invention also provides sequences of isolated polypeptide comprising SEQ ID NO: 51, as well as sequences having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

[00010] The present invention also relates to food or animal feed prepared from a seed provided by the invention with increased protein and/or amino acid content, or a processed product from such a seed, for example, a flour. Thus, the present invention also comprises a corn seed containing an asparagine synthetase enzyme produced through the expression of a heterologous DNA construct comprising a DNA molecule encoding a corn asparagine synthetase enzyme. One embodiment of such a seed is harvested grain, the present invention also comprises a flour, gluten and other corn products made from such grain.

[00011] The present invention includes isolated nucleic acid primer sequences comprising one or more SEQ ID NOs: 52-59 or a complement thereof. The present invention includes a method for detecting or identifying, in the genome of a transformed plant or progeny thereof, a heterologous polynucleotide molecule encoding a plant AsnS polypeptide, or a plant polypeptide having AsnS activity of the present invention, comprising a polynucleotide molecule selected from the group consisting of SEQ ID NOs: 52-59, wherein said polynucleotide molecule is used as a DNA primer in a DNA amplification method.

BRIEF DESCRIPTION OF THE FIGURES

[00012] Figure 1 illustrates the plasmid map of pMON79706.

[00013] Figure 2 illustrates the plasmid map of pMON66229.

[00014] Figure 3 illustrates the plasmid map of pMON66230.

[00015] Figure 4 illustrates the plasmid map of pMON66231.

[00016] Figure 5 illustrates the plasmid map of pMON66239.

[00017] Figure 6 Transgene expression in pMON79706 events. Error bars represent 95% safety interval, with n=5 for transgenic events and n=10 for inbred control.

[00018] Figure 7 Transgene expression in pMON92870 events. Error bars represent 95% safety interval, with n>3 plants for transgenic events and n=8 plants for inbred control.

DESCRIPTION OF NUCLEIC ACID AND POLYPEPTIDE SEQUENCES

[00019] SEQ ID NO: 1 is a polynucleotide sequence encoding a Zea mays Asn1.

[00020] SEQ ID NO: 2 is a Zea mays AsnS1 polypeptide.

[00021] SEQ ID NO: 3 is a polynucleotide sequence encoding AsnS2 from Zea mays.

[00022] SEQ ID NO: 4 is an AsnS2 polypeptide from Zea mays.

[00023] SEQ ID NO: 5 is a polynucleotide sequence encoding AsnS3 from Zea mays.

[00024] SEQ ID NO: 6 is an AsnS3 polypeptide from Zea mays.

[00025] SEQ ID NO: 7 is a polynucleotide sequence encoding AsnS from Glycine max.

[00026] SEQ ID NO: 8 is a Glycine max AsnS polypeptide.

[00027] SEQ ID NO: 9 is a polynucleotide sequence encoding an AsnS from Xylella fastidiosa.

[00028] SEQ ID NO: 10 is a polynucleotide sequence encoding an AsnS from Xanthomonas campestris.

[00029] SEQ ID NO: 11 is a polynucleotide sequence encoding an AsnS from Bacillus halodurans.

[00030] SEQ ID NO: 12 is a polynucleotide sequence encoding an AsnS from Oryza sativa.

[00031] SEQ ID NO: 13 is a polynucleotide sequence encoding an AsnS from Galdieria sulphuraria.

[00032] SEQ ID NO: 14 is a polynucleotide sequence encoding an AsnS from Galdieria sulphuraria.

[00033] SEQ ID NO: 15 is a polynucleotide sequence encoding an AsnS from Galdieria sulphuraria.

[00034] SEQ ID NO: 16 is a polynucleotide sequence encoding an AsnS from Galdieria sulphuraria.

[00035] SEQ ID NO: 17 is a polynucleotide sequence encoding an AsnS CGPG3913 from Saccharomyces cerevisiae.

[00036] SEQ ID NO: 18 is a forward AsnS PCR primer sequence (f).

[00037] SEQ ID NO: 19 is a forward AsnS PCR primer sequence (f).

[00038] SEQ ID NOs:20-43 are primary and secondary AsnS forward (f) and reverse (r) PCR primer sequences used in a Gateway cloning procedure.

[00039] SEQ ID NO: 44, a forward AsnS PCR primer sequence (f).

[00040] SEQ ID NO: 45, a forward AsnS PCR primer sequence (f).

[00041] SEQ ID NO: 46 sensorZmAS initiator.

[00042] SEQ ID NO: 47 ZmAS antisense primer.

[00043] SEQ ID NO: 48 maize AsnS3 forward primer

[00044] SEQ ID NO: 49 corn AsnS3 reverse primer

[00045] SEQ ID NO: 50 is a polynucleotide sequence encoding AsnS4 from Zea mays.

[00046] SEQ ID NO: 51 is an AsnS4 polypeptide from Zea mays.

[00047] SEQ ID NO: 52 is an advanced PCR primer, Zm-AsnS1_for1.

[00048] SEQ ID NO: 53 is a reverse PCR primer, Zm-AsnS1_rev1.

[00049] SEQ ID NO: 54 is an advanced PCR primer, Zm-AsnS3_for2.

[00050] SEQ ID NO: 55 is a reverse PCR primer, Zm-AsnS3_rev2.

[00051] SEQ ID NO: 56 is an advanced PCR primer, Zm-AsnS4_for3.

[00052] SEQ ID NO: 57 is a reverse PCR primer, Zm-AsnS4_rev3.

[00053] SEQ ID NO: 58 is an advanced PCR primer, Zm-AsnS2_for4.

[00054] SEQ ID NO: 59 is a reverse PCR primer, Zm-AsnS2_rev4. DETAILED DESCRIPTION OF THE INVENTION

[00055] What follows is a detailed description of the invention provided to assist those skilled in the art in the practice of the present invention. Unless otherwise defined herein, the terms are to be understood in accordance with conventional usage by those skilled in the relevant art. Definitions of common terms in molecular biology can also be found in Rieger et al., 1991; and Lewin, 1994. The nomenclature for DNA bases as shown in 37 CFR § 1.822 is used. The standard one-letter and three-letter nomenclature for amino acid residues is used. Modifications and variations in the embodiments described herein may be made by those skilled in the art without departing from the spirit or scope of the present invention.

[00056] The present invention provides a method for increasing the protein content in a corn plant by introducing into the genome of a corn plant cell a heterologous polynucleotide that expresses an AsnS polypeptide in the transgenic plant cell. The present invention provides DNA constructs comprising (comprising means "including, but not limited to") polynucleotide molecules, or segments of a polynucleotide molecule encoding an AsnS polypeptide, optionally operably linked to a chloroplast transit peptide.

[00057] Polynucleotide molecules encoding an AsnS polypeptide or analogue or allele thereof or polynucleotide molecules encoding a transit peptide or marker/reporter gene are "isolated" by the fact that they were at least partially prepared in vitro, e.g. isolated from their native state, from a cell, purified and amplified, for example, they are in combination with genetic elements heterologous to those normally found associated with them in their native state. As used herein, a heterologous DNA construct comprising a polynucleotide molecule encoding AsnS that has been introduced into a host cell is preferably non-identical to any polynucleotide molecule present in the cell in its native, untransformed state, and is isolated with respect to to other DNA molecules that occur in the host cell's genome.

[00058] As used herein, "altered or increased" levels of asparagine in a plant, plant tissue or transformed plant cell are levels that are greater than the levels found in the corresponding plant, plant tissue or plant cells not containing the DNA constructs of the present invention.

[00059] The agents of the present invention can also be recombinant. As used herein, the term recombinant means any agent (e.g., DNA, peptide, etc.) that is, or results, even indirectly, from human manipulation of a polynucleotide molecule.

[00060] As used herein in a preferred aspect, an increase in the nutritional quality of a seed, e.g., increased seed protein content, is determined by the ability of a plant to produce a seed having a greater yield of protein or a component nutritional value than a seed without such an increase in protein or nutritional quality. In a particularly preferred aspect of the present invention, the increase in nutritional quality is measured with respect to a plant with a genetic basis similar to the nutritionally higher plant except that the plant of the present invention expresses or overexpresses a protein or fragment thereof described herein. heterologous DNA constructs.

Polynucleotide Molecules

[00061] The present invention includes and provides transgenic corn plants and seeds that comprise in their genome a transgene comprising a heterologous DNA molecule encoding a corn asparagine synthetase enzyme (Zm.AsnS4), the DNA molecule, for example, comprising SEQ ID NO: 50 and sequences having at least 90%, 95% or 99% identity with such sequences with asparagine synthetase activity.

[00062] A further aspect of the invention is a method for increasing protein in a corn plant by introducing into a corn cell a DNA construct that provides heterologous polynucleotide molecules, for example, SEQ ID NOs: 1, 3 , 5, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 50 which encode an asparagine synthetase enzyme. The polynucleotide may differ from any of these examples without changing the polypeptide it encodes. For example, it is understood that codons capable of encoding such conservative amino acid substitutions are known in the art. Additionally, the invention comprises that polypeptides where one or more amino acids have been deleted, replaced or added without altering the function of asparagine synthetase can be used in the invention.

[00063] In one aspect of the present invention, the polynucleotides of the present invention are said to be introduced polynucleotide molecules. A polynucleotide molecule is said to be "introduced" if it is inserted into a cell or organism as a result of human manipulation, no matter how indirect. Examples of introduced polynucleotide molecules include, without limitation, polynucleotides that have been introduced into cells through transformation, transfection, injection and projection and those that have been introduced into an organism through conjugation, endocytosis, phagocytosis, etc. Preferably, the polynucleotide is inserted into the genome of the cell.

[00064] A subset of the polynucleotide molecules of the present invention are fragment polynucleotide molecules. Fragmented polynucleotide molecules may consist of a significant portion(s) of, or indeed the majority of, the polynucleotide molecules of the present invention, such as those specifically described. Alternatively, the fragments may comprise smaller oligonucleotides (having from about 15 to about 400 nucleotide residues and more preferably about 15 to about 30 nucleotide residues or about 50 to about 100 nucleotide residues or about from 100 to about 200 nucleotide residues or from about 200 to about 400 nucleotide residues or from about 275 to about 350 nucleotide residues). A fragment of one or more polynucleotide molecules of the present invention can be a probe and specifically a PCR primer molecule. A PCR primer is a polynucleotide molecule capable of initiating a polymerase activity while in a double-stranded structure with another polynucleotide. Various methods for determining the structure of PCR probes and PCR techniques exist in the art.

[00065] As used herein, two polynucleotide molecules are said to be capable of specifically hybridizing to each other if the two molecules are capable of forming an antiparallel, double-stranded polynucleotide structure.

[00066] A polynucleotide molecule is said to be the "complement" of another polynucleotide molecule if they exhibit complete complementarity. As used herein, molecules are said to exhibit "complete complementarity" when each nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be "minimally complementary" if they can hybridize to each other with sufficient stability to allow them to remain annealed to each other at least under conventional "low stringency" conditions. Similarly, molecules are said to be "complementary" if they can hybridize to each other with sufficient stability to allow them to remain annealed to each other under conventional "high stringency" conditions. Conventional stringency conditions are described by Sambrook et al. (2001) and by Haymes et al. (1985). Departures from complete complementarity are then permissible, as long as such departures do not completely impede the ability of the molecules to form a double-stranded structure. Thus, in order for a polynucleotide molecule to serve as a primer or probe it needs to be just sufficiently complementary in sequence to be capable of forming a double-stranded structure under the particular solvent and salt concentrations employed.

[00067] Appropriate stringency conditions that promote DNA hybridization are, for example, 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0X SSC at 20-25 °C, are known to those skilled in the art or can be found in Ausubel et al., Eds. (1989), section 6.3.1-6.3.6. For example, the salt concentration in the washing step can be selected from a low stringency of about 2.0 X SSC at 50 ° C to a high stringency of about 0.2 X SSC at 65 ° C. Furthermore, the temperature in the washing step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. Both the temperature and the salt can be varied or the temperature or salt concentration can be kept constant so that nucleic acid hybridizes specifically to one or more polynucleotide molecules provided herein, for example, as shown in: SEQ ID NOs: 1 , 3, 5, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18-45 and 50, and complements thereof, under moderately stringent conditions, for example, at about 2.0 X SSC and about 65°C.

[00068] In one embodiment of a method of the present invention, any of the polynucleotide sequences or polypeptide sequences, or fragments of any, of the present invention can be used to search for related sequences. As used herein, "search for related sequences" means any method of determining the relationship between two sequences including, but not limited to, searches that compare sequence homology: for example, a PBLAST search of a database for relationship to a unique polypeptide sequence. Other research can be conducted using profile-based methods, such as HMM (Hidden Markov model) META-MEME, which is maintained by South Dakota State University, SD, and PSI-BLAST, which is maintained by the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health (NCBI).

[00069] A polynucleotide molecule can encode a substantially identical or substantially homologous polypeptide molecule. Degrees of identity or homology can be determined through the use of computer software such as the Gap WISCONSIN PACKAGE Program. The Gap program in WISCONSIN PACKAGE version 10.0-UNIX from Genetics Computer Group, Inc. is based on the method of Needleman and Wunsch, 1970. Using the TBLASTN program in the BLAST 2.2.1 software suite (Altschul et al., 1977) or using the BLOSUM62 matrix (Henikoff and Henikoff, 1992). A polynucleotide molecule of the present invention may also encode a homologous polypeptide. As used herein, a homologous polypeptide molecule or fragment thereof is a counterpart protein molecule or fragment thereof in a second species (e.g., maize rubisco small subunit is a homologue of the Arabidopsis rubisco small subunit). A homologue can also be generated through molecular evolution or DNA shuffling techniques, so that the molecule retains at least one functional or structural characteristic of the original polypeptide (see, for example, U.S. Patent 5,811,238).

[00070] In a preferred embodiment, any of the polynucleotide molecules of the present invention can be operably linked to a promoter region that functions in a plant cell to cause the production of an mRNA molecule, where the polynucleotide molecule that is linked to the promoter is heterologous with respect to that promoter. As used herein, "heterologous" DNA is any DNA sequence that is not naturally found in proximity to adjacent DNA. "Native" refers to a naturally occurring nucleic acid sequence. "Heterologous" sequence often originates from a foreign source or species or, if from the same source, is modified from its original form and/or location in the genome.

[00071] As used herein, the term "protein", "peptide molecule" or "polypeptide" includes any molecule comprising five or more amino acids. It is well known in the art that protein, peptide or polypeptide molecules can undergo modification, including post-translational modifications, such as, but not limited to, disulfide bond formation, glycosylation, phosphorylation or oligomerization. Therefore, as used herein, the term "protein", "peptide molecule" or "polypeptide" includes any protein that is modified by any biological or non-biological process. The terms "amino acid" and "amino acids" refer to all naturally occurring L-amino acids. The definition is intended to include norleucine, norvaline, ornithine, homocysteine, and homoserine.

[00072] A "protein fragment" is a peptide or polypeptide molecule whose amino acid sequence comprises a subset of the amino acid sequence of that protein. A protein or fragment thereof that comprises one or more additional peptide regions not derived from that protein is a "fusion" protein. Such molecules can be derivatized to contain carbohydrate or other moieties (such as hemocyanin from the keyhole limpet clam). Fusion protein or peptide molecules of the present invention are preferably produced by recombinant means.

Plant Constructs and Plant Transformants

[00073] One or more DNA constructs of the present invention that encode an asparagine synthetase can be used in plant transformation or transfection. Exogenous genetic material can be transferred into a plant cell and the plant cell regenerated into a whole, fertile or sterile plant. Exogenous genetic material is any genetic material, whether naturally occurring or otherwise, from any source that is capable of being inserted into any organism.

[00074] In a further aspect of the present invention, polynucleotide sequences of the present invention also encode peptides involved in intracellular localization, export or post-translational modification, for example, chloroplast transit peptides.

[00075] As used herein, the term "gene" includes a nucleic acid molecule that provides transcriptional regulation that includes a promoter that functions in plants, 5' untranslated molecules, for example, introns and leader sequences, a molecule of transcribed nucleic acid and a 3' transcriptional termination molecule.

[00076] The nucleic acid molecules encoding a polypeptide of the present invention can be combined with other non-native or heterologous sequences in various ways. By "heterologous" sequences is meant any sequence that is not naturally found joined to a polypeptide-encoding nucleotide sequence of the present invention including, for example, combinations of nucleotide sequences from the same plant that are not naturally found together, or both sequences originate from two different species. The term "operatively linked" as used in reference to the physical arrangement and function of regulatory and structural polynucleotide molecules that cause regulated expression of an operably linked structural polynucleotide molecule.

[00077] Expression of a DNA construct or transgene means the transcription and stable accumulation of sense or antisense RNA or protein derived from the polynucleotide molecule of the present invention or translation thereof. "Sense" RNA means an RNA transcript that includes mRNA and can then be translated into a polypeptide or protein by the cell."antisense RNA" means an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Patent 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA can be with any part of the specific gene transcript, i.e., in the 5' non-coding sequence, 3' non-translated sequence, introns or the coding sequence. "RNA transcript" means the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be an RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA.

[00078] As used herein, the term plant expression cassette refers to a construct comprising the necessary DNA regulatory molecules operably linked to the target molecule to provide expression in a plant cell.

[00079] The DNA construct of the present invention may, in one embodiment, contain a promoter that causes overexpression of the polypeptide of the present invention, where "overexpression" means the expression of a polypeptide either not normally present in the host cell or present in said host cell at a level greater than normally expressed from the endogenous gene encoding said polypeptide. Promoters, which can cause overexpression of the polypeptide of the present invention, are generally known in the art, examples of such which provide constitutive expression pattern include 19S cauliflower mosaic virus promoter and 35S cauliflower mosaic virus promoter. (U.S. Patent 5,352,605), scrofularia mosaic virus 35S promoter (U.S. Patent 6,051,753), sugarcane bacilliform virus promoter (U.S. Patent 5,994,123), comelina yellow mottle virus promoter ( Medberry et al., 1992), small subunit of ribulose-1,5-bisphosphate carboxylase promoter, rice cytosolic triosephosphate isomerase promoter, adenine phosphoribosyltransferase promoter, rice actin 1 promoter (U.S. Patent 5,641,876), maize ubiquitin, mannopine synthase promoter and octopine synthase promoter.

[00080] Such genetic constructs can be transferred to either monocotyledonous or dicotyledonous plants including, but not limited to, alfalfa, apple, Arabidopsis, banana, Brassica campestris, canola, castor bean, coffee, corn, cotton, cottonseed, chrysanthemum, crambe , cucumber, Dendrobium spp., Dioscorea spp., eucalyptus, fescue, flax, gladiolus, liliaceae, flax seed, millet, cantaloupe melon, mustard, oats, oil palms, oilseed rape, peanuts, perennial ryegrass, Phaseolus, rape seed, rice, sorghum, soybeans, rye, wheatgrass, lawn, wheat, saffron, sesame, sugar beet, sugar cane, blackberry, papaya, saffron and sunflower (Christou, 1996). In a preferred embodiment, genetic material is transferred to a corn cell.

[00081] Transfer of a polynucleotide molecule that encodes a protein can result in expression or overexpression of that polypeptide in a transformed cell or transgenic plant. One or more proteins or fragments thereof encoded by polynucleotide molecules of the present invention can be overexpressed in a transformed cell or transformed plant.

[00082] In one embodiment, DNA constructs of the present invention comprise a polynucleotide molecule encoding a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 50. The invention provides transformed corn cells where, relative to an untransformed corn plant without such a DNA construct, the cell has an increased level of asparagine.

[00083] In another embodiment, the DNA constructs of the present invention comprise a heterologous DNA molecule operably linked to a maize asparagine synthetase coding sequence, e.g., SEQ ID NO.: 1, 3, 5 or 50, and the DNA construct is transformed corn cell. In one embodiment, DNA constructs of the present invention comprising SEQ ID NO: 50 or related sequences described herein are provided in a transformed corn cell, and expression of the DNA construct provides an asparagine-enhanced corn plant tissue or a seed of corn plant with increased protein compared to the corn plant not transformed with the DNA construct.

[00084] In some embodiments, the levels of one or more AsnS products can be increased throughout an entire plant or localized in one or more specific organs or tissues of the plant. Without limiting the scope of the present invention, various promoter sequences are useful for expressing the above enzyme gene. For example, maize C4-type PPDK promoter (Glackin et al., 1990), maize C4-type PEPC promoter (Hudspeth and Grula, 1989), rice Rubisco small subunit promoter (Kyozuka et al., 1993), and light-harvesting chlorophyll a/b binding protein (Sakamoto et al., 1991), P-FDA promoter (US20040216189A1, the polynucleotide sequence of which is incorporated herein by reference), and P-RTBV promoter (U.S. Pat. 5,824,857), the polynucleotide sequence of which is incorporated herein by reference). For example, asparagine or protein levels may be increased in one or more tissues and organs of a plant including without limitation: roots, tubers, stems, leaves, stalks, fruits, berries, nuts, bark, pods, seeds and flowers. A preferred organ is a seed.

[00085] For the purpose of expression in plant source tissues, such as the leaf, seed, root or stem, it is preferred that promoters used have relatively high expression in these specific tissues. Tissue-specific expression of a protein of the present invention is a particularly preferred embodiment.

[00086] DNA constructs or vectors may also include, with the coding region of interest, a polynucleotide sequence that acts, in whole or in part, to terminate transcription of that region. Several such sequences have been isolated, including the 3' T-NOS region (Ingelbrecht et al., 1989; Bevan et al., 1983). Regulatory transcription termination regions can be provided in plant expression constructs of the present invention as well. Transcript termination regions may be provided by the DNA sequence encoding the gene of interest or a convenient transcription termination region derived from a different gene source, for example, the transcript termination region that is naturally associated with the initiation region. of transcript. One skilled in the art will recognize that any convenient transcript termination region that is capable of terminating transcription in a plant cell can be employed in the constructs of the present invention.

[00087] A vector or construct may also include regulatory elements, such as introns. Examples of such include the Adh 1 intron (Callis et al., 1987), the sucrose synthase intron (Vasil et al., 1989), hsp70 intron (U.S. Patent 5,859,347), and the TMV omega element (Gallie et al., 1989). . These and other regulatory elements may be included where appropriate.

[00088] A vector or construct may also include a selectable marker. Selectable markers can also be used to select plants or plant cells that contain exogenous genetic material. Examples of such include, but are not limited to: a neo gene (Potrykus et al., 1985), which encodes kanamycin resistance and can be selected using kanamycin, nptII, G418, hpt, etc.; a bar gene, which encodes resistance to bialaphos; a mutant EPSP synthase gene (Hinchee et al., 1988; Reynaerts et al., 1988; Jones et al., 1987), which encodes glyphosate resistance; a nitrilase gene that confers resistance to bromoxynil; a mutant acetolactate synthase (ALS) gene that confers resistance to imidazolinone or sulfonylurea (U.S. Patent 4,761,373); D'Halluin et al., 1992); and a methotrexate-resistant DHFR gene (Thillet et al., 1988).

Plant Transformation

[00089] The most commonly used methods for plant cell transformation are the Agrobacterium-mediated DNA transfer process and the biolistics-mediated process or microprojectile bombardment (i.e., the gene gun). Typically, nuclear transformation is desired, but if it is desired to specifically transform plasmids, such as chloroplasts or amyloplasts, plant plastids can be transformed using a microprojectile-mediated delivery of the desired polynucleotide.

[00090] Methods for introducing transgenes into plants through Agrobacterium-mediated transformation utilize a T-DNA (transfer DNA) that incorporates the genetic elements of the transgene and transfers these genetic elements to the genome of a plant. In general, the transgene(s) having around a right border (RB) DNA molecule and a left border (LB) DNA molecule is (are) transferred into the plant genome in a unique location. The "T-DNA" molecule refers to a DNA molecule that integrates into a plant genome through an Agrobacterium-mediated transformation method. The ends of the T-DNA molecule are defined in the present invention as being flanked by the border regions of the T-DNA of Agrobacterium Ti plasmids. These border regions are generally referred to as the right border (RB) and left border (LB) regions and exist as variations in sequence and nucleotide length depending on whether they are derived from nopaline- or octopine-producing strains of Agrobacterium. The border regions commonly used in DNA constructs designed for transgene transfer into plants are often several hundred polynucleotides in length and comprise a cutting site where an endonuclease digests the DNA to provide a site for insertion into the genome of a plant. T-DNA molecules generally contain one or more plant expression cassettes.

[00091] In connection with microprojectile bombardment (U.S. Patents 5,550,318; 5,538,880; and 5,610,042; each of which is specifically incorporated herein by reference in its entirety), particles are coated with polynucleotides and applied to cells through a propelling force. Exemplary particles include those comprised of tungsten, platinum and, preferably, gold. A useful method for delivering DNA to plant cells through particle acceleration is the Biolistics Particle Delivery System (BioRad, Hercules, Calif.), which can be used to propel DNA-coated particles or cells through a screen, such as a stainless steel screen or Nytex screen, on a filter surface covered with cultured monocot plant cells in suspension. Microprojectile bombardment techniques are widely applicable, and can be used to transform virtually any plant species. Examples of species that have been transformed through microprojectile bombardment include monocot species such as corn (PCT Publication WO 95/06128), barley, wheat (U.S. Patent 5,563,055, incorporated herein by reference in its entirety), rice, oats , rye, sugar cane and sorghum; as well as various dicotyledons including tobacco, soybeans (U.S. Patent 5,322,783, incorporated herein by reference in its entirety), sunflower, peanuts, cotton, tomatoes and legumes in general (U.S. Patent 5,563,055, incorporated herein by reference in its entirety).

[00092] To select or register transformed plant cells regardless of the transformation methodology, the DNA introduced into the cell contains a gene that functions in a regenerable plant tissue to produce a compound that confers the plant tissue resistance to a compound of otherwise toxic. Genes of interest for use as a selectable, classifiable, or registerable marker would include, but would not be limited to, GUS, green fluorescent protein (GFP), luciferase (LUX), and antibiotic or herbicide tolerance genes. Examples of antibiotic resistance genes include those that confer resistance to kanamycin (and neomycin, G418) and bleomycin.

[00093] The regeneration, development and cultivation of plants from various transformed explants are well documented in the art. This process of regeneration and growth typically includes the steps of selecting transformed cells and culturing these individualized cells through common stages of embryonic development through the rooted seedling stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are then planted in an appropriate plant growth medium such as soil. Cells that survive exposure to the selective agent, or cells that are recorded positive in an evaluation assay, can be cultured in medium that supports plant regeneration. Developing seedlings are transferred to soilless plant growth mix, and acclimatized, before transfer to a greenhouse or growth chamber for maturation.

[00094] The present invention can be used with any transformable cell or tissue. By transformable as used herein is meant a cell or tissue that is capable of further propagation to give rise to a plant. Those skilled in the art recognize that various plant cells or tissues are transformable where after insertion of exogenous DNA and appropriate culture conditions the plant cells or tissues can form into a differentiated plant. Suitable tissue for these purposes may include, but are not limited to, immature embryos, scutellar tissue, suspension cell cultures, immature inflorescence, shoot meristem, nodal explants, callus tissue, hypocotyl tissue, cotyledons, roots and leaves.

[00095] Any suitable plant culture medium can be used. Examples of suitable media would include, but are not limited to, MS-based media (Murashige and Skoog, 1962) or N6-based media (Chu et al., 18:659, 1975) supplemented with additional plant growth regulators including , but not limited to, auxins, cytokines, ABA and gibberellins. Those skilled in the art are familiar with the variety of tissue culture media, which, when supplemented appropriately, support plant tissue growth and development and are suitable for plant transformation and regeneration. These tissue culture media can either be purchased as a commercial preparation or prepared as usual and modified. Those skilled in the art are aware of media and media supplements such as nutrients and growth regulators for use in transformation and regeneration and other culture conditions such as light intensity during incubation, pH and incubation temperatures that can be optimized for the variety. particular interest.

[00096] Any of the polynucleotide molecules of the present invention can be introduced into a plant cell in a permanent or transient manner in combination with other genetic elements such as vectors, promoters, enhancers, etc. Furthermore, any of the polynucleotide molecules of the present invention can be introduced into a plant cell in a manner that allows expression or overexpression of the protein or fragment thereof encoded by the polynucleotide molecule.

[00097] The present invention also provides parts of the plants, particularly reproductive or storage parts, of the present invention. Plant parts, without limitation, include seed, endosperm, ovule, pollen or tubers. In a particularly preferred embodiment of the present invention, the plant part is a corn seed. In one embodiment, corn seed (or grain) is a constituent of animal feed.

[00098] In a preferred embodiment, corn or corn flour or corn seed protein feed is prepared for farm animals or humans or both. Methods for producing feed, flour and protein are known in the art. See, for example, U.S. Patents 4,957,748; 5,100,679; 5,219,596; 5,936,069; 6,005,076; 6,146,669; and 6,156,227. In a preferred embodiment, the protein preparation is a protein-rich preparation. Such a high protein preparation preferably has a protein content greater than about 5% (w/v), more preferably 10% (w/v) and most preferably 15% (w/v).

[00099] Descriptions of breeding methods that are generally used for different traits and cultures can be found in one of several reference books (e.g., Hayward, 1993; Richards, 1997; Allard, 1999).

Other Organisms

[000100] A polynucleotide of the present invention can be introduced into any cell or organism such as a mammalian cell, mammalian cell, fish cell, fish cell, bird cell, avian cell, algae cell, algae, fungus cell, fungus cell or bacterial. A protein of the present invention can be produced in an appropriate cell or organism. Preferred hosts and transformants include: fungal cells such as Aspergillus, yeast, mammals, particularly bovine and porcine, insects, bacteria and algae. Particularly preferred bacteria are Agrobacterium tumefaciens and E. coli.

[000101] In one aspect of the present invention, one or more nucleic acid molecules of the present invention are used to determine the level of expression (i.e., the concentration of mRNA in a sample, etc.) in a plant (preferably canola, corn, Brassica campestris, oilseed rapeseed, rapeseed, soybean, crambe, mustard, castor, peanut, sesame, cottonseed, linseed, safflower, oil palm, hemp or sunflower) or standard (i.e. the expression kinetics, decay rate, stability profile, etc.) of the expression of a protein encoded in part or in full by one or more of the polynucleotide molecule of the present invention. Several methods can be used to compare expression between two or more cell or tissue samples. These methods include hybridization assays such as northerns, RNAse protection assays, and in situ hybridization. Alternatively, the methods include PCR-type assays. In a preferred method, expression is assessed by hybridizing polynucleotides from two or more samples to a polynucleotide array. The array contains a plurality of suspect sequences known or suspected to be present in the cells or tissues of the samples.

[000102] The following examples are included to demonstrate aspects of the invention, those skilled in the art should, in light of the present report, understand that many changes can be made to the specific aspects that are described and still obtain the same or similar result without depart from the spirit and scope of the invention.

EXAMPLES

[000103] Those skilled in the art will understand the many advantages of the methods and compositions provided by the present invention. The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques described in the examples that follow represent techniques found by the inventors to work well in the practice of the invention, and so may be considered to constitute preferred modes of practice thereof. However, those skilled in the art should, in light of the present report, understand that many changes can be made to the specific embodiments that are described and still obtain the same or similar result without departing from the spirit and scope of the invention. All references cited here are incorporated herein by reference to the extent that they supplement, explain, provide a basis for or teach methodology, techniques or compositions employed herein.

Example 1 Construction of corn and soybean plant cDNA and genomic libraries

[000104] This example describes the production of cDNA libraries made from corn and soybean plant tissues from which the corn and soybean AsnS polynucleotide sequences of the present invention were isolated. cDNA libraries were generated from Zea mays and Glycine Max tissue using techniques known in the field, e.g., Alba, 2004. Maize cDNA libraries were made from two different tissues. A library was made from incipient seeds collected in the slow phase of the inbred line 90DDD5. A second corn cDNA library was made from corn hair tissue at the corn hair growth stage of the corn inbred line H99 and germinating pollen from the corn inbred line MO17. To construct a soybean cDNA library (Glycine Max), meristematic tissue and part of the hypocotyl were excised from rehydrated dry soybean seeds of the A3237 variety (Asgrow). Explants were prepared by first germinating surface-sterilized seeds in solid tissue culture medium for 6 days at 28°C on 18 hours of light/day, and then transferring the germinated seeds to 4°C for at least 24 hours. For the tissue used in library preparation the cotyledons were removed to enrich the specific tissue of interest. 0.5 to 2 grams of tissue was used for preparation of total RNA and poly A+ RNA. For all cDNA libraries, plant tissues were collected and immediately frozen in liquid nitrogen. The collected tissue was stored at −80°C until preparation of total RNA. Total RNA was purified using Invitrogen Corporation's Trizol reagent (Invitrogen Corporation, Carlsbad, Calif., U.S.A.), essentially as recommended by the manufacturer. Poly A+ RNA (mRNA) was purified using dT oligo magnetic beads essentially as recommended by the manufacturer (Dynabeads®, Dynal Biotech, Oslo, Norway).

[000105] Construction of plant cDNA libraries is well known in the art and several cloning strategies exist. Several cDNA library construction kits are commercially available. cDNA libraries were prepared using the Superscript® Plasmid System for DNA Synthesis and Plasmid Cloning (Invitrogen Corporation) as described in the Superscript II cDNA Library Synthesis Protocol. The cDNA libraries were checked to confirm an appropriate insert:vector ratio.

[000106] A genomic DNA library was constructed using genomic DNA isolated from Zea mays using a modified genomic DNA isolation protocol described below (Dellaporta et al., 1983). Corn seedlings were grown in soil or in Petri dishes, harvested and kept frozen in liquid nitrogen until extraction. The tissue was ground into a fine powder using a pestle while keeping the tissue frozen with liquid nitrogen. The powdered tissue was transferred to a Waring mixer containing 200 mL of cold (0°C) DNA extraction buffer (350 mM sorbitol; 100 mM Tris; 5 mM EDTA; pH to 7.5 with HCl; bisulfite sodium, 3.8 mg/mL) which was added just before use and homogenized at high speed for 30-60 seconds. The homogenate was filtered through a layer of cotton gauze and collected in a centrifuge bottle. The samples were then centrifuged at 2500 x g for 20 minutes and the supernatant and any loose green material were discarded. The pellet was then resuspended in 1.25 mL of DNA extraction buffer and transferred to a 50 mL polypropylene tube. Core lysis buffer (1.75 mL containing 200 mM Tris; 50 mM EDTA; 2 M NaCL; 2.0% (w/v) CTAB; pH adjusted to 7.5 with HCl) was then added, followed by the addition of 0.6 mL of 5% sarcosyl (w/v). The tubes were gently mixed and the samples were incubated at 65°C for 20 minutes. An equal volume of chloroform:isoamyl alcohol (24:1) was added and the tubes were again gently mixed. The tubes were then centrifuged at 2500xg for 15 minutes and the resulting supernatant was transferred to a clean tube. An equal volume of ice-cold isopropanol was poured into the sample, and the sample was inverted several times until a precipitate formed. The precipitate was removed from the solution using a glass pipette and residual alcohol removed by allowing the precipitate to air dry for 2-5 minutes. The precipitate was resuspended in 400 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH adjusted to 8.0).

Example 2 Isolation of AsnS polynucleotide sequences via ligation-independent cloning and Gateway® methods and maize transformation

[000107] This example illustrates the isolation of polynucleotide molecules encoding AsnS using ligation and Gateway® independent cloning methods and the construction of DNA constructs of the present invention comprising polynucleotide molecules encoding AsnS polypeptides isolated from various sources of plants and microorganisms as described in Table 1. The promoter molecules used to direct expression of the linked AsnS-encoding polynucleotide molecules are the rice actin promoter 1, P-Os.Act1 (US Patent 5,641,876, incorporated herein for reference purposes); the Zea mays PPDK (Matsuoka et al., 1993), P-RTBV-1 (US Patent No. 5,824,857, incorporated herein by reference) and P-Zm.NAS (genomic region promoter molecule encoding a polypeptide of maize nicotianamine synthase 2).Table 1. Source of AsnS coding sequence, promoter and DNA constructs

[000108] Ligation-independent cloning was developed to clone PCR products and is based on the annealing of non-palindromic single-strand ends. LIC is an efficient cloning method that is not limited by restriction sites or the need for restriction of enzyme digestion or ligation reactions and leaves junctions unstitched (Aslanidis and de Jong, 1990).

[000109] Terminal, single-stranded DNA segments are produced in the vector through the use of a "cutting endonuclease" and restriction endonuclease. A nicking endonuclease is an endonuclease that cuts one strand of the polynucleotide duplex to create single strands in the cloning vector. The vector is first linearized with a standard restriction endonuclease. This is then followed by digestion with a nicking endonuclease. After heat treatment, single-stranded DNA segments are produced in the vector. A GC content of plus or minus 55% is recommended for downstream PCR amplification and efficient annealing. The promoter, tag, or other sequence element can be added to the 5' and 3' ends of the PCR-amplified product to create a linear construct that can be used in downstream applications.

[000110] The DNA construct pMON92870 was assembled from the base vector, pMON82060, and a maize AsnS3 polynucleotide molecule encoding an AsnS polypeptide provided as SEQ ID NO: 5. The backbone of the plasmid pMON82060 was linearized using the restriction endonuclease, HpaI. The plasmid backbone was then treated with the nicking endonuclease, N.BbvC IA (New England Biolabs, Beverly, MA). After digestion, the reaction was heated to 65°C. This causes the cut strands of DNA to dissociate from their complementary DNA strands. The resulting linearized plasmid backbone was left with two terminal, single-stranded DNA segments available for assembly.

[000111] The polymerase chain reaction was employed to produce the terminal single-stranded DNA segments in the DNA molecule encoding AsnS. The maize AsnS3 polynucleotide sequence (SEQ ID NO: 5) encoding the AsnS polypeptide was used to prepare the forward PCR primer (SEQ ID NO: 48) and reverse PCR primer (SEQ ID NO: 49). SEQ ID NO: 48: GCAGTCGCTGTCGTTACCCGGCATCATGGTTGGC ATC SEQ ID NO: 49: GCGAGTACCGCTGGGTTCTAACGTACTCTCGTCAG AC CGCG

[000112] Polymerase chain reaction amplification was performed using the high-fidelity thermal polymerase, KOD hot start DNA polymerase (Novagen, Madison, WI). The polymerase chain reaction was performed in a 25 μL volume containing: 1X KOD hot start DNA polymerase buffer, 1 M betaine (Sigma, St. Louis, MO), 1 mM MgSO4, 250 μM dNTPS, 5 pmols of each primer and 1 unit of KOD hot start DNA polymerase. The polymerase chain reaction was performed on a PTC-225 DNA Engine Tetrad® thermal cycler (MJ Research Inc., Waltham, MA) using the following cycler parameters: 1. 94°C for 2 minutes 2. 94°C for 15 seconds 3. 70°C for 30 seconds (-1°C per cycle) 4. 72°C for 5 minutes 5. Go to step 2, 9 times 6. 94°C for 15 seconds 7. 60°C for 30 seconds 8. 72°C for 5 minutes 9. Go to step 6, 24 times 10. 72°C for 10 minutes 11. maintain 10°C 12. end

[000113] A second round of polymerase chain reaction was performed to introduce uridine residues into the region where the terminal, single-stranded DNA segments were produced. Many DNA polymerases are unable to read uridine residues on the DNA template strand or are unable to polymerize strands using uridine residues. Polymerase chain reaction was then performed using an enzyme capable of incorporating and reading uridines (Expand High Fidelity® plus PCR System; Roche, Indianapolis, IN). Modification of this method and use of other methods that provide the expected result are known to those skilled in the art.

[000114] The assembled DNA construct was transformed into E. coli ElectroMAX® DH10B competent cells (Invitrogen, Carlsbad, CA). A 0.5 μL (microliter) aliquot of the assembly reaction was mixed with 20 μL of ElectroMAX® DH10B competent cells on ice and loaded into a 0.2 mm MicroPulser electroporation crucible (Bio-Rad Laboratories, Inc., Hercules , CA) for electroporation. Cells were electroporated at 1.8 kV using a 165-2100 MicroPulser Electroporator (Bio-Rad Laboratories, Inc.). The electroporated cells were incubated in 180 μL of SOC medium (Invitrogen, Inc.) at 37°C for 1 hour. Cells were then plated on LB agar plates containing spectinomycin (75 mg/mL) and cultured overnight at 37°C. Colonies were selected and grown in LB medium overnight at 37°C. The plasmid DNA construct was isolated using the QIAprep® Spin Miniprep Kit (QIAgen Sciences, Valencia, CA). DNA sequencing was performed on an ABI 3730x1 DNA Analyzer, using BigDye® terminator (Applied Biosystems, Foster City, CA).

[000115] Cloning of polynucleotide sequences encoding corn AsnS AsnS2, soybean AsnS and yeast AsnS1 was performed using the Gateway® cloning method as described by the manufacturer (Invitrogen Corp.). The goal of the Gateway® cloning method is to make an expression clone. This two-step process involves first cloning the gene of interest into an input vector, followed by subcloning the gene of interest from the input vector into a destination vector to produce an expression vector. Cloning technology is based on the site-specific recombination system used by lambda phage to integrate its DNA into the E. coli chromosome.

[000116] DNA constructs for use in subsequent recombination cloning, two attB or attR recombination sequences were cloned into a recombinant vector flanking a Spectinomycin/Streptomycin resistance gene (SPC/STR) and a polynucleotide sequence encoding AsnS. The polynucleotide sequences encoding AsnS were isolated from cDNA or genomic libraries made from their respective species using the primary and secondary primer sequences (SEQ ID NOs: 20-43). The contiguous attB1/R1, SPC/STR gene, AsnS gene and att-B2/R2 sequences were moved as a single polynucleotide molecule into a recombinant construct for expression in plant cells, double-stranded DNA plasmids called pMON79706 (AsnS2 from Zea mays), pMON79700 (AsnS from Glycine Max) or pMON79653 (AsnS from Saccharomyces cerevisiae). These DNA constructs comprise the right border regions (O-OTH.-RB) and the left border regions (LB) of Agrobacterium, and others described by Herrera-Estrella et al. 1983; Bevan, 1984; Klee et al., 1985, the e35S promoter (P-CAMV.35S, tandem duplicated enhancer U.S. Patent 5,322,938), the attB1/R1 genetic element (O-Lam.attB1/R1), the SPC/STR gene, the respective AsnS coding region (CR), the attB2/R2 genetic element (O-Lam.attB2/R2), potato protease inhibitor terminator II (St.Pis), the Agrobacterium NOS promoter (P-AGRtu ..nos, Fraley et al., 1983), the left edge of Agrobacterium (O-OTH.-LB), the kanamycin resistance gene (CR-OTH.Kan, U.S. Patent 6,255,560), and the origin of replication of E. coli (Ec.ori.ColE).

[000117] The DNA constructs were amplified in Library Efficiency® DB3.1® cells (Invitrogen Corporation) under chloramphenicol selection (25 μg/ml) and kanamycin selection (50 μg/mL) for pMON79706, pMON79700 or pMON79653. Vector DNA was purified from bacterial cultures using a QIAGEN Plasmid Kit (QIAGEN Inc.).

[000118] DNA for pMON79700, pMON79706 and pMON79653 was introduced into corn embryos as described in U.S. Patent No. 5,015,580 using the electrical discharge particle acceleration gene delivery device. For microprojectile bombardment of pre-cultured immature embryos of LH59, 35% to 45% maximum voltage was preferably used. Following microprojectile bombardment, maize tissue was cultured in the dark at 27°C. Transformation methods and materials for manufacturing transgenic plants of the present invention, e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants are described in U.S. Patents 6,194,636 and 6,232,526 and Publication of U.S. Patent Application 20040216189, which are incorporated herein by reference.

[000119] Fertile transgenic corn plants were produced from transformed corn cells by cultivating transformed callus in the appropriate regeneration medium to initiate shoot development and seedling formation. Seedlings were transferred to soil when they were about 7.62 cm (3 inches) tall and had roots (about four to 6 weeks after transfer to medium). Plants were maintained for two weeks in a growth chamber at 26°C, followed by two weeks on a mist bench in a greenhouse. Plants were subsequently transplanted into 18.93 L (5 gallon) pots and grown to maturity in the greenhouse. Reciprocal pollinations were performed with the maize inbred line LH59. Seed was collected from corn plants and used for protein analysis and further breeding activities.

Example 3 Vector construction and maize transformation with AsnS polynucleotide sequences

[000120] Maize AsnS2 (SEQ ID NO: 3, pMON79706, Figure 1) was amplified using PCR (polymerase chain reaction). Reaction conditions for PCR reaction followed the manufacturer's protocol (PE Applied Biosystems, Foster City, CA). Approximately 100 ng of maize DNA, prepared as described above, was amplified using 30 nmols each of forward primer (f) (SEQ ID NO: 32) and reverse primer (r) (SEQ ID NO: 33) and 10 micromoles of each dATP, dCTP, dGTP and TTP, 2.5 units of TaKaRaLa Taq in 1X LA PCR Buffer II (Takara Bio INC., Shiga, Japan). After initial incubation at 94°C for 1 minute, 35 cycles of PCR were performed at 94°C for 45 seconds, followed by annealing at 60°C for 45 seconds, 72°C for 1 minute and 15 seconds, followed by 1 cycle 72°C for 7 minutes.

[000121] Five AsnS2 DNA constructs were made. The first maize AsnS2 construct was made by isolating a 1821 base pair AsnS fragment from pMON79706 via PCR as described above, followed by restriction digestion with XbaI and EcoRI restriction enzymes. The resulting AsnS2 gene was ligated into pMON61560, which had also been digested with XbaI and EcoRI. The resulting scramble vector (pMON66246) was digested with NotI and the insert containing the AsnS2 gene, in operable linkage with the PPDK promoter and RGLUT1 terminator, was ligated into pMON30167, which had also been digested with NotI. Plasmid pMON30167, which contains the EPSPS gene, provides selection with glyphosate. The resulting final plasmid was called pMON66230 (Figure 3).

[000122] A second AsnS2 construct was made using the AsnS2 gene (pMON79706). The construct was made by inserting the XbaI/EcoRI-digested AsnS2 gene into pMON61562, which had also been digested with XbaI and EcoRI, resulting in the AsnS2 gene being in operable linkage with the NAS promoter and RGLUT1 terminator. The resulting plasmid was digested with NotI and ligated into NotI-digested pMON30167. The resulting plasmid was called pMON66229 (Figure 2).

[000123] A third AsnS2 construct was made using the aforementioned AsnS2 gene (pMON79706). The P-FDA promoter used in this construct was isolated from pMON78810 through digestion with NotI and XbaI restriction enzymes. The P-FDA promoter was then ligated into pMON66246, which was previously digested with NotI and XbaL to remove its PPDK promoter. The resulting plasmid was digested with NotI and ligated into NotI-digested pMON30167. The resulting plasmid was called pMON66231 (Figure 4).

[000124] A fourth AsnS2 construct was made using the aforementioned AsnS2 gene (pMON79706). The P-RTBV promoter to be used in this construct was generated through PCR from pMON74576. The 721 bp fragment was digested with NotI and XbaI and ligated into pMON66246, which was previously digested with NotI and XbaI. The resulting plasmid, containing the AsnS2 gene in operable linkage with the P-RTBV promoter and RGLUTI terminator was digested with NotI and ligated to NotI-digested pMON30167. The resulting plasmid was called pMON66239 (Figure 5).

[000125] A fifth AsnS2 construct was made using the aforementioned AsnS2 gene (pMON79706). A primer pair of ZmASense, 5'TCCTAGACATGTCCGGCATACTTGCTG3' (SEQ ID NO: 46), and ZmASantisense, 5'TGCAGAATTCTATCCCTCGATGG; (SEQ ID NO: 47), was used to amplify maize AsnS2 from pMON66240. PCR adjustment was as follows: in a total volume of 50 μl of PCR reaction, 1 μl of 10 mM each of ZmASense and ZmASantisense primers, 0.2 to 0.5 μg (1 μl) of pMON66240 plasmid DNA , 5 μl of 10X AccuPrime® Pfx Reaction Mix, 1 μl of ACCuPrime® Pfx DNA Polymerase (Invitrogen), and 41 μl of distilled water. The PCR reaction was performed with the following cycling parameters: 94°C for 1 minute, followed by 30 cycles of 94°C for 15 seconds for denaturation; 58°C for 15 seconds of annealing and 68°C for 4 minutes; followed by 10 minutes of extension at 68°C. The PCR product was purified using a QIAGEN PCR purification kit (QIAGEN Inc.). An aliquot of the maize AsnS2 PCR product was digested with restriction enzyme NcoI and EcoRI and another aliquot of the PCR product was digested with AflIII and NcoI. The NcoI and EcoRI fragment was then cloned into the NcoI and EcoRI sites of pMON94901. The 5' end fragment of AflIII and NcoI of maize AsnS2 was cloned into the NcoI and EcoRI of the maize AsnS2 fragment at the NcoI site. The resulting plasmid (pMON74940), containing maize AsnS2 in operable linkage to the e35S promoter and Hsp17 terminator, was digested with NotI and ligated to NotI-digested pMON53616 to construct pMON74946.

[000126] Each construct described above contained an expression cassette for expression of a glyphosate-insensitive Type II EPSPS as a means for selection of transgenic events (U.S. Patent 5,633,435). The nucleic acid sequence of each construct was determined using standard methodology as described by PE Applied Biosystems BigDye terminator v.3.0 (PE Applied Biosystems, Foster City, CA) and the integrity of the cloning junctions confirmed. The vectors pMON66229, pMON66230, pMON66231, pMON66239 and pMON74946 were used in the subsequent transformation of maize cells and regeneration of these cells into intact maize plants. Constructs of interest were introduced into immature embryos of the corn line LH244 via an Agrobacterium-mediated transformation method, for example, as described in U.S. Published Patent Application 20050048624.

Example 4 Protein and amino acid analysis of corn seed samples

[000127] This example shows a protein and amino acid analysis method for selecting seed of the present invention with increased asparagine and protein using HPLC and near-infrared measurements. For seed protein analysis, small raw samples consisting of 50 to 100 seeds for each treatment were measured using near-infrared transmittance spectroscopy (model Infratec 1221, Tecator, Hoganas Sweden). This procedure was based on the observation that a linear relationship exists between the absorption of near-infrared radiation and the amount of chemical constituents comprised in a typical seed sample. Prior to analysis of unknown samples, spectral data were collected with calibration samples that were subsequently analyzed using a primary analysis technique. The primary technique used was nitrogen combustion (Murray and Williams, 1987). A multivariate model was developed using the spectrometer spectral data and the primary data. In the present case, a multivariate PLS-1 (Partial Least Squares Regression Type I) model was constructed using 152 calibration samples. Each unknown sample was scanned on the spectrometer at least five times and its protein content predicted with each scan. Each time the sample was swept, it was added back to the sample pan to provide an accurate representation of the sample tested. Predicted protein values were averaged across multiple scans and then reported for each sample.

[000128] Free amino acid analysis was performed on corn tissues using HPLC. For each sample, 20-50 mg of lyophilized tissue was extracted with 1.5 mL of 10% trichloroacetic acid in 2 mL microfuge tubes. Samples were extracted at room temperature overnight with gentle shaking. The extracted samples were cleaned by centrifugation and the supernatant was removed for further analysis. Free amino acid analysis was performed via HPLC on an Agilent Series 1100 HPLC with a fluorescence detector and 96-well plate autosampler equipped with a Zorbax Eclipse AAA C18 column (4.6 x 75 mm, 3.5 microns, Agilent Technologies , Palo Alto, CA) and Zorbax Eclipse AAA analytical guard column (4.6 x 12.5 mm, 5 microns). Samples were pre-derivatized with o-phthalaldehyde immediately before separation. Free amino acids were separated with a 40 mM phosphate buffer, pH 7.6/Methanol/Acetonitrile gradient followed by fluorescence detection at 340 nm/450 nm (excitation/emission). Free amino acids were quantified based on external amino acid standards and peaks were integrated with ChemStation software (Agilent). Relative standard deviations were typically less than 8%.

Example 5 Field evaluation of asparagine levels and grain protein content in transgenic corn plants

[000129] This example shows the result of a field evaluation of the effects of corn AsnS constructs (pMON79706 and pMON92870) on asparagine and protein levels in transformed corn plants and seed; the effects of maize AsnS constructs (pMON79700 and pMON79653) on grain protein content. The relative concentration of free asparagine in maize tissues was obtained from inbred lines of R0 maize plants transformed with pMON79706 or pMON92870. For pMON79706, R0 transformants were backcrossed to the parent inbred, LH59, to create BC1 seed. The BC1 seed, which segregates with the transgene, was planted in a field nursery and individual plants were scored for the presence of the NPTII marker gene. Leaf tissue was collected for amino acid analysis for transgene-positive and transgene-negative plants for each transgenic event for free amino acid analysis. Leaf-free amino acids from pMON79706 transgenic plants were compared to isoline-negative plants within each event and statistically analyzed using the Student's T test with JMP 5.1 software (SAS Institute, Cary, NC). For pMON92870, R0 transformants were backcrossed to the parent inbred, LH244, to create BC1 seed. The BC1 seed was planted in a field nursery and self-pollinated to create the BC1S1 seed, which was subsequently planted in a second inbred nursery. Transgene-positive plants were identified for each transgenic event following classification for the presence of the NPTII marker gene. Leaf tissue was collected from transgene-positive BC1S1 plants and parent inbred plots planted at regular intervals in the nursery. Sheet-free amino acids for pMON92870 were statistically analyzed by performing analysis of variance and comparing transgenic releases with source control by conducting Student's T test using SAS 9.1 software. For free amino acid analyzes for both constructs, leaf tissue was collected by removing a fully opened upper leaf at anthesis followed by freezing on dry ice. Leaf samples were frozen ground, freeze-dried and measured for amino acid content via HPLC.

[000130] Multiple transgenic events of pMON79706 and pMON92870 were observed to show substantial increases in leaf asparagine content (Table 2). Four of seven pMON79706 events tested showed significant increases in leaf asparagine concentration, as indicated by a p value of 0.05 or less. In transgenic events of pMON92870, expression of a second maize asparagine synthetase gene, four of five events showed significant increases in leaf asparagine levels (Table 2). These data show that transgenic expression of maize AsnS2 and maize AsnS3 under rice actin promoter in pMON79706 and pMON92870, respectively, can result in a specific increase in free asparagine, which is in accordance with overexpression of active asparagine synthetase.

[000131] The relative concentration of protein in corn seed was obtained from inbred lines derived from R0 corn plants transformed with pMON79706 or pMON92870. BC1 transgenic plants from pMON79706 (described above) were self-pollinated and the resulting BC1S1 grain was grown to maturity and measured for protein content via single ears. Protein was measured as a percentage of dry weight at 0% moisture. Grain protein for pMON79706 transgenic plants was compared to isoline-negative plants within each event and statistically analyzed using the Student's T test with SAS 9.1 software. For pMON98270, BC1S1 plants were self-pollinated to grow to maturity and measured for protein content via single ears. Grain protein for pMON92870 was statistically analyzed with a spatial method developed as usual by conducting "by-location" analysis. "By-location" analysis is a two-step process. The first step in the analysis involved estimating spatial autocorrelation in the field by fitting an anisotropic spherical semivariogram model using all spatial check plots that were systematically placed in the field (every 6th plot). The second stage of analysis involved adjusting the transgenic release values for spatial variability using the spatial autocorrelation structure estimated in the first stage of the analysis. Following adjustment for spatial autocorrelation, mean comparison was performed where the mean value of a transgene release was compared to the source control to test the statistical significance of the difference between the mean of the transgene and the control.

[000132] Multiple events of both pMON79706 and pMON92870 showed significant increases in protein content in the inbred grain (Table 3). Three of five pMON79706 events that were statistically analyzed showed significant increases in grain protein content (p <0.05) and two other events showed a trend towards significant increases (p <0.15). Two events did not return sufficient numbers of ears for statistical analysis. Three of four pMON92870 transgenic events showed significant increases in grain protein content (p < 0.1), with one event not tested due to insufficient numbers of ears for analysis. These data confirm that pMON79706 and pMON92870 produce transgenic events that increase grain protein content in maize in addition to increasing leaf asparagine content.

[000133] pMON79706 with a phenotype of high asparagine and protein in the grain was confirmed in multiple tissues in a second test. After the BC1 generation, five pMON79706 events were self-pollinated in the next two nurseries to generate BC1S3 seed that was homozygous for the transgene. The relative concentration of asparagine resulting from expression of the pMON79706 construct was determined in a study on the V8 growth stage of maize by comparing homozygous BC1S3 plants and a LH59 maize variety control (Table 4). Transgenics and controls were planted in a randomized complete block design with 5 replicate blocks in a field plot. The fully opened upper leaves and stem sections from two plants were sampled and pooled, placed on dry ice, ground, freeze-dried, and measured for amino acid content via HPLC. Values followed by "*" indicate a significant difference from the LH59 control (Dunnet's one-sided test; (SAS 9.1, Cary, NC). Asparagine measurements obtained at both the V8 growth stage and the R1 generation showed that plants from five growth events pMON79706 had significant increases in free asparagine. Relative free asparagine levels in V8 leaf tissue were increased to up to 13.9% compared to 3.4% in the LH59 control, and stem asparagine was increased to 39% compared to LH59. 9.6 in the control (Table 4). For grain protein analysis, 10 ears were sampled per batch, scaled and analyzed for grain protein concentration also increased significantly in five pMON79706 events (Table 4). The results show that, as a general trend, events producing a significant increase in asparagine also produced a significant increase in seed protein (Tables 2-4). Table 4. Relative asparagine concentrations at the V8 growth stage and protein concentration. in the grain at maturity in BC1S3 maize plants transformed with the maize AsnS2 gene (pMON79706).

[000134] Significant increases in hybrid grain protein were observed for three different constructs expressing asparagine synthetase genes under the rice actin promoter. Homozygous inbred corn lines were produced from R0 transgenic events of pMON79706 (corn AsnS2), pMON79700 (soybean AsnS), and pMON79653 (yeast AsnS1) through the first R0 backcross event to the recurrent origin, LH59, followed by self-pollinations of transgene-positive selections in two subsequent inbreeding nurseries using the NPTII selectable marker to classify zygosity. The homozygous events for each construct were then used as a male pollen donor in a cross with a female inbred line to create the F1 hybrid. The F1 hybrid seed was planted in a multiple location test and transgenic events for each construct were analyzed for protein in the final grain and compared to the control hybrid of recurrent origin following a spatial correction analysis based on protein in the grain in control hybrids. which were planted at regular intervals across the field. Grain was collected from each batch, flaked and analyzed for protein content. Data were analyzed using a spatial method developed as usual by conducting a "by-location" and an "across location" analysis. "By-location" analysis is a two-step process. The first step in the analysis involved estimating spatial autocorrelation in the field by fitting an anisotropic spherical semivariogram model using all spatial check plots that were systematically placed in the field (every 3rd plot). The second stage of analysis involved adjusting GMO release values for spatial variability using the spatial autocorrelation structure estimated in the first stage of analysis. Following adjustment for spatial autocorrelation at each location separately, an "across-location" analysis was conducted where the mean value of a transgenic release was compared with the source control to test for statistical significance (P=0.20) between the mean of the transgene and the control. All five pMON79706 events showed significant increases in grain protein in the hybrid test, consistent with the observation that grain protein was increased in the inbred lines of transgenic events of this construct (Table 5). Two other asparagine synthetase constructs, pMON79700 (soybean AsnS) and pMON79653 (soybean AsnS), also showed significant increases in grain protein levels in two of five events and two of two events, respectively.

Example 6 Field evaluation of transgene expression and asparagine synthetase enzyme activity due to pMON79706 and pMON92870

[000135] Transgene expression was confirmed in pMON79706 and pMON92870 transgenic events. For pMON79706, tissue used for determining leaf asparagine content in the field test with BC1S3 homozygous inbred lines was also used for determining transgene expression based on measurement of expression from the 3' terminator sequence (St.Pis4) of pMON79706 at anthesis. Two leaf samples were collected and pooled from each of 5 replicate batches (10 for inbred control) and frozen on dry ice. Leaf samples were then frozen ground for expression analysis. For RNA extraction, 50 mg of frozen tissue was aliquoted into 96-well plates. Each sample was extracted with 500 μl of lysis buffer containing a 1:1 solution of ABI nucleic acid lysis solution (Applied Biosystems, Foster City, CA) to 1X PBS pH 7.4 (no MgCl or CaCl ). RNA was extracted from freshly frozen tissue samples using filter plates to capture nucleic acids from crude lysates, and 50 μl of ABI elution buffer was used to elute bound RNA. Quantitative PCR was performed using an RNA template with 5 μl of ABI one-step RT-PCR reagent. Reactions were performed for 40 PCR cycles on an ABI Taqman 7900 PCR instrument, with cyclization parameters of 48°C for 30 minutes, 95°C for 10 minutes, 95°C for 10 seconds, 60°C for 1 minute . Fluorescent measurements were obtained from each well in each of 40 cycles for both the terminator sequence derived from potato protease inhibitor II (St.Pis4) and the endogenous control (ubiquitin). A subset of samples was administered without reverse transcriptase to monitor DNA contamination. Samples were scored for relative expression by subtracting the cycle threshold values for St.Pis4 from the cycle threshold value of the endogenous control. The cycle threshold (Ct) was determined, and the Ct delta was calculated from the St.Pis4 minus endogenous control value. An in situ wild type was created by calculating the average endogenous control signals and setting the St.Pis4 signal value to 40. The Ct delta of the unknown samples was subtracted from the Ct delta of the in situ wild type. Final data were reported as expression of pinII (St.Pis4) relative to wild type. Quantitative RT-PCR analysis confirmed transgene overexpression from six of six pMON79706 events (Figure 6).

[000136] Transgene expression was also confirmed in inbred events comprising pMON92870. RNA expression was determined from leaf tissue at anthesis of inbred plants in a field nursery by first collecting an open upper leaf from each plant (4-8 plants per event) and freezing on dry ice. Transgene-positive plants were previously identified based on the presence of the NPTII marker gene. Leaf tissue was ground while frozen and analyzed for expression of the 3' terminator sequence (St.Pis4) of pMON92870. Quantitative RP-PCR analysis showed that five of six events comprising pMON92870 showed increased transgene expression compared to an inbred control (Figure 7). The low RNA expression in pMON92870 event ZM_M103316 is in accordance with the low leaf asparagine content and low grain protein content in this event.

[000137] The effect of expression of asparagine synthetase genes on asparagine synthetase activity was measured in transgenic events of pMON79706 and pMON92870. Frozen, ground leaf tissue was separated into aliquots (200-400 mg) into wells of a pre-chilled deep 96-well plate. Protein was extracted in Buffer A (100 mM Hepes-OH, pH 8.0, 0.1 mM EDTA, 10 mM MgCl2, 2 mM aspartate, 0.5 mM DTT, 67 mM mercaptoethanol, glycerol 20% (v/v), 0.1 mM ATP, 1% (v/v) P9599 (Sigma Company), 25 mM KCl). A small amount of sand was added to each cavity. Buffer A was then added to the leaf tissue in the wells at a ratio of 4:1 (buffer:tissue). The plates were then shaken on a paint shaker for 2 minutes to mix the sample and then centrifuged at 5000 x g for 10 minutes. The supernatant (100-200 μL) was desalted in a 96-well macrospinning plate (SNS S025L, The Nest Group Inc., Southboro, MA) equilibrated in buffer A. The supernatant was then either assayed immediately or frozen in liquid nitrogen and kept at -80°C until used. To assay asparagine synthetase activity, desalted protein extracts (10-50 μL) were added to wells containing 100 μL of assay solution (100 mM Hepes, pH 8.0, 10 mM MgCl2, 2 mM aspartate, DTT at 5 mM, 10 mM ATP, 1 mM amino(oxy)acetic acid (aspartate amino transferase inhibitor), 1 mM aspartic semialdehyde (asparaginase inhibitor). To initiate the reaction, glutamine (final concentration of 2 mM for). standard assay) was added to the solution, which was then mixed. The assay mixture was then incubated for 1 to 2 hours. The reaction was then stopped by adding an equal volume of 20% (w/v) trichloroacetic acid. The mixture was then filtered to remove precipitate and asparagine was measured via HPLC. Sample size was increased from 0.5 μl to 2.5 μL for HPLC, excitation wavelength was reduced from 340 nm to 235 nm and gain. fluorometer was increased from 10 to 13. This results in a detection sensitivity of 0.5 to 100 mM asparagine and allows measurement of activity levels in the 100s of microunits.

[000138] For pMON79706, tissue used to determine the activity of the asparagine synthetase enzyme in the leaf was from a field test with BC1S3 homozygous inbred lines collected at the V7 growth stage. Events from pMON79706 were shown to exhibit increased leaf asparagine synthetase activity (Table 6). Asparagine synthetase activity was increased up to 5-fold over the inbred control. Asparagine synthetase enzyme activity was also determined for pMON92870 transgenic events in an inbred field nursery at the time of anthesis. Four of five pMON92870 events also showed increased enzyme activity (Table 6). The increased asparagine synthetase enzyme activity in maize plants expressing maize AsnS2 (pMON79706) or maize AsnS3 (pMON92870) under the rice actin promoter is in agreement with the increase in gene expression and increases in leaf asparagine observed. with these constructs. Table 6. Asparagine synthetase activity in inbred lines from pMON79706 and pMON92870a transgenic events. a Enzyme activities for pMON79706 and pMON92870 were determined from two different field experiments.

Example 7 Field evaluation of the effects of pMON66231, pMON66239 and pMON74946 on asparagine and protein content in grain

[000139] The relative content of free asparagine in corn tissues was obtained from hybrid lines derived from R0 corn plants (LH244 base) transformed with pMON66231 (Figure 4), where corn AsnS2 is under the control of the corn FDA promoter. Hybrids were made by crossing R0 plants with the male inbred line LH59, which creates a segregating F1 population (1:1). The resulting F1 seed was planted at three locations in the Midwest with replicates at each location. Batches were sprayed with glyphosate at the V3 growth stage to eliminate null segregants. A hybrid control was planted on the perimeter and comparisons were made with the hybrid control. Top leaves were collected and pooled from the three plants within each plot at the time of anthesis, two hours after sunset, at all three locations. The leaves were immediately placed on dry ice and then stored at -80°C until processing. The leaves were ground frozen, and a portion was lyophilized for free amino acid analysis by HPLC. Data were first evaluated for outliers with the two-step method for deleted studentized residuals using Bonferroni-adjusted p-values. Externals were identified and removed from data fitting before analysis of variance calculations began. Data were analyzed according to a randomized full block design "across-locations". Construct-event combinations were modeled with fixed effects, and sites and replicates within sites were modeled with random effects. Treatment comparisons were made by contrasting the least squares means of the construct-event combinations. Relative leaf asparagine was significantly increased in 11 of 12 pMON66231 events, with asparagine levels as high as 16% compared with 3% in the control (Table 7). Mature grain protein was also measured following the collection of 10 ears per batch followed by hulling and seed grouping for each batch, which was then measured for protein content in the grain. Nine of 12 events were found to significantly increase the protein content in mature grain over the LH244/LH59 hybrid control. Table 7. Relative leaf asparagine and mature grain protein content in pMON66231 transgenic events. a Relative free asparagine measured as a percentage of total free amino acids in leaf tissue b Compared to hybrid control.

[000140] The relative content of free asparagine in corn tissues was obtained from hybrid lines derived from R0 corn plants (LH244 base) transformed with pMON66239 and pMON74946, in which corn AsnS2 is under control of the RTBV or e35S promoter, respectively. The hybrids were made by crossing the R0 plants with the male inbred line, LH59, which creates a segregating F1 population (1:1). The resulting F1 seed was planted at a site in Hawaii with three replicates for each transgenic event. Batches were sprayed with glyphosate at the V3 growth stage to eliminate null segregants. A hybrid control lacking the maize AsnS2 gene was included for comparison. Top leaves were collected and pooled from three plants within each plot at the time of anthesis, two hours after sunset. The leaves were immediately placed on dry ice and then stored at -80°C until processing. The leaves were ground frozen and a portion was lyophilized for free amino acid analysis using HPLC. Data were first evaluated for outliers with the two-step method for deleted studentized residuals using Bonferroni-adjusted p-values. Externals were identified and removed from the data fit before the analysis of variance calculations was initiated. Data were analyzed according to a randomized complete block design. Construct-event combinations were modeled with fixed effects, and replicates were modeled with random effects. Treatment comparisons were made by contrasting the least square means of the construct-event combinations. Relative leaf asparagine was significantly increased in 10 of 13 pMON74946 events, with asparagine levels as high as 16% compared with 2% in the control (Table 8). Mature grain protein was also measured following the collection of all ears per batch followed by flaking and seed grouping for each batch, which was then measured for protein content in the grain and statistically analyzed as the asparagine trait in the leaf. Ten of thirteen events were found to have significantly higher protein content in the mature grain compared to the hybrid control, and the same 10 events with more asparagine in the leaf also showed more protein in the hybrid test. For pMON66239 transgenic events, 11 of 15 events showed increases in leaf asparagine content and 3 of 15 events showed significant increases in grain protein at the alpha 0.05 level, although five more transgenic events showed more protein at p<0. 15, indicating that expression of maize AsnS2 under the RTBV promoter (pMON66239) can increase leaf asparagine content and seed protein content, but to a lesser degree than under the e35s promoter (pMON74946) (Table 8). Table 8. Relative leaf asparagine and mature grain protein content in transgenic events pMON74946 and pMON66239 a Relative free asparagine measured as a percentage of total free amino acids in leaf tissue b Compared to hybrid control.

Example 8 Bacterial expression vectors, purification and kinetics of AsnSI, AsnS2, AsnS3 and AsnS4 isoforms

[000141] This example describes the cloning of the nucleotide sequence for AsnS1 (SEQ ID NO: 1), AsnS2 (SEQ ID NO: 3), AsnS3 (SEQ ID NO: 5) and AsnS4 (SEQ ID NO: 50) into vectors of E. coli expression, as well as the expression, purification and kinetics of recombinant forms of the four AsnS isoforms.

[000142] Bioinformatics searches using a published maize AsnS gene sequence (Chevalier et al., 1996; gi984262) resulted in the identification of four full-length cDNA sequences in the proprietary's internal cDNA collections that were identified as sharing similarity of significant sequence with the published maize AsnS gene. Two of the full-length cDNAs that share sequence identity with the public AsnS sequence were named Zm-AsnS1 and Zm-AsnS3. The two other genes were called Zm-AsnS2 and Zm-AsnS4 (SEQ ID NO: 50), respectively. Cloning of Zm-AsnS1, Zm-AsnS2 and Zm-AsnS3 sequences into plant expression vectors is described in Examples 2 and 3 above. These three genes as well as Zm-AsnS4 were cloned into bacterial expression vectors as follows:

[000143] The full-length coding sequences Zm-AsnS1, Zm-AsnS3 and AsnS4 were amplified via polymerase chain reaction (PCR) from cDNA clones 700151670_FLI, LIB5399-001-A2 and LibLIB3732-039-F9_FLI, respectively , in the owner's internal cDNA collections. Sequences of the forward and reverse primers for PCR-amplified fragments encoding Zm-AsnS1 were: Zm_AsnS1_for1 5'-ggaattccatATGTGTGGCATCTTAGC-3' (SEQ ID NO: 52) and Zm_AsnS1_rev1 5'-ataagaatgcggccgcGACCGCGATCGCGATT GCGA CA-3' (SEQ ID NO: 53 ) Forward and reverse primer sequences used for PCR amplification of Zm-AsnS3 were: Zm_AsnS3-for2 5'-ctagctagctagATGTGCGGCATCCTC-3' (SEQ ID NO: 54) and Zm_AsnS3-rev2 5'-ccgctcgagGACAGCTGTGGCTGAAGCAACG-3' (SEQ ID NO: 55). A PCR-amplified fragment encoding the full length of AsnS4 (SEQ ID NO: 50) was obtained with the following primer pair: Zm-AsnS4_for3 5'-gggaattccatATGTGTGGCATCTTAGC-3' (SEQ ID NO: 56) and Zm-AsnS4_rev3 5 '-ataagaatgcggccgcCACCGCGATCGCGACAG CGA- 3' (SEQ ID NO: 57). The full-length coding sequence of Zm-AsnS2 was amplified via polymerase chain reaction (PCR) from pMON79706 (Figure 1; SEQ ID NO: 3). Primer sequences for PCR amplification of Zm-AsnS2 were: Zm-AsnS2_for4 5'-tatgtgcggcatacttgctggctcgggt-3' (SEQ ID NO: 58) and Zm-AsnS2_rev4 5'-gctatccctcgatggcaacgccagat-3' (SEQ ID NO: 59).

[000144] PCR was performed in a total volume of 50 μL using the Expand® High Fidelity PCR kit (Boehringer Mannheim, Germany). The reaction was carried out in a PTC-200 Peltier thermal cycler (MJ Research Inc., Watertown, Massachusetts) under the following conditions: Initial incubation: 5 minutes at 95°C 27 cycles of: 1 minute at 95°C annealing of 1 minute at 56°C followed by: 2 minute extension at 72°C. incubation for 10 minutes at 72°C.

[000145] The resulting PCR fragments were subcloned into one of the expression plasmids pET30a(+) or pET21(+) (Novagen, San Diego, CA), giving plasmids pET30a-Zm-AsnS1, pET30a-ZmAsnS2, pET30a-Zm- AsnS3 and pET21d-Zm-AsnS4. Plasmid sequences were confirmed through DNA sequence analysis. These vectors are expected to produce recombinant proteins with C-terminal histidine tags.

[000146] The E. coli expression vectors described in the previous paragraph were used to transform E. coli Rosetta DE5® cells (Novagen). Transformed cells were transferred from solid LB media plates and cultured in liquid LB media containing standard concentrations of kanomycin (50 μM) and chloramphenicol (25 μM) at 37°C. An overnight culture was used to inoculate 25 mL of culture, which was grown at 37°C to semi-log phase (OD600 = 0.4 - 0.6). Cells were then transferred to 20°C for 30 min, after which IPTG was added to a final concentration of 0.5 mM. Cells were then cultured at 20°C for 12 hours and cell pellets were collected via centrifugation.

[000147] The protein was recovered from the insoluble fraction while attached to a nickel column. Buffers used for the purification of recombinant AsnS were:

[000148] Buffer A: 100 mM Hepes-OH, pH = 7.6, 0.1 mM EDTA, 10 mM MgCl2, 2 mM aspartate, 0.5 mM DTT, 20% glycerol (v/ v), 0.1 mM ATP, 1% (v/v) protease inhibitor cocktail (Sigma Product No. P9599), 25 mM KCl. Buffer B: (Buffer A + 6 M Guanidine-HCl) Buffer C: (Buffer A + 6 M Urea) Buffer D: (Buffer D + 4 M Urea) Buffer E: (Buffer A + 2 M Urea) Buffer F: (Buffer A + 1 M Urea) Buffer G: (Buffer A + 500 mM imidazole)

[000149] All purification procedures were carried out at 4°C unless otherwise stated. E. coli pellets were solubilized in Buffer A and passed through a French Press three times at 110.32 MPa (16,000 PSI). The sample was then centrifuged at 75,000 x g for at least one hour. The pellet (inclusion body) was frozen in liquid N2 and then stored at -80°C until use.

[000150] All routine kinetic measurements for plant AsnS enzymes were performed on his-tagged forms of plant AsnS that had been refolded on the nickel column during purification. An E. coli pellet resulting from bacteria expressing one of the AsnS forms in 200 mL of LB media as described here was used as the starting material. Inclusion bodies were isolated using the method described in the Pierce Pro-Matrix® Protein Refolding Guide (Thermo Fisher Scientific, Rockford, IL, Product No. 89867; Appendix A) and then solubilized using the Pierce Pro-Matrix Protein Refolding Guide protocol ( Product No. 89867; Appendix B) except that buffer B was used in place of 6-8 M GdnHCl, 50 mM Tris. The solubilized sample was then applied at a flow rate of 1 mL/min to a 3-5 mL Nickel NTA agarose column (Qiagen) previously equilibrated in Buffer B. The column was then successively washed at 1 mL/min with at least minus two column bed volumes of Buffers B, C, D, and E and F. The column was then washed with Buffer A until the OD280 of the column eluent was less than 0.05. The entire washing process was completed within 1-2 hours. The protein was then eluted with Buffer G at 1 mL/min. 2 mL fractions were obtained and the three fractions with the highest amounts of protein were pooled, frozen in liquid N2, and stored at -80°C until assayed as described in Example 6. The levels of each substrate (ATP, glutamine, NH4+ and aspartate) were titrated to determine individual Km and Vmax values. Kinetic parameters measured were Km (Asp), Km (Gln), Km (NH4+) and Vmax, for each substrate, and for some enzymes. The results are reported in Table 9.

[000151] All four genes encode sequences that give rise to polypeptides with AsnS activity. AsnS1, AsnS2 and AsnS3 appear to be kinetically distinct since Km (Gln) for AsnS2 is several times lower than the other enzymes; and the Vmax of AsnS1 is several times lower than the other enzymes. Table 9. Kinetic comparison of corn asparagine synthetase

[000152] All publications, patents and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

BIBLIOGRAPHIC REFERENCE

[000153] The following references, to the extent that they provide procedural or other details supplementary to those shown here, are specifically incorporated herein by reference: U.S. Patent 4,761,373 U.S. Patent 4,957,748 U.S. Patent 5,100,679 U.S. Patent 5,219,596 U.S. Patent 5,322,783 U.S. Patent 5,322,938 U.S. Patent 5,538,880 U.S. Patent 5,550,318 U.S. Patent 5,563,055 and U.S. 5,936,069 U.S. Patent 6,005,076 U.S. Patent U.S. Patent 6,146,669 81-808, 2004. Allard , In: Principles of Plant Breeding, 2nd Ed., John Wiley & Sons, ISBN: 0471023094, 1999. Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997. Aslanidis and de Jong, Nucleic Acids Res., 18(20):6069-6074, 1990. Ausubel et al., eds. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989. Bevan, Nucleic Acids Res., 12:8711-8721,1984. Chu et al., Scientia Sinicα, 18:659, 1975. Dellaporta et al., Plant Molecular Biology Reporter, 1:19-21, 1983. D'Halluin et al., Bio/Technology, 10:309-314, 1992. Fraley et al. others., Proc. Natl. Academic. Sci USA, 80:4803-4807, 1983. Haymes et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC, 1985. Hayward, In: Plant Breeding: Principles and Prospects, Vol. 1, Chapman & Hall , ISBN: 0412433907, 1993. Henikoff and Henikoff, Proc. Natl. Academic. Sci USA, 89: 10915 10919, 1992. Herrera-Estrella et al., Nature, 303:209, 1983. Hinchee et al., Bio/Technology, 6:915-922, 1988. Jones and Shenk, Cell, 13: 181- 188, 1978. Jones et al., MoI Gen. Genet, 210(l): 1-4, 1987. Klee et al., Bio-Technology, 3(7):637-642, 1985. Lewin, In: Genes V, Oxford University Press, NY, 1994. Matsuoka et al., Proc. Natl. Academic. Sci USA, 90:95869590, 1993. Murashige and Skoog, Physiol. Plant, 15:473-497, 1962. Murray and Williams, In: Chemical Principles of Near-Infrared Technology, Near-Infrared Technology in the Agricultural and Food Industries, Williams and K. Norris (Eds.,), 1987. Request for PCT Patent WO 95/06128. Reynaerts et al., In: Selectable and Screenable Markers, Gelvin and Schilperoort (Eds.), Plant Molecular Biology Manual, Kluwer, Dordrecht, 1988. Richards,In: Plant Breeding Systems, Stanley Thornes Pub Ltd; 2nd Ed., ISBN: 0412574500, 1997. Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, New York, 2001. Stalker et al., J. Biol. Chem., 263:6310-6314, 1988. Thillet et al., J. Biol. Chem., 263:12500-12508, 1988.

Claims (7)

1. DNA construct, characterized by the fact that it comprises the nucleic acid sequence comprising the sequence of SEQ ID NO: 50; wherein the nucleic acid sequence is operably linked to a heterologous regulatory element or a heterologous promoter functional in plants. 2. DNA construct according to claim 1, characterized by the fact that the nucleic acid sequence encodes an asparagine synthetase polypeptide. 3. DNA construct according to claim 1, characterized by the fact that said heterologous promoter is a rice actin 1 promoter. 4. Method of producing a transgenic corn plant with high asparagine content, characterized by the fact that it comprises: a) introducing into a corn plant a DNA construct as defined in claim 1; b) cultivation of the plant until maturity; and c) collecting the plant seed. 5. Method according to claim 4, characterized by the fact that said nucleic acid sequence encodes a heterologous asparagine synthetase. 6. Method according to claim 4, characterized by the fact that it further comprises the step of: f) selecting a seed with increased protein content. 7. Method of producing food or feed, characterized by the fact that it comprises obtaining the transgenic plant comprising the DNA construct as defined in claim 1, and producing food or feed from the plant or a part thereof.