MXPA06009201A - Transgenic corn seed with enhanced amino acid content - Google Patents

Abstract

Anti-sense-oriented RNA gene suppression agents in the form of a loop of anti--sense-oriented RNA is produced in cells of transgenic organisms, e.g. plants, by transcription from a recombinant DNA construct which comprises in 5'to 3'order a promoter element operably linked to an anti-sense-oriented DNA element and a complementary DNA element.

Description

TRANSGENIC CORN SEED WITH IMPROVED AMINO ACID CONTENT CROSS REFERENCE TO RELATED REQUESTS This request claims priority under 35 U.S.C. 119 (e) of provisional applications Serial No. 60 / 543,157, filed on February 10, 2004, No. 60 / 543,187, filed on February 10, 2004 and No. 60 / 600,859, filed on August 11, 2004, the descriptions of which are incorporated herein by reference in their entirety.

Incorporation of the sequence listing A computer-readable form of the sequence listing is contained in the file named "53490.ST25.txt", which is 10.7 kb (measured in the MS-Windows Explorer), and was created in February 9, 2005 and is located on a CDROM, which is presented with the present and is incorporated herein as a reference.

FIELD OF THE INVENTION Transgenic maize seeds having a high amino acid level, recombinant DNA constructs to produce turns that suppress the antisense RNA gene and a method for making and using such transgenic constructs and plants expressing the turns that suppress the gene are described herein. gene of an antisense RNA.

BACKGROUND OF THE INVENTION Certain plants have low levels of specific amino acids compared to other plants, for example, corn has low levels of lysine, methionine and tryptophan. Efforts to increase the levels of amino acids in transgenic plants include expressing the recombinant DNA encoding the proteins in a pathway of amino acid synthesis at higher levels than the native genes. One such gene for producing improved levels of lysine in corn is a bacterial dihydropicolinic acid synthase, as described in U.S. Pat. 5,288,300 (Glassman et al.), 6,459,019 (Falco et al.) And the U.S. Patent Application Publication. 2003/0056242 A1, each of which is hereby incorporated by reference in its entirety. A concept for more augmented levels of amino acids includes the suppression of the genes that encode proteins in the catabolic trajectories of amino acids. Deletion of the gene includes any of the well-known methods for suppressing the transcription of a gene or the accumulation of the mRNA corresponding to the gene, thus preventing the transcription of the transcript in the protein. More particularly, the suppression of the gene mediated by inserting a recombinant DNA construct with antisense oriented DNA to regulate the expression of the gene in the cells of the plant, is described in the patent of E.U.A. 5,107,065 (Shewmaker et al.) And the U.S. Patent. 5,759,829 (Shewmaker et al.). Transformed plants using such antisense oriented DNA constructs for gene deletion, may comprise integrated DNA, arranged as an inverted repeat resulting from the co-replacement of several copies of the transferred DNA (T-DNA) in plants by Agrobacterium-mediated transformation, as described by Redenbaugh et al. in "Safety Assessment of Genetically Engineered Flavr Savr ™ Tomato, CRC Press, Inc. (1992)." Repeated inverse inserts may comprise part or all of the T-DNA, eg, contain an inverted repeat of a complete construct or construct. partial antisense The screening for the inserted DNA comprising inverted repeat elements can improve the efficiency of identification of the effective transformation events to silence the gene when the transformation construct is a simple antisense DNA construct.The suppression of the unchained gene inserting a recombinant DNA construct with a sense oriented DNA to regulate gene expression in plants, is described in US Patent 5,283,184 (Jorgensen et al.) and US Patent 5,231,020 (Jorgensen et al.) The inserted T-DNA that provides the suppression of the gene in plants transformed with such constructs sense by Agrobacterium, it is predominantly organized in inverted repeated structures, as described by Jorgensen et al., Mol. Gen. Genet., 207: 471-477 (1987). See also Stam et al., The Plant Journal, 12: 63-82 (1997) and De Buck et al., Plant Mol. Biol. 46 433-445 (2001), which used segregation studies to support Jorgensen's findings that in many events, the silencing of the gene is mediated by a T-DNA of the multimeric transgene, where the T-DNAs are arranged in inverted repetitions. Scanning for the inserted DNA comprising the inverted repeat elements can improve the silencing efficiency of the gene when it is transformed with single sense oriented DNA constructs. Gene silencing can also be effected by transcribing the sense and antisense oriented DNA RNA using two separate transcription units, for example, as described by Shewmaker et al., In the U.S. Patent. 5,107,065, wherein in Example 1, a binary vector was prepared with both sense and antisense aroA genes. Similar constructs are described in International Publication No. WO 99/53050 (Waterhouse et al.). See also the U.S. Patent. 6,326,193, wherein the DNA selected by the gene is operably linked to opposing promoters. Deletion of the gene can be achieved in plants by providing transformation constructs that are capable of generating an RNA that can form double-stranded RNA along at least a part of its length. The deletion of the gene in plants is described in EP 0426195 A1 (Goldbach et al.), Wherein the recombinant DNA constructs for transcription in the hairpin RNA provide transgenic plants with resistance to tobacco blight virus. See also Sijen et al., The Plant Cell, Vol. 8, 2277-2294 (1996), which describes the use of constructs carrying inverted repeats (sense, followed by antisense) of a Chinese vine mosaic virus gene in transgenic plants to mediate the resistance of the virus. See also International Publication No. 98/53083 (Grierson et al.) And the Publication of the Patent Application of E.U.A. Related No. 2003/0175965 A1 (Lowe et al.), which describes the deletion of the gene, using a double-stranded RNA construct, comprising a sequence encoding a gene, preceded by an inverted 5 'UTR repeat. The constructs for the suppression of the posttranscriptional gene in plants by the double-stranded RNA of the target gene are also described in International Publication No. WO 99/53050 (Waterhouse et al) and International Publication No. WO 99/49029 (Graham et al.). See also the Publication of the Patent Application of E.U.A. No. 2002/0048814 A1 (Geller), wherein the DNA constructs are transcribed to sense or antisense RNA with a poly (T) -poly (A) tail forming the hairpin. See also the Publication of the Patent Application of E.U.A. No. 2003/0018993 A1 (Gutterson et al.), Wherein the sense or antisense DNA is followed by an inverted repeat of the 3 'untranslated region of the NOS. See also the Publication of the Patent Application of E.U.A. No. 2003/0036197 A1 (Glassman et al.), Wherein the RNA for reducing the expression of the target mRNA, comprises a part with homology to the target mRNA and a part with complementary regions of the RNA that are not related to the endogenous RNA. The production of dsRNAs in plants to inhibit the expression of the gene, for example, in a nematode that is fed into the plant, is described in the patent of E.U.A. 6,506,559 (Fire et al.). The suppression vectors of multiple genes for use in plants are described in the Application for Patent of E.U.A. No.10 / 465,800 (Fillatti). Transcriptional suppression such as suppression of the trans promoter can be effected by the expression of a DNA construct comprising a promoter operably linked to inverted repeats of the promoter DNA of a target gene. Useful constructs for such gene suppression mediated by suppression of the trans promoter are described by Mette et al., The EMBO Journal, Vol. 241-148, (1999) and by Mette et al., The EMBO Journal, Vol. 19, pp. 5194-5201-148, (2000), both of which are incorporated herein by reference. All the patents, applications and international publications described above, which describe materials and methods for the suppression of the gene in plants, are incorporated herein by reference.

BRIEF DESCRIPTION OF THE INVENTION This invention provides a seed for transgenic corn that has an improved amino acid content. Such transgenic maize with high amino acids in its grains has integrated in its genome a recombinant DNA construct that transcribes an antisense oriented RNA that suppresses the production of a protein in a catabolic pathway of the amino acid. In one aspect of the invention, the seed has recombinant DNA to suppress a gene encoding a protein in a catabolic pathway of lysine, for example, the lysine pre-polymer ketoglutarate reductase / saccharopine dehydrogenase. A useful target protein for suppression is ketoglutarate reductase. The improved amino acid content can also be achieved by concurrently expressing a gene in a pathway of amino acid synthesis, for example, an exogenous gene encoding dihydrodipicolinate synthase in the path of lysine synthesis. Thus, this invention also provides seeds and methods in which recombinant DNA is used to suppress a protein in a catabolic pathway of the amino acid and expresses, for example, overexpressed, a protein in an amino acid synthesis path. Another aspect of the invention provides methods for increasing the content of an amino acid, for example, the lysine content in corn kernels, by expressing in the developing maize seed a recombinant DNA construct to suppress the expression of a protein in a catabolic path of the amino acid, and optionally, to express a protein in the path of synthesis of the amino acid.

The recombinant DNA constructs of this invention comprise an antisense oriented DNA element of a gene selected for deletion. The constructs also comprise sense oriented DNA that transcribes DNA that is complementary to at least a portion of the antisense oriented RNA. In a preferred aspect of the recombinant DNA constructs of this invention, the DNA element oriented in the sense that it is shorter than the antisense oriented DNA element and the RNA oriented in the transcribed sense of the sense oriented DNA element, is complementary to the nearly 5 'part of the antisense oriented RNA transcribed from the antisense oriented DNA element. Such transcribed RNA forms a loop of antisense oriented RNA to suppress at least one target gene for a protein in a catabolic pathway of the amino acid. Recombinant DNA constructs comprise a promoter, eg, a seed-specific promoter, operably linked to DNA that is transcribed to antisense oriented RNA, for example, which forms an antisense oriented RNA loop. Such recombinant DNA is useful for producing a corn seed having a high amino acid content, as compared to the seed progeny of the control corn plants, in which the production of a protein in the catabolic path of the amino acid is not suppressed, for example, an ancestral maize plant of the wild type, or the negative segregant of the transgenic maize plant.

In preferred aspects of the invention, the seed-specific promoter is a specific promoter of the embryo or an endosperm-specific promoter and the recombinant DNA construct produces antisense-oriented RNA to suppress a gene encoding a protein in the catabolic path of the lysine, for example, lysine ketoglutarate reductase and / or saccharopine dehydrogenase. In another aspect of the invention, the amino acid content is improved in the transgenic maize which also has a recombinant DNA integrated in its genome, which expresses a protein in an amino acid synthesis pathway, for example, dihydropicolinate synthase. Thus, a unique aspect of this invention provides for transgenic seeds and methods that use a recombinant DNA construct to produce in a plant, an antisense oriented RNA loop for the deletion of the lysine ketoglutarate reductase gene and / or the saccharopine dehydrogenase, as well as as to express an exogenous gene encoding dihydrodipicolinate synthase. Such constructs comprise, in the order of 5 'to 3', a seed-specific promoter element operably linked to an antisense oriented DNA element and a DNA element oriented in the direction of the gene encoding the lysine pre-protein ketoglutarate reductase / saccharine dehydrogenase. The sense oriented DNA element is shorter than the antisense oriented DNA and the sense oriented RNA transcribed by the sense oriented DNA element is complementary to the nearly 5 'segment of the antisense oriented RNA, transcribed by the targeted DNA element in antisense. The elements of the DNA are transcribed as RNA that form an antisense oriented RNA loop to suppress the expression of the native gene encoding the lysine ketoglutarate reductase.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic illustration of a recombinant DNA construct useful in this invention, to produce an antisense oriented RNA loop. Figure 2 is a Western analysis indicating the suppression of the gene using a construct of this invention.

DETAILED DESCRIPTION OF THE INVENTION SEQ ID NO: 1 is a nucleotide sequence of a recombinant DNA construct, useful for transcribing RNA that can form an antisense oriented RNA loop to suppress one or multiple genes in transgenic plants. See Table 1 for the description of the elements. As used herein, "complementary" refers to polynucleotides that are capable of hybridizing, for example, strands of sense and antisense DNA or self-complementary strands of RNA, due to the complementarity of the aligned nucleotides that allow CG binding and AT or AU. As used herein, "vector" means a DNA molecule capable of replicating in a host cell and / or to which another DNA segment can be operatively linked to cause replication of the attached segment. A plasmid is an exemplary vector. As used herein, a "transgenic" organism, for example, a plant or seed, is one whose genome has been altered by the incorporation of recombinant DNA comprising exogenous genetic material or additional copies of the native genetic material, for example, by the transformation or recombination of the organism or an ancestral organism. Transgenic plants include progeny plants of an original plant derived from a transformation process that includes the progeny of crossing transgenic plants with wild type plants or other transgenic plants. Crop plants of particular interest in the invention include, but are not limited to, corn, soybean, cotton, canola (rapeseed), wheat, rice, sunflower, safflower and flax. Other crops of interest include plants that produce vegetables, fruits, grasses and wood.

Recombinant DNA constructs for the transformation of the plant The recombinant DNA constructs for producing the spiral RNA, antisense, gene suppression agents in transgenic plants can be easily prepared by those skilled in the art. Typically, the DNA construct comprises as a minimal active promoter in the tissue selected for deletion, a transcribable DNA element having a sequence that is complementary to the nucleotide sequence of a gene selected for deletion and a terminator element of transcription . The selected gene element copied for use in the transcribable DNA in the gene suppression construct can be a promoter element, an intron element, an exon element, a 5 'UTR element, or a 3' UTR element. Although it is believed that the minimum size of DNA copied for the sequence of a gene selected for deletion is approximately 21 or 23 nucleotides; longer nucleotide segments are preferred, for example, up to the total length of a selected gene. The DNA element can comprise multiple parts of a gene, for example, nucleotides that are complementary to contiguous or separate UTR gene elements, exons and introns. Such constructs may also comprise other regulatory elements, transient peptides encoding the DNA, signal peptides, selective markers and selectable markers are desired. To form a spire of antisense oriented RNA, the conveniently complementary DNA element is no more than about half the length of the antisense oriented DNA element, often no more than one third of the length of the targeted DNA element. antisense, for example, no more than a quarter of the length of the antisense oriented DNA element. The total lengths of the combined DNA elements may vary. For example, the antisense oriented DNA element may consist of 500 to 5000 nucleotides and the complementary DNA element may consist of 50 to 500 nucleotides. The antisense transcription unit can be designed to suppress multiple genes, wherein the DNA is arranged with two or more antisense oriented elements of different genes selected for deletion, followed by an element oriented in a complementary sense, for example, complementary to at least one part of the antisense element almost 5 '. With reference to Figure 1, a recombinant DNA construct comprising a promoter element, an antisense oriented DNA element (denoted "DNA a / s"), a DNA element oriented in a complementary sense (denoted "DNA") is shown schematically. ") and a DNA that provides signals and a polyadenylation site (denoted" polyA site "). The DNA construct is transcribed into RNA comprising an antisense oriented RNA segment and a complementary RNA segment, which is complementary to the near 5 'end of the antisense oriented RNA segment. The 5 'and 3' ends of the antisense RNA can self-hybridize to form a double-stranded RNA segment that encloses an antisense oriented RNA loop. For example, if the nucleotide sequence of the nearly 5 'end of the strand of the transcribed antisense oriented DNA is 5'-CGGCATA -, the sequence of the nearly 3' end of the transcribed strand of the inverted repeat DNA will be - TATGCCG-3 ', which it is easily cloned from the source DNA, providing the antisense element. With such sequences, the antisense oriented RNA loop will be extended from one side of the dsRNA segment, for example, 5'-GCCGUAU 3'-CGGCAUA. The antisense oriented DNA and its self-complementary DNA can be contiguous or separated by a vector DNA, for example, up to about 100 nucleotides or the like of a vector DNA that separates the restriction sites used for the assembly of the vector. Recombinant DNA constructs can be assembled using commercially available materials and methods known to those of ordinary skill in the art. A useful technology for constructing DNA constructs and vectors for transformation is the GATEWAY ™ cloning technology (available from Invitrogen Life Technologies, Carisbad, California), which utilizes the cloning reaction of the site-specific LR recombinase. Full coverage of bacteriophage lambda vector construction, rather than endonucleases and restriction ligases. The LR cloning reaction is described in U.S. Pat. 5,888,732 and 6,277,608, the Publications of the Patent Application of E.U.A. 2001283529, 2001282319 and 20020007051, all of which are incorporated herein by reference. The GATEWAY ™ Cloning Technology Instruction Manual, which is also provided by Invitrogen, also provides concise directions for the routine direction of any desired DNA in a vector comprising operable plant expression elements. An alternate method of manufacturing a vector, employs independent cloning of the ligand, as described by Aslanidis, C. et al., Nucleic Acids Res., 18, 6069-6074, 1990 and Rashtchian, A. et al., Biochem., 206, 91-97, 1992, wherein a DNA fragment with 5 'and 3' ends of a single strand is ligated into a desired vector that can then be amplified in vivo. Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in the genomes of the plant, as well as promoters from other sources, including the promoter of nopaline synthase (nos) and octopine synthase (oes) promoters carried in plasmids that induce a tumor of Agrobacterium tumefaciens, promoters of the colimoviruses, such as promoters of the cauliflower mosaic virus or the scrapie mosaic virus. For example, see the Patents of E.U.A. 5,322,938 and 5,858,742, which describe versions of a constitutive promoter derived from the cauliflower mosaic virus (CaMV35S), the Patent of E.U.A. 5,378,619, which describes the 35S promoter of the Escrafularia Mosaic Virus (FMV), the U.S. Patent. 5,420,034, which describes a napine promoter, the U.S. Patent. 6,437,217, which describes an RS81 promoter of maize, the U.S. Patent. 5,641, 876, which describes a rice actin promoter, the U.S. Patent. 6,426,446, which describes an RS324 corn promoter, the U.S. Patent. 6,429,362, which describes a corn PR-1 promoter, the U.S. Patent. 6,232,526, which describes a maize A3 promoter, the U.S. Patent. 6,177,611, which describes corn constitutive promoters, the U.S. Patent. 6,433,252, which describes an oleic acid promoter L3 of maize, the Patent of E.U.A. 6,429,357, which describes a promoter and an intron 2 of rice actin, the Patent of E.U.A. 5,837,848, which describes a specific promoter of the root, the Patent of E.U.A. 6,084,089, which describes cold-inducible promoters, the U.S. Patent. 6,294,714, which describes light-inducible promoters, the U.S. Patent. 6,140,078, which describes saline-inducible promoters, the U.S. Patent. 6,252,138. describing promoters inducible by pathogens, the U.S. Patent. 6,175,060, which describes promoters inducible by phosphorus deficiency, Patent of U.S.A. 6,635,806, which describes a coixin promoter, U.S. 2002 / 0192813A1, which describes 5 ', 3' and intron elements useful in the design of effective plant expression vectors, U.S.2004 / 0216189 A1, which describes a promoter of corn chloroplast aldolase, and U.S. 2004 / 0123347A1, which describes promoters inducible by a water deficit, all of which are incorporated herein by reference. These promoters and numerous others that function in plant cells are known to those skilled in the art, and are available for use in the recombinant polynucleotides of the present invention to provide expression of the desired genes in the cells of transgenic plants.

In addition, promoters can be altered to contain multiple "enhancer sequences" to help elevate gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein can be improved. These enhancers are often at the 5 'position at the start of transcription in a promoter that functions in eukaryotic cells, but can be inserted frequently upstream (5') or downstream (3 ') to the coding sequence. In some cases, these elements that improve 5 'are introns. Particularly useful as enhancers are the 5 'introns of rice actin 1 (see US Patent 5,641, 876), and the rice actin 2 genes, the corn alcohol dehydrogenase gene intron, and the gene intron. of heat shock protein 70 of corn (US Patent 5,593,874), and the shrunken corn 1 gene. In other aspects of the invention, sufficient expression in the tissues of the seed of the plant is desired to effect improvements in the composition of the seed. Exemplary promoters to be used for modification of the seed composition include promoters of seed genes such as napin (US 5,420,034), corn oleosin L3 (US 6,433,252), zein 227 (Russell et al. (1997) Transgenic Res 6 (2): 157-166), globulin 1 (Belanger et al (1991) Genetics 129: 863-872), glutelin 1 (Russell (1997) supra), and peroxyrredoxin antioxidant (Perl) (Stacy et al. (1996) Plant Mol Biol. 31 (6): 1205-1216).

Recombinant DNA constructs prepared according to the invention often include a 3 'element that typically contains a signal from a polyadenylation site, especially if the recombinant DNA is intended for protein expression as well as for deletion of the gene. Well-known 3 'elements include those from genes of Agrobacterium tumefaciens, such as nos 3', tml 3, tmr 3 ', tms 3', oes 3 ', tr7 3', described, for example, in the U.S. 6,090,627, incorporated herein by reference; the 3 'elements of plant genes such as wheat heat shock protein 17 (Triticum aesevitum) (Hsp17 3'), a wheat ubiquitin gene, a wheat fructose-1, 6-biphosphatase gene, a rice glutelin gene, a rice lactate dehydrogenase gene, and a rice beta-tubulin gene, all of which are described in the published US patent application 2002/0192813 A1, incorporated herein by reference; and the ribulose biphosphate carboxylase gene from the pea (Pisum sativum) (rbs 3 '), and the 3' elements of the genes within the host plant. The recombinant DNA construct that suppresses the gene can also be stacked with DNA that imparts other traits of agronomic interest, including DNA that provides resistance to herbicides or resistance to insects, such as using a Bacillus thuringienis gene to provide resistance. against lepidoptera, coleoptera, homoptera, hemiptera and other insects. The herbicides for which resistance in a plant is useful include glyphosate herbicides, phosphinothricin herbicides, oxyinyl herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and herbicides. of glufosinate. Those of ordinary skill in the art are capable of providing stacked features with reference to the publications of the U.S. patent applications. 2003 / 0106096A1 and 2002 / 0112260A1, and the Patents of E.U.A. 5,034,322; 5,776,760; 6,107,549 and 6,376,754, and resistance to insects / nematodes / viruses with reference to the Patents of E.U.A. 5,250,515; 5,880,275; 6,506,599; 5,986,175 and the Publication of the Patent Application of E.U.A. 2003/0150017 A1, all of which are incorporated herein by reference.

Methods of transformation Numerous methods for transforming plant cells with recombinant DNA are known in the art, and can be used in the present invention. Two methods commonly used for the transformation of plants is a transformation mediated by Agrobacterium and bombardment with microprojectiles. Methods of bombardment with microprojectiles are illustrated in the Patents of E.U.A. 5,015,580 (soybean); 5,550,318 (corn); 5,538,880 (corn); 5,914,451 (soybean); 6,160,208 (corn); 6,399,861 (corn) and 6,153,812 (wheat) and the transformation mediated by Agrobacterium is described in the Patents of E.U.A. 5,159,135 (cotton); 5,824,877 (soy); 5,591, 616 (corn); and 6,384,301 (soy), all of which are incorporated herein by reference. For the plant transformation system based on Agrobacterium tumefaciens, additional elements present in the transformation constructs will include left and right T-DNA border sequences to facilitate the incorporation of the recombinant polynucleotide into the plant genome. In general, it is useful to introduce a recombinant DNA in a random manner, ie, not in a specific location, in the genome of a target plant line. In special cases it may be useful to direct the insertion of the recombinant DNA in order to achieve site-specific integration, for example, to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to inserting a recombinant polynucleotide at a predetermined site known to be active for gene expression. There are several site-specific recombination systems, which are known to function as implants, including cre-lox, as described in the U.S. Patent. 4,959,317 and FLP-FRT as described in the U.S. Patent. 5,527,695, both incorporated herein by reference. The transformation methods of this invention are preferably practiced in tissue culture in a medium and in a controlled medium. "Medium" refers to the numerous mixtures of nutrients that are used to grow cells in vitro, that is, outside the living organism intact. The targets of the recipient cells include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant can be regenerated is useful as a recipient cell. Calluses can be initiated from tissue sources including, but not limited to, immature embryos, atypical meristems of seedlings, microspores and the like. The cells capable of proliferating as callus are also receptor cells for genetic transformation. Methods and materials for the practical transformation to make the transgenic plants of this invention, for example, various means and recipient target cells, the transformation of immature embryos and the subsequent regeneration of fertile transgenic plants, are described in U.S. Pat. 6,194,636 and 6,232,526, which are incorporated herein by reference. The seeds of transgenic plants can be harvested from fertile transgenic plants and used to culture generations of progeny of the transformed plants of this invention, including a line of hybrid plants to select plants having an improved agronomic trait. In addition to the direct transformation of a plant with recombinant DNA, the transgenic plants can be prepared by crossing a first plant having a recombinant DNA with a second plant lacking the DNA. For example, the recombinant DNA can be introduced into a first plant line that is susceptible to transformation to produce a transgenic plant that can be crossed with a second plant line to introduce the recombinant DNA into the second plant line.

A transgenic plant with recombinant DNA that provides an improved agronomic trait, eg, improved performance, can be crossed with a transgenic plant line having another recombinant DNA that confers another trait, eg, herbicide resistance or resistance to pesticides, to produce progeny plants that have a recombinant DNA that confers both traits. Typically, at such a crossing to combine the traits, the transgenic plant that donates the additional trait is the male line and the transgenic plant that carries the basic traits is the female line. The progeny of this cross will segregate so that some of the plants will carry the DNA for both original traits and some will carry the DNA for an original trait; such plants can be identified by markers associated with the original recombinant DNA. Progeny plants that carry the DNA for both original traits can cross back into the original female line multiple times, for example, usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as a primary transgenic primary line, but for the recombinant DNA of another original transgenic line. In the practice of transformation, DNA is typically introduced into only a small percentage of target cells in any transformation experiment. The marker genes are used to provide an efficient system for the identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the herbicides for which the plants of this invention can be resistant, are useful agents for selective markers. Transformed cells are potentially exposed to the selective agent. In the population of surviving cells, the cells will be those in which, generally, the gene that confers the resistance is integrated and expressed at sufficient levels to allow cell survival. The cells can be further tested to confirm the stable integration of the exogenous DNA. Selective marker genes commonly used include those that confer resistance to antibiotics, such as kanamycin and paromomycin (nptll), hygromycin B (aph IV) and gentamicin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (aroA or EPSPS). Examples of such selectable ones are illustrated in the Patents of E.U.A. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Selectable markers that provide an ability to visually identify transformants can also be employed, for example, a gene that expresses a colored or fluorescent protein such as luciferase or green fluorescent protein (GFP) or a gene that expresses a beta-glucuronidase or uidA gene (GUS) for which several chromogenic substrates are known. Cells that survive exposure to the selective agent or cells that have been labeled as positive in a selection assay can be cultured in a regeneration medium and allowed to mature in plants. Developing seedlings can be transferred to a plant growth mixture, and hardened, for example, in an environmentally controlled chamber at approximately 85% relative humidity, 600 ppm C02, and 25-250 microeinsteins m "2s" 1 of light, before transferring to a greenhouse or growth chamber for ripening. Plants regenerate from about 6 weeks to 10 months after the transformant is identified, depending on the initial tissue. The plants can be pollinated using conventional plant crossing methods known to those skilled in the art and the seed produced, for example, self-pollination is commonly used with transgenic corn. The regenerated transformed plant or its seed or progeny plants can be tested for the expression of the recombinant DNA and selected for the presence of the improved agronomic trait.

Transgenic Plants and Seeds The seed of the transgenic plant provided by this invention is grown to generate transgenic plants that have an improved trait compared to a control plant. Such seeds for plants with an improved agronomic trait are identified by selecting the plants or seeds of the transformed progeny for the improved trait. For efficiency, a selection program is designed to evaluate multiple transgenic plants (events), which comprise the recombinant DNA, for example, multiple plants of 2 to 20 or more transgenic events. Transgenic plants growing from a transgenic seed provided herein, demonstrate improved agronomic traits that contribute to increasing yield or other trait that provides an increased value to the plant, including, for example, improved seed quality such as a level increased of certain amino acids; for example, lysine. Many transgenic events that survive the fertile transgenic plants that produce seeds and progeny plants will not exhibit an improved agronomic trait. The selection is necessary to identify the transgenic plant having the improved agronomic traits of transformed plant populations, as described herein, evaluating the transgenic plants for the improved trait and a minimal effect on other agronomic traits. These assays can also take many forms, including non-exclusively, analysis to detect changes in chemical composition, biomass, physiological properties, plant morphology. The methods of this invention provide a means for a person of ordinary skill in the art to design recombinant DNA constructs, make transgenic plants, select an improved amino acid level in the seeds and a minimal adverse effect on other agronomic traits, to provide a transgenic seed of this invention. Such seed can be used to produce a transgenic maize plant that has a recombinant DNA construct that transcribes antisense-oriented RNA, which suppresses the level of a protein in an amino acid catabolic pathway, integrated into its genome. The following examples illustrate aspects of the invention.

EXAMPLE 1 This example illustrates the preparation of a transformation vector useful for inserting a recombinant DNA construct of this invention in a transgenic plant, to practice a method of this invention. The LKR / SDH gene encodes a preprotein for lysine ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are enzymes in a catabolic pathway of lysine. The deletion of LKR is manifested in the modification, for example, increase in the lysine content.

The deletion of LKR is carried out by expressing in a plant a construct of Recombinant DNA that produces a stabilized antisense RNA transcribed from the LKR DNA oriented antisense and LKR DNA oriented in sense, which forms an antisense oriented RNA loop. A transformation vector is prepared, comprising two transcription units between the left and right borders of Agrobacterium tumefaciens. A transcription unit for a marker comprises: (a) DNA from a rice actin promoter and a rice actin intron, (b) DNA from a transient chloroplast peptide from Arabidopsis EPSPS (c) A. aroA DNA. tumefaciens (a marker resistant to glyphosate), and (or) DNA from a NOS terminator of A. tumefaciens, The other transcription unit for the deletion of the LKR gene comprises: (a) DNA from a GLB1 promoter from Zea mays, (b) DNA of an ADH1 intron of Zea mays, (c) an antisense oriented DNA fragment of Zea mays LKR, (d) a DNA fragment oriented in the sense of LKR of Zea mays, and (e) DNA of a GLB1 terminator of Zea mays. SEQ ID NO: 1 is a DNA sequence of a transformation vector comprising the marker and the gene deletion transcription units, described above. See Table 1 below for a description of the elements of the transformation vector contained within SEQ ID NO: 1.

TABLE 1 A vector prepared with the elements listed in Table 1 was used to transform the tissue of corn plants. The transgenic maize plants were obtained by transformation mediated by Agrobacterium. The transgenic plants of two separate transgenic insertion events were cultured to produce the F1 seed. Six mature seeds from each event were analyzed to determine the success of the transformation and the deletion of LKR. The mature transgenic seeds were dissected to extract the protein that was analyzed by Western analysis. With reference to Figure 2, the seed of one of the events showed no reduction in LKR compared to the wild type; and the seed of the other event showed to be segregating (1: 1 hemocigoto: wild type), since three of the six seeds showed a substantial reduction in LKR in comparison with the wild type.

EXAMPLE 2 This example illustrates transgenic maize with improved lysine. The transformation vector prepared in Example 1 was modified by inserting a transcription unit comprising a seed-specific promoter operably linked to DNA encoding dihydrodipicolinate synthase. More specifically, the transcription unit comprises the DNA of the corn globulin 1 promoter (bp 48 to 1440; Kriz, Biochem. Genet., 27: 239-251, 1989; Belanger and Kriz, Genetics, 129: 863- 872, 1991, and U.S. Patent No. 6329574), an intron of rice actin 1 (bp 1448 to 1928; McEIroy et al., Plant Cell, 2: 163-171, 1990), a transient peptide of Chloroplast DHDPS from corn (bp 1930 to 2100, Frisch et al., Mol.Gen. Genet., 228: 287-293, 1991), a DHDPS gene from Corynebacterium (pb 2101-3003, Bonnassie et al., Nucleic Acids Research , 18: 6421, 1990; Richaud et al., J. Bacteriol., 166: 297-300, 1986), a 3 'untranslated region of corn globulin 1 (bp 3080 to 4079; Belanger and Kriz, 1991). Promoters for the suppression of lysine ketoglutarate synthase and the expression of dihydrodipicolinate synthase are adjacent to the RNA transcribed in opposite directions. Corn produced from transgenic plants has high levels of lysine, for example, in the range of 3000 to 4000 ppm, compared to essentially no lysine in corn type. All materials and methods described and claimed herein may be made and used without undue experimentation as instructed by the foregoing description. Although the materials and methods of this invention have been described in terms of the preferred embodiments and illustrative examples, it will be apparent to those skilled in the art that variations may be applied to the materials and methods described herein, without departing from the concept, spirit and scope of the invention. All substitutes and similar modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the invention, as defined by the appended claims.

Claims (18)

NOVELTY OF THE INVENTION CLAIMS

1. - A seed to produce a transgenic corn with an improved amino acid content, which has a recombinant DNA construct integrated in its genome that transcribes the antisense oriented RNA that suppresses the production of a protein in a catabolic pathway of the amino acid, where the DNA The recombinant comprises a seed-specific promoter operably linked to the DNA that is transcribed into the RNA, and wherein the seed has a high content of amino acids, as compared to the progeny seed of control maize plants, in which the Protein production is not suppressed.

2. The seed according to claim 1, further characterized in that the DNA that is transcribed to the RNA comprises an antisense oriented DNA element and a sense oriented DNA element, wherein the DNA element oriented in the sense is shorter that the antisense oriented DNA element, wherein the RNA oriented in sense transcribed by the sense oriented DNA is complementary to the nearly 5 'end of the antisense oriented RNA transcribed by the antisense oriented DNA element, wherein the transcribed RNA forms a loop of antisense oriented RNA to suppress the protein in a catabolic pathway of the amino acid.

3. - The seed according to claim 1, further characterized in that the seed-specific promoter is a specific promoter of the embryo or an endosperm-specific promoter.

4. The seed according to claim 1, further characterized in that the recombinant DNA construct produces RNA to suppress a gene encoding a protein in the catabolic path of lysine.

5. The seed according to claim 4, further characterized in that the protein in the catabolic pathway of lysine is lysine ketoglutarate reductase, saccharopine dehydrogenase or both.

6. The seed according to claim 1, further characterized in that it has integrated in its genome a recombinant DNA that expresses a protein in a pathway of synthesis of amino acids.

7. The seed according to claim 6, further characterized in that the protein in a pathway of synthesis of the amino acids is dihydropicolinate synthase.

8. The seed according to claim 3, further characterized in that the amino acid is lysine, the protein in a catabolic pathway of the amino acids is lysine ketoglutarate and the protein in a pathway of synthesis of the amino acids is dihydropicolinate synthase.

9. - A recombinant DNA construct for producing, in a plant, an antisense oriented RNA loop for the suppression of the gene, wherein the construct comprises, in the order 5 'to 3', a seed-specific promoter element, operably linked to an antisense oriented DNA element and a sense oriented DNA element, wherein the sense oriented DNA element is shorter than the antisense oriented DNA element, wherein the RNA oriented in sense transcribed by the targeted DNA element in sense it is complementary to the nearly 5 'segment of the antisense oriented ANR transcribed by the antisense oriented DNA element, wherein the DNA elements are transcribed as RNA that forms an antisense oriented RNA loop to suppress the expression of at least one gene; wherein the gene selected for deletion expresses lysine ketoglutarate reductase.

10. A method, further characterized in that it is for increasing the level of lysine in corn seed by expressing a recombinant DNA construct according to claim 9 in a developing corn seed.

11. A method for producing corn seeds with an improved amino acid level, which comprises cultivating the corn plants of transgenic seeds that have a recombinant DNA construct integrated in their genome to suppress the expression of a protein in a catabolic path of the amino acids, wherein the recombinant DNA construct comprises a seed-specific promoter operably linked to the DNA that is transcribed to the antisense-oriented RNA complementary to the messenger RNA of the protein, and wherein the transgenic corn has a high content of amino acids in the its grains, in comparison with the control corn plant, in which the protein is not suppressed.

12. The method according to claim 11, further characterized in that the recombinant DNA construct comprises in the order 5 'to 3' the seed-specific promoter, operably linked to an antisense oriented DNA element and an element of sense-oriented DNA, wherein the sense-oriented DNA element is shorter than the antisense oriented DNA element, wherein the RNA oriented in sense transcribed by the sense-oriented DNA element is complementary to the nearly 5 'segment of the DNA. Antisense oriented RNA transcribed by the antisense oriented DNA element, and wherein the DNA elements are transcribed as RNA that forms a loop of antisense oriented RNA to suppress the expression of the protein.

13. The method according to claim 12, further characterized in that the seed-specific promoter is a specific promoter of the embryo or an endosperm-specific promoter.

14. The method according to claim 12, further characterized in that the recombinant DNA construct produces RNA to suppress the expression of a protein in the catabolic pathway of lysine.

15. The method according to claim 14, further characterized in that the protein gene in the catabolic pathway of lysine is lysine ketoglutarate reductase, saccharopine dehydrogenase or both.

16. The method according to claim 12, further characterized in that it has integrated in its genome a recombinant DNA that expresses a protein in a pathway of synthesis of amino acids.

17. The method according to claim 16, further characterized in that the protein in the path of the synthesis of the amino acid is dihydropicolinate synthase.

18. The method according to claim 12, further characterized in that the amino acid is lysine, the protein in a catabolic path of the amino acid is lysine ketoglutarate and the protein in a path of amino acid synthesis is dihydropicolinate synthase.