MXPA06006574A - High lysine maize compositions and methods for detection thereof - Google Patents

Abstract

Disclosed herein are assays for detecting the presence of a lysine-increasing transgenic event based on the DNA sequence of the exogenous DNA construct inserted into the maize genome and of genomic sequences flanking the insertion site. Also provided are transgenic plants having a novel exogenous DNA construct that expresses a dihydrodipicolinic acid synthase, the activity of which results in increased lysine in a plant or plant product.

Description

COMPOSITIONS OF CORN OF HIGH LYSINE CONTENT, AND METHODS FOR THE DETECTION OF THE SAME This application claims the benefit under 35 USC § 119 (e) of the United States provisional application serial number 60 / 529,182, filed on December 11, 2003, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION The present invention relates to the field of molecular biology of plants. More specifically, the present invention relates to transgenic maize with increased lysine content, and to tests and methods for the identification of specific exogenous DNA that provides increased lysine content.

BACKGROUND OF THE INVENTION The Zea mays species, commonly known as corn, is a grain that is widely used in animal feed. The grain, that is, the seed, is a source of protein, starch and oil for pigs, livestock and poultry.

Of the ten amino acids that are considered essential in a mixed grain source, corn is particularly limiting in lysine, threonine and methionine.

The lack of these amino acids, especially lysine, requires that corn or whole grain corn meal used as food be supplemented with these nutrients, often provided by the addition of whole soybean meal. It would be of benefit for the technique to increase the level of lysine in the corn kernel as a means to make the whole seed and flour more nutritious as a feed. To increase lysine levels using a molecular biology method, a version insensitive to the feedback of at least one of the enzymes of the lysine pathway, namely dihydrodipicolinic acid synthase (referred to herein as DHDPS). It has been shown in vitro that a bacterial DHDPS gene isolated from E. coli is approximately 200 times less sensitive to inhibition by increases in lysine levels and, when introduced into transgenic tobacco, the overexpression of the DHDPS gene of E coli resulted in increased levels of lysine in the leaf tissue (Glassman et al., U.S. Patent No. 5,288,300). Falco et al., Describe transgenic plants with increased levels of lysine in the seed, and genes useful for the production of said transgenic plants (U.S. Patent No. 5,773,691 and 6,459,019, U.S. Patent Application Publication 2003/0056242, each of which is incorporated herein by reference in its entirety). In these reports, Falco et al., Describe the isolation and use of DHDPS from E. coli insensitive to feedback, and DHDPS from Corynebacterium (also known as cordapA), to generate transgenic rape, tobacco, corn and soybean plants with increased levels of lysine in the seed. For corn, Falco e al. report an increase of approximately 130% in free lysine in grains transformed with the cordapA gene with respect to non-transformed grains. It would be advantageous to be able to detect the presence or absence of a particular transgene in a plant or seed, or progeny of said plants or seeds, not only with respect to the transgene itself, but also with respect to its location in the genome of a host plant or seed . The identification with respect to the location also provides the identification of the transgenic event by which a genetic engineer inserted the transgene into the parent plant of the plant or seed.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides constructs useful for the generation of transgenic events, and materials and methods useful for the identification of particular transgenic events that resulted in transgenic plants that accumulate higher levels of lysine than closely related plants that do not include construction. In particular, this invention comprises a marker-free transgenic corn line comprising a specific exogenous DNA that was introduced by means of standard corn transformation, referred to herein as "event LY038" or "event LY038". The present invention further provides a method for detecting the presence or absence of the LY038 event in DNA obtained from samples of plants, seeds or corn tissue. The corn plant of the present invention comprising event LY038 exhibits increased lysine content in the grain relative to a parent or other substantially related plant. In addition, the present invention provides an exogenous DNA construct comprising a corn globulin 1 promoter, an intron of rice actin 1, a DNA molecule encoding the transit peptide of dihydrodipicolinate synthase in corn chloroplasts, a molecule of DNA encoding Corynebactepum dihydrodipicolinate synthase, and a 3 'untranslated region of corn globulin 1 which, when operably linked and expressed in transgenic plant and plant cells, results in increased lysine content in the grain or parts of the same, or a processed product derived from the grain or plant. In another embodiment, the construct further comprises a lox site, present as a component of a mechanism for the removal of the marker gene used to identify successful transformants. With respect to the identification of a plant or seed derived from a particular transgenic event, compositions and methods are provided for the detection of the presence of the genomic insertion region of a novel maize plant comprising the event LY038, ie the site in the genome where the construction resides. DNA molecules comprising at least a portion of the exogenous DNA inserted in the genome, and a portion of the corn genome DNA flanking the insertion site (referred to herein as a "binding sequence") are provided. In another embodiment of the present invention, novel DNA molecules comprising at least about 20 base pairs of SEQ ID NO: 1 or 2 are provided, wherein the base pairs 1781-1782 or 200-201 are included, respectively . Further provided by the present invention, they are DNA molecules comprising the sequence of an amplicon having the sequence of SEQ ID NO: 6 which is obtained by a DNA amplification method using the primers of SEQ ID NOs: 3 and 4; and a hybridization probe complementary to said amplicon, having the sequence of SEQ ID NO: 5, and complements thereof. DNA molecules having the sequence of SEQ ID NO: 5 or 6, encompass the junction between exogenous DNA and maize flanking genomic DNA, and serve as diagnostics for the DNA of event LY038 when used in suitable analytical tests. Other preferred DNA molecules of the present invention which encompass the binding of the exogenous DNA / genomic insertion region of the LY038 event, are molecules having the sequence of SEQ ID NOs: 1, 2 and 11, and complements thereof. A stably transformed corn plant or seed comprising these molecules is another aspect of this invention. The primers are said to be of "sufficient length", when they are of a length that allows the primer to function in a PCR reaction and specifically amplify an objective sequence; a length of about 11 nucleotides or more is sufficient, more preferably about 18 nucleotides or more, still more preferably about 24 nucleotides or more, even more preferably about 30 nucleotides, is sufficient to specifically yield and amplify an objective sequence. The person skilled in the art would know that an initiator of even greater length of about 30 nucleotides can be usefully used in a PCR reaction and, consequently, is of sufficient length. PCR primers useful for the identification of the LY038 event comprise a sufficient length of the portion of a transgene of the DNA sequence of SEQ ID NO: 1, and a sufficient length of a 5 'flanking DNA sequence of the SEQ maize. ID NO: 1, or a sufficient length of the portion of a transgene of the DNA sequence of SEQ ID NO: 2 and a sufficient length of a 3 'flanking DNA sequence of the maize of SEQ ID NO: 2. These primers are useful in PCR methods to provide a DNA amplicon product that serves as a diagnostic for the LY038 event and its progeny. PCR primers homologous or complementary to any suitable length of SEQ ID NOs: 1 and 2 that can produce an amplicon or probe that serves as diagnostics for the LY038 event, are another aspect of the present invention. For example, without limitation, preferred primers that serve as diagnostics for event LY038 include those having at least about 18 contiguous nucleotides of any of the sequences of SEQ ID NO: 3 or 4. Amplicons produced using DNA primers that serve as diagnostics for the event LY038 and its progeny, are an aspect of this invention. A preferred amplicon that serves as diagnostics for event LY038 has the sequence of SEQ ID NO: 6. Another aspect of the present invention provides methods for detecting the presence or absence of DNA corresponding to event LY038 in a sample. Said methods comprise obtaining DNA from a plant, seed or corn tissue by contacting the DNA of the sample with a group of PCR primers, performing PCR and detecting the presence or absence of an amplicon. Preferred PCR primers that serve as diagnostics for event LY038, include primer oligonucleotides having the sequence of SEQ ID NOs: 3 and 4, which produce a specific amplicon from event LY038 having the sequence, for example, of SEQ ID NO: 6, which is detectable by a probe specific for the event LY038 having the sequence, for example, of SEQ ID NO: 5. Hybridization of a probe indicating the presence of the event LY038 with an amplicon comprising DNA specific for the LY038 event can be detected by any suitable means available for the 1 nucleic acid manipulation techniques, including TaqMan® tests and Southern blot methods among other methods known to those skilled in the art. molecular biology. The person skilled in the art would know that detection of the amplicon can be carried out by detection that does not include hybridization of a probe with an amplicon, such as by analysis in acrylamide gel or agarose gel. The person skilled in the art would also know that the length and sequence of the initiator and the probe can be varied from the exemplified sequences presented in SEQ ID NOs: 3, 4 and 5, and that they still produce a PCR amplicon, or amplicon group and probe, which serves as a diagnostic for event LY038. In another aspect, the present invention provides a method for producing progeny plants comprising DNA from event LY038. Progeny plants can be inbred or hybrid plants. In another application, the present invention provides a method for carrying out DNA-assisted genetic enhancement of event LY038. According to another aspect of the present invention, there is provided a stably transformed corn plant comprising DNA from event LY038 and also comprising increased content of lysine in the grain or parts thereof. The present invention further relates to DNA detection equipment comprising at least one DNA molecule of sufficient length of contiguous nucleotides homologous or complementary to SEQ ID NO: 1 or 2, which functions as a specific DNA primer or probe for event LY038 or its progeny. The present invention further relates to the plants and seeds and processed products thereof of high maize corn (Zea mays) comprising the event LY038, and the progeny derived therefrom having representative seed deposited as access number of ATCC PTA-5623. Further provided by the present invention, it is a corn plant or a part thereof which includes, for example, pollen or seed, which is produced by developing a plant comprising DNA from event LY038. The corn seed and plant comprising DNA from the LY038 event for which the DNA primer molecules of the present invention are usefully used for the detection of event-specific sequences, are other aspects of this invention. A processed product of event LY038 comprises a part of a grain of corn, for example, the endosperm. A whole corn flour of the present invention can be obtained from the grain comprising the transgenic DNA molecule of LY038, wherein the wholemeal flour has high lysine content with respect to other whole corn flours that do not contain the DNA molecule. The foregoing and other aspects of the present invention will become more apparent from the detailed description, examples and accompanying figures given below. The examples given below are included to demonstrate examples of certain preferred embodiments of the present invention. Those skilled in the art should appreciate that the techniques described in the examples below represent procedures that the inventors have found to work well in the practice of the present invention, and thus can be considered to constitute examples of preferred modes for their practice. However, those skilled in the art should appreciate, in light of the present disclosure, that many changes can be made in the specific embodiments described, and that an equal or similar result is obtained without departing from the spirit and scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a plasmid map of pMON55221. Figure 2A is a schematic of the exogenous DNA insert of event LY038. The exogenous DNA and the relevant base pairs are indicated in italics, and the maize genomic DNA and the relevant base pairs are indicated in normal letters. Figure 2B is the sequence at the 5 'junction. Figure 2C is the sequence at the 3 'junction.

BRIEF DESCRIPTION OF THE SEQUENCES Molecules having defined sequences used in the context of the present invention are set forth in the sequence listing presented concomitantly with this application. A summary of the sequence listing is given below: SEQ ID NO: 1 is a 1961 base pair (bp) polynucleotide sequence of the 5 'DNA comprising the genomic portion of maize (bp 1-1781) flanking the side 5 'of the insertion site and insertion portion of the transgene (bp 1782-1961) of the DNA of event LY038.

SEQ ID NO: 2 is a polynucleotide sequence of 867 base pairs of 3 'DNA comprising the 3' genomic portion of maize (bp 201-887) flanking the 3 'side of the insertion site and the transgene insertion sequence. (pb 1-200) of the DNA of event LY038. SEQ ID NOs: 3 and 4 are polynucleotide sequences of PCR primers useful for producing an amplicon that serves as a diagnostic for DNA of event LY038. SEQ ID NO: 5 is a polynucleotide sequence of an oligonucleotide probe useful for hybridizing with an amplicon to detect DNA from event LY038. SEQ ID NO: 6 is a polynucleotide sequence of an amplicon that serves as a diagnostic for DNA of the LY038 event. SEQ ID NO: 7 is a polynucleotide sequence of a corn globulin 1 promoter (bp 48 to 1440; Kritz, Biochem. Genet., 27: 239-251, 1989; Belanger and Kriz, Genetics, 129: 863-872. , 1991; U.S. Patent No. 6,329,574, incorporated herein by reference in its entirety), an intron of rice actin 1 (bp 1448 to 1928; McEIroy et al., Plant Cell, 2: 163-271, 1990) , a corn chloroplast DHDPS transit peptide (bp 1930 to 2100; Frisch et al., Mol. Gen. Genet. 228: 287-293, 1991), a Corynebacterium DHDPS gene (bp 2101 to 3003; Bonnassie et al., Nucleic Acids Research, 18: 6421, 1990); Richaud et al., J. Bacterio !., 166: 297-300, 1986), a 3 'untranslated region of corn globulin 1 (bp 3080 to 4079; Belanger and Kritz, cited above), and a lox P site. (bp 4091 to 4124; Russell et al., Mol. Gen. Genet., 234: 45-59, 1992). SEQ ID NO: 8 is a polynucleotide sequence of a Corynebacterium DHDPS gene (Bonnassie et al., Nucleic Acids Research, 18: 6421, 1990; Richaud et al., J. Bacterio!., 166: 297-300, 1986). SEQ ID NO: 9 is a 1736 base pair polynucleotide sequence of additional genomic DNA from corn flanking the 5 'side of the insertion site of event LY038 (see Figure 2A). SEQ ID NO: 10 is a polynucleotide sequence of 359 base pairs of additional genomic DNA from corn flanking the 3 'side of the insertion site of event LY038 (see Figure 2A). SEQ ID NO: 11 is a polynucleotide sequence of 20 base pairs consisting of 10 contiguous DNA nucleotides of the transgene insert and 10 contiguous nucleotides of maize genomic DNA of the binding sequence illustrated in Figure 2C.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term "exogenous DNA" refers to DNA that does not naturally originate from the particular construct, cell or organism in which that DNA is found. The exogenous DNA may include a DNA or RNA sequence native to a genome, but at a new site in the genome or linked to other sequence elements not naturally associated with the exogenous DNA in its native state.

Recombinant DNA constructs used to transform plant cells comprise exogenous DNA, and usually other elements as discussed below. As used herein, the term "transgene" means an exogenous DNA that has been incorporated into the genome of a host, or that is capable of displaying autonomous replication in a host cell, and is capable of causing the expression of one or more cellular products. Examples of transgenes provide the host cell or regenerated plants thereof, with a novel phenotype with respect to the corresponding non-transformed parent cell or plant, or a corresponding transformed progenitor cell or plant comprising other transgenes, but not the particular transgene in question. Transgenes can be directly introduced into a plant by genetic transformation, or they can be inherited from a plant of any previous generation that was transformed with the exogenous DNA. As used herein, "gene" or "coding sequence" means a DNA sequence from which an RNA molecule is transcribed. The RNA may be a messenger RNA that codes for a protein product, an RNA that functions as an antisense molecule, or a structural RNA molecule such as a tRNA, rRNA, snRNA, or other RNA. As used herein, the term "expression" refers to the combination of intracellular processes, including transcription and translation, by which a DNA molecule, such as a gene, is used to produce a polypeptide or a molecule of RNA An example of a coding sequence is a Corynebacterium dihydrodipicolinate synthase gene (DHDPS); Bonnassie et al., Nucleic Acids Research, 18: 6421, 1990; Richaud et al., J. Bacterio!., 166: 297-300, 1986; bp 2101 to 3003 of SEQ ID NOs: 7 and 8), useful for the production of corn grains with increased lysine content. A corn plant, transformed to contain and express a Corynebacterium DHDPS gene, which results in increased lysine content in the grain tissue, is also referred to as a high lysine corn plant. As used herein, the term "promoter" means a region of DNA sequence that is essential for the initiation of DNA transcription, which results in the generation of an RNA that is complementary to the transcribed DNA; this region can also be referred to as a "regulatory region 5 '". The promoters are located towards the 5 'end of the coding sequence to be transcribed, and have regions that act as binding sites for RNA polymerase, and have regions that function with other factors that promote RNA transcription. Promoters useful in plants, include those that are constitutive, inducible, tissue-specific, temporarily regulated, regulated by the circadian rhythm, inducible by drought, inducible by stress, regulated by development, cold-inducible, inducible by light, and the like. Of particular importance to the present invention, it is a specific promoter of the embryo such as, without limitation, the corn globulin 1 promoter (Kriz, Biochem. Genet., 27: 239-251, 1989; Belanger and Kriz, Genetics, 129; : 863-872, 1991; bp 48 to 1440 of SEQ ID NO: 7). As is well known in the art, recombinant DNA constructs also typically comprise other regulatory elements in addition to a promoter such as, but not limited to, 3 'untranslated regions (such as polyadenylation sites or transcription termination signals). , transit or signal peptides, introns, and marker gene elements. A 3 'untranslated region (3' UTR) useful in the practice of the present invention is the 3 'UTR of globulin 1 (Kriz, Biochem. Genet., 27: 239-251, 1989; Belanger and Kriz, Genetics, 129: 863-872, 1991; bp 3080 to 4079 of SEQ ID NO: 7). A particularly useful transit peptide is the corn DHDPS transit peptide (Frisch et al., Mol.Gen. Genet., 228: 287-293, 1991; bp 1930 to 2100 of SEQ ID NO: 7). An intron useful in the context of the present invention is intron 1 of rice actin 1 (McEIroy et al., Plant Cell, 2: 163-171, 1990; pb 1448 to 1928 of SEQ ID NO: 7). As used herein, the term "corn" means Zea mays, and includes all plant varieties that can be multiplied with corn, including wild corn species. Methods and compositions for the transformation of plants by introducing an exogenous DNA into the genome of a plant in the practice of this invention may include any of the well-known and demonstrated methods. To date, the supply of genes mediated by Agrobacterium and by microparticles, are the two methods of transformation of plants that are used more commonly. Microparticle mediated transformation refers to the delivery of coated DNA onto microparticles that are driven into target tissues by various methods. The transformation mediated by Agrobacterium is achieved through the use of a genetically engineered soil bacterium that belongs to the genus Agrobacterium. Several species of Agrobacterium mediate the transfer of a specific DNA known as "T DNA," which can be genetically engineered to carry any desired piece of DNA in many plant species. Preferred plant transformation methods are bombardment of microprojectiles, as illustrated in the U.S. Patents. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865; and transformation mediated by Agrobacterium, as illustrated in the patents of E.U.A. 5,635,055; 5,824,877; 5,591, 616; 5,981, 840; and 6,384,301, which are incorporated herein by reference. As used herein, a "transgenic" organism is one whose genome has been altered by the incorporation of foreign genetic material or additional copies of native genetic material, for example, by transformation or recombination. The transgenic organism can be a plant, mammal, fungus, bacteria or virus. As used herein, the term "transgenic plant" means a plant or plant of stably transformed progeny of any subsequent generation derived therefrom, wherein the DNA of the plant or progeny thereof contains an introduced exogenous DNA not present originally in a non-transgenic plant of the same strain. The transgenic plant may also contain sequences that are native to the plant that is being transformed, but where the exogenous DNA has been altered to alter the level or pattern of expression of the gene. As used herein, a "stably" transformed plant is a plant in which the exogenous DNA is inheritable. The exogenous DNA can be inheritable as a fragment of DNA maintained in the plant cell, and not inserted into the genome of the host. Preferably, the stably transformed plant comprises the exogenous DNA inserted into the chromosomal DNA in the nucleus, mitochondria or chloroplast, more preferably in the nuclear chromosomal DNA. As used herein, a "R0 transgenic plant" is a plant that has been directly transformed with an exogenous DNA, or has been regenerated from a cell or group of cells that have been transformed with an exogenous DNA. As used herein, the term "progeny" means any subsequent generation, including seeds and plants thereof, which is derived from a particular progenitor plant or group of progenitor plants; the resulting line of progeny can be inbred or hybrid. The progeny of a transgenic plant of the present invention can be, for example, cross-linked, crossed with a transgenic plant, crossed with a non-transgenic plant, and / or backcrossed. The seeds of the plants of the present invention can be harvested from fertile transgenic plants, and can be used to develop generations of progeny plants of the present invention, including a line of hybrid plants comprising the exogenous DNA of event LY038, which provides the benefit of the increased content of lysine in the corn kernel. The corn kernel can be processed into wholemeal flour and oil products, or the grain can be fed to animals without processing. The whole flour product contains in particular an enhanced agronomic trait, increased lysine content. The present invention contemplates an integral maize flour with increased lysine content with respect to other whole corn flours, where the whole corn meal comprises the exogenous DNA of the LY038 event. The term "event DNA LY038" refers to a DNA segment comprising an exogenous DNA inserted at a particular site in a genome, and adjacent flanking genomic DNA that would be expected to be transferred to a progeny plant of a progenitor plant that it contains exogenous DNA. More specifically, the DNA of the LY038 event also refers to each of the DNA regions that include an interface of the genomic DNA and the exogenous DNA inserted in the genome of the Ro transformant, for example, a region around an interface where the 5 'end is in genomic DNA, and the 3' end is in exogenous DNA. In addition, the exogenous DNA sequence comprising an event DNA can be altered while it is in its particular location in the genome of a host, for example, a portion of the sequence can be changed, deleted or amplified, and still constitute said DNA of the event, provided that said exogenous DNA continues to reside in the same place in the genome. A transgenic "event" occurs by transforming a plant cell with an exogenous DNA construct, the regeneration of a plant resulting from the insertion of exogenous DNA into the genome of the plant, and selection of a particular plant characterized by DNA from the event. Typically, many plant cells are transformed, producing a population of plants from which a particular plant is selected. The term "event" refers to the original transformant Ro and progeny of the transformant that includes the exogenous DNA inserted in a particular and unique site in the genome, ie, DNA of the event. The term "event" also refers to progeny that is produced by repeated sexual exogamy, cross-hatching or backcrossing, where at least one of the plants used in breeding is from any generation of the original R0 transformant that contains DNA of the event. In this way, a transgenic "event" is a plant that understands and is defined by a "DNA of the event". In this way, "event LY038" includes "DNA of event LY038". A plant can comprise two or more DNA molecules of different events, and can thus comprise two or more different events. In addition, a plant that lacks a given transgenic event X does not include that DNA of event X in question. The DNA of the event can be transferred from plant to plant, generation to generation, by any genetic improvement scheme, method or tool known to those skilled in the corn breeding technique. Plant transformation typically uses a selectable marker and selection method to distinguish transformed cells from the culture of untransformed cells. In some cases, the selectable marker gene remains in the transgenic plant; in other cases, it is desirable to remove the selectable marker gene or other sequences introduced into the exogenous DNA. Homologous recombination is a useful method for the deletion of marker genes that reside within a transgenic plant (see U.S. Patent No. 6,580,019, incorporated herein by reference in its entirety). Another useful tool for removing sequences from a plant, includes the use of site-specific recombinase enzymes, and their respective site-specific target sites. Many different site-specific recombinase systems could be used in accordance with the present invention and include, but are not limited to, the Cre / lox system of bacteriophage P1, and the FLP / FRT system of yeast. The Cre / lox system of bacteriophage P1 and the FLP / FRT yeast system are two particularly useful systems for integration or excision of site-specific transgenes. In these systems, a recombinase (Cre or FLP) will interact specifically with its respective site-specific recombination sequence (lox or FRT, respectively), to invert or separate the intervening sequences. The sequence for each of these two systems is relatively short (34 bp for lox, and 47 bp for FRT) and, therefore, convenient for use with transformation vectors. It has been shown that the FLP / FRT and Cre / lox recombinase systems work efficiently in plant cells. In a preferred embodiment, a Cre / lox recombinase system is used for removed selectable marker sequences, in particular a NPT II marker gene (see Figure 1) flanked by lox P recombination sites (bp 4091 to 4124 of SEQ ID NO. : 7, Russell et al., Mol. Gen. Genet. 234: 45-59, 1992). A transgenic plant, seed or parts thereof, which shows an intensified desired trait, for example "increased lysine content", is a plant comprising an exogenous DNA that imparts a desired measurable change in a trait as compared to a plant. substantially the same genotype that lacks the desired exogenous DNA. Preferably, the intensified desired trait is measured by comparing the trait in a transgenic plant possessing the exogenous DNA associated with the intensified desired trait, with the trait in a plant of substantially the same genotype, but lacking the exogenous DNA. Said plant lacking the exogenous DNA may be a wild type natural plant or a transgenic plant, preferably of the same species as the transgenic plant. Preferably, the plant lacking the exogenous DNA is a co-descendant lacking the desired exogenous DNA of the plant comprising the desired exogenous DNA. Said co-descendant plant may comprise other exogenous DNA molecules. The increased content of lysine can be exhibited by the plant by the accumulation of increased amounts of the amino acid in the grain, and can be measured by any suitable method, as is the case of mass spectrometry or liquid chromatography of high-performance tissue extracted properly. As used herein, a "probe" is an isolated oligonucleotide to which a detectable label or reporter molecule can be attached, eg, a radioactive isotope, ligand, chemiluminescent agent, dye or enzyme. Said probe is complementary to a chain of a target nucleic acid. In the case of the present invention, said probe is complementary to a genomic DNA strand of event LY038, for example, genomic DNA from a maize plant or seed or other part of the plant of event LY038. The probes according to the present invention are materials, including DNA, RNA and polyamides, that specifically bind to a target DNA sequence, and can be used to detect the presence of that target DNA sequence. "Initiators" are isolated oligonucleotides that can bind to a complementary target DNA chain by nucleic acid hybridization, and which can then be extended along the target DNA chain by the action of a polymerase, for example, a DNA polymerase. As used herein, the primers of the present invention are used for DNA amplification of a target nucleic acid sequence, for example, by the polymerase chain reaction (PCR), and can also be referred to as "DNA primers". PCR. " The probes and primers are of sufficient nucleotide length to bind to the target DNA sequence specifically under the conditions of hybridization or reaction conditions determined by those skilled in the art. This length can be any length that is of sufficient length to be useful in the detection method of choice. In general, about 11 nucleotides or more in length, preferably about 18 nucleotides or more, more preferably about 24 nucleotides or more, and most preferably about 30 nucleotides or more are used. Said probes and primers specifically hybridize with a target. Preferably, the probes and primers according to the present invention have complete similarity of nucleotide DNA sequences contiguous with the target sequence, although probes differ from the target DNA sequence and retain the ability to hybridize with DNA sequences objective, can be designed by conventional methods. Methods for the preparation and use of probes and primers are known to those skilled in the art, using published protocols, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, 1989 , and similar. The identification of flanking genomic DNA sequences around the insertion site of transgenic events allows the design of detection methods that are specific for a particular transgenic event inserted at a particular site in a genome. Said detection method, which can differentiate between the same transgene or similar transgenes located at different insertion sites in a genome, is called an event-specific DNA detection method, for example, an event-specific test. Event-specific tests have been described, for example, for event nk603 of glyphosate-tolerant corn (see U.S. Patent Application Publication 2002/0013960, incorporated herein by reference in its entirety). In a preferred embodiment, a nucleic acid probe of the present invention hybridizes specifically with the specific amplicon of the LY038 event having the nucleic acid sequence of SEQ ID NOs: 3-6, or complements thereof, more preferably SEQ ID NO. : 5, or complements thereof. In another aspect of the present invention, a preferred nucleic acid probe molecule of the present invention shares between about 80%, preferably about 90%, more preferably about 95%, even more preferably about 98%, and most preferably about 99 % sequence identity with the nucleic acid sequence set forth in one or more of SEQ ID NOs: 3-6, or complements or fragments thereof. Examples of probes that serve as diagnostics for event LY038, have the sequence of SEQ ID NO: 6. Those skilled in the art can use such probe molecules as markers in breeding methods to identify the progeny of genetic crosses. Hybridization of the probe with the target DNA molecule can be detected by any of many methods known to those skilled in the art. These detection methods may include, but are not limited to, fluorescent labels, radioactive labels, antibody-based labels, and chemiluminescent labels. As used herein, the term "homologous" refers to a nucleic acid sequence that will specifically hybridize to the complement of the nucleic acid sequence with which it is being compared under conditions of high stringency. Appropriate severity conditions, which include conditions of time, temperature and salt condition, which promote DNA hybridization, are known to those skilled in the art. Both the temperature and the concentration of salts can be varied, or the temperature or concentration of salts can be kept constant, while changing the other variable. In a preferred embodiment, a polynucleic acid of the present invention will hybridize specifically to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1 or 2, or complements thereof or fragments of any thereof, under conditions of strongly to moderately severe. In a particularly preferred embodiment, a nucleic acid of the present invention will hybridize specifically to one or more of the nucleic acid molecules set forth in SEQ ID NO: 1 or 2, or complements or fragments of any of the same under conditions of high stringency. Hybridization of the probe with the target DNA molecule can be detected by any of many methods known to those skilled in the art.; such methods may include, but are not limited to, fluorescent labels, radioactive labels, antibody-based labels, and chemiluminescent labels. As used herein, the term "amplicon" refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether the maize plant resulting from a sexual cross contains genomic DNA from the transgenic event of the maize plant comprising exogenous DNA from LY038, the DNA extracted from a tissue sample from the maize plant can be subjected to a nucleic acid amplification method using a pair of DNA primers including a first primer derived from the flanking sequence in the plant genome adjacent to the insertion site of the inserted heterologous DNA, and a second primer derived from the heterologous DNA inserted for produce an amplicon that serves as a diagnostic for the presence of the DNA of the event. The amplicon is of a length and has a sequence that also serves as a diagnostic for the event. The amplicon can vary in length from the combined length of the primer pairs plus a pair of nucleotide bases, preferably more about 20 base pairs of nucleotide, more preferably more about 50 base pairs of nucleotide, and even more preferably more approximately 150 nucleotide base pairs and more, depending on the method used to detect the amplicon. Alternatively, a pair of primers can be derived from the flanking sequence on both sides of the inserted DNA, to produce an amplicon that includes the nucleotide sequence of the entire insert. A member of a pair of primers derived from the genomic sequence of the plant can be located at a distance from the inserted DNA sequence. This distance may vary from a pair of nucleotide bases to the limits of the amplification reaction, or about 20,000 base pairs of nucleotide. The use of the term "amplicon" specifically excludes primer dimers that can be formed in the thermal amplification reaction of the DNA. Amplification of the nucleic acid can be achieved by any of several nucleic acid amplification methods that are known in the art, including the polymerase chain reaction (PCR). The sequence of the heterologous DNA insert or flanking DNA sequence of event LY038 can be verified (and corrected if necessary), amplifying said sequences from DNA extracted from the seed or plants of the ATCC deposit accession number PTA-5623, using DNA primers derived from the sequences provided herein, followed by standard DNA sequencing of the PCR amplicon or cloned DNA. Probes and primers based on the insertion sequences and the flanking genomic DNA that are described herein, can be used to confirm (and, if necessary, to correct) the described DNA sequences by conventional methods, for example, by recloning and sequencing of said DNA molecules. The amplicons produced by the amplification methods can be detected by a plurality of techniques including, but not limited to, gel-based analyzes, analysis of genetic fragments (Nikiforov et al., Nucleic Acid Res., 22: 4167-4175, 1994), pyrosequencing (Winge, M., Pyrosequencing - a new approach to DNA analysis, (2000), Innovations in Pharmaceutical Technology, vol. 00, 4, p18-24), fluorescence polarization (Chen et al., Genome Res. ., 9: 492-498, 1999) and molecular guides (Tyangi et al., Nature Biotech., 14: 303-308, 1996). Of particular interest for the present invention is detection by Taqman® test (available from Applied Biosystems, Foster City, California). The Taqman® test is a method of detecting and quantifying the presence of a DNA sequence that is well known in the art., and is fully described in the instructions provided by the manufacturer. This method includes the use of PCR amplification and detection of the amplification product by hybridization, using a special FRET oligonucleotide probe. The FRET oligonucleotide probe is designed to have a 5 'fluorescent reporter dye and a 3' quench dye covalently linked to the 5 'and 3' ends of the probe. The probe is designed to overlap the junction of the genomic DNA and the inserted DNA. The FRET probe and the PCR primers (one primer in the exogenous DNA sequence of the transgene and one in the flanking genomic sequence) are passed through a cycle in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in digestion and release of the fluorescent portion away from the extinction portion in the FRET probe. A fluorescent signal indicates the presence of the transgene / flanking insertion sequence due to successful amplification and hybridization. PCR primers that are preferred for use with the Taqman® test are designed (a) to have a length in the size range of 18 to 25 bases, and mating sequences in the flanking genomic DNA and the transgene insert , (b) to have a melting temperature calculated on the scale from about 57 to about 60 ° C, for example, which corresponds to an optimal PCR binding temperature of about 52 to about 55 ° C, and (c) to obtain a product that includes the junction between the flanking genomic DNA and the transgene insertion, and has a size scale length of about 75 to about 250 base pairs. PCR primers are preferably located at the locus, so that the binding sequence is at least one base away from the 3 'end of each PCR primer. PCR primers should not contain regions that are extensively self-complementary or intercomplementary. FRET probes are designed to span the sequence of the binding sequence. In a preferred embodiment, the FRET probes will have incorporated at their 3 'end a chemical portion which, when the probe is attached to the template DNA, joins the minor DNA groove, thereby enhancing the stability of the probe-template complex . The probes preferably have a scale length of about 12 to about 17 bases, and with the minor slot junction portion 3 ', have a calculated melting temperature of about 5 to about 7 ° C above the temperature of PCR primers. The design of the probe is described in the patents of E.U.A. Nos. 5,538,848; 6,084,102; and 6,127,121. Another test using the sequences of the present invention is a proof of zygosity. A zygosity test is useful to determine if a plant comprising an event is homozygous for the event DNA, that is, it comprises the exogenous DNA at the same site on each chromosome of a chromosome pair, or heterozygous for the event DNA , that is, it comprises the exogenous DNA in only one chromosome of a chromosomal pair. In one embodiment, a three-primer test is used, wherein the primer 1 hybrid and extends specifically from the inserted exogenous DNA, the hybrid primer 2 and extends specifically from the DNA flanking the 5 'side of the exogenous DNA. inserted, and the hybrid primer 3 extends specifically from the DNA flanking the 3 'side of the inserted exogenous DNA. The three initiators serve as a diagnostic for the event. Typically, the exogenous DNA is such a size, for example, about 3 to about 7 kilobases or more, that initiator 1 and initiator 3 fail to produce an amplicon in the PCR reaction. When the three primers are mixed together in a PCR reaction with DNA extracted from a homozygous plant for a given event, an individual amplicon is produced by primer 1 and primer 2, whose size and sequence will be indicative of the event DNA, and will serve as a diagnostic for it. When the three primers are mixed together in a PCR reaction with DNA extracted from a plant that does not comprise the determined event, an individual amplicon is produced by primer 1 and primer 3, whose size and sequence will be indicative of corn genomic DNA , and will serve as a diagnostic for it, which lacks an exogenous DNA. When the three primers are mixed together in a PCR reaction with DNA extracted from a plant that is heterozygous for a given event, 2 amplicons are produced: 1) an amplicon is produced by primer 1 and primer 3, whose size and sequence they will be indicative of maize genomic DNA, and serve as a diagnostic for it, lacking an exogenous DNA, and 2) an amplicon is produced by primer 1 and primer 2, whose size and sequence will be indicative of event DNA, and will serve as a diagnostic for it. Methods for the detection of various amplicons produced by the zygosity test are known to those skilled in the art and include, but are not limited to, gel electrophoresis, Taqman® tests, Southern blot, Invader technology, sequencing, molecular guides, pyrosequencing, and the like. DNA detection equipment can be developed, using the compositions described herein and methods well known in the art of DNA detection. The kits are useful for the identification of DNA from the LY038 event of maize in a sample, and can be applied to methods to genetically improve maize plants that contain DNA from event LY038. The kits contain DNA sequences that are useful as primers or probes, and that are homologous or complementary to any portion of SEQ ID NO: 1 or 2, or to DNA sequences homologous or complementary to DNA contained in any of the genetic elements of the transgene. from pMON55221 (figure 1), which have been inserted into the genome of a corn plant to form event LY038 (figure 2A). These DNA sequences can be used in DNA amplification methods (PCR), or as probes in polynucleic acid hybridization methods, i.e., Southern analysis or Northern analysis. The DNA molecule (SEQ ID NO: 7) contained in the genome of event LY038 comprises the heterologous genetic elements of the transgene, which include a corn globulin 1 promoter (P-ZM globl), an intron of rice actin 1 ( l-Os actin), a DNA molecule that codes for dihydrodipicolinate synthase transit peptide in corn chloroplasts (Zm DHDPS CTP), a DNA molecule that codes for dihydrodipicolinate synthase (DHDPS) from Corynebacterium, a 3 'untranslated region of corn globulin 1 (T-Zm globl) and a lox P site, and can be used as a template for DNA amplification, or to select homologous or complementary DNA molecules that can be used as an initiator or DNA probe in a DNA detection method. The present invention contemplates that one skilled in the art of DNA detection may select one or more DNA molecules homologous or complementary to the transgenic DNA of SEQ ID NO: 7 that are useful in a method for detecting transgenic DNA in the genome of LY038 , and progeny thereof. The following examples are included to demonstrate examples of certain preferred embodiments of the present invention. Those skilled in the art should appreciate that the techniques described in the following examples represent methods that the inventors have found to work well in the practice of the present invention, and thus can be considered to constitute examples of preferred modes for their practice. However, those skilled in the art should appreciate, in light of the present disclosure, that many changes can be made in the specific embodiments described, and that an equal or similar result is obtained without departing from the spirit and scope of the invention.

EXAMPLE 1 Preparation of transgenic plants Immature embryos of the corn line H99 were isolated for transformation. Adhered to gold particles, cassette DNA isolated from vector pMON55221 (see Figure 1) comprising a corn globulin 1 promoter (Kriz (1989), cited above; Belanger and Kriz (1991), cited above; US No. 6,329,574, incorporated herein by reference in its entirety, bp 48 to 1440 of SEQ ID NO: 7), an intron of rice actin 1 (McEIroy et al. (1990), cited above, pp. 1448 to 1928 of SEQ ID NO: 7), a DNA molecule encoding DHDPS transit peptide in corn chloroplasts (Frisch et al. (1991), cited above, pb 1930 to 2100 of SEQ ID NO: 7), a molecule of DNA encoding Corynebacterium dihydrodipicolinate synthase (Bonnassie et al. (1990), cited above; Richaud et al. (1986), cited above; pb 2101-3003 of SEQ ID NO: 7), a 3 'untranslated region of corn globulin 1 (Belanger and Kriz (1991), cited above, bp 3080 to 4079 of SEQ ID NO: 7), a lox P site (patent of E.U.A. No. 5,658,772, specifically incorporated herein by reference in its entirety; bp 4091 to 4124 of SEQ ID NO: 7), as well as a 35S promoter (Kay et al., Science, 236: 1299-1302, 1987; U.S. Patent No. 5,164,316), a DNA molecule encoding selectable marker from NPTII (Potrykus et al. (1985), cited above), a UTR 3 'nos (Fraley et al., Proc. Nati. Acad. Sci. (USA), 80: 4803-4807 (1983), cited above) and a lox P site (U.S. Patent No. 5,658,772, specifically incorporated herein by reference in its entirety). Microprojectile bombardment was used to introduce the exogenous DNA to the immature embryos of the corn, using methods known to those skilled in the art. Transformed cells were selected using a kanamycin selection scheme. Callus resistant to kanamycin was obtained, and regenerated in several fertile R0 transgenic plants using standard methods.

EXAMPLE 2 Scheme of genetic improvement and analysis of lysine Table 1 summarizes the genetic improvement scheme and free lysine data used for the development of the maize LY038 event, which exhibits high lysine content in corn grain tissue. Plants produced by the transformation method described in Example 1, were initially selected for the presence or absence of the Corynebacterium DHDPS sequence using PCR. Taqman® test technology was used to determine the number of copies of transgenic inserts. Plants comprising a DNA molecule with the Corynebacterium DHDPS sequence, operably linked as described in Example 1 and Figure 1, were allowed to reach maturity and produce F1A seed. For the production of F1A seed, the transgenic primary Ro plants were crossed with an inbred line of non-transgenic elite corn. The F-? A plants were selected for the presence or absence of the NPTII sequence, as evidenced using a field-evaluable kanamycin resistance test. The lack of sensitivity to kanamycin indicated that a plant comprised and was expressing the NPTII marker gene as shown in Figure 1 and described in Example 1; these plants are later referred to as NPTII +. NPTII + plants were crossed with a transgenic corn line that expressed a bacterial recombinase Cre to produce FIB seed. The levels of free lysine were determined in a sample of the resulting F1B seed collected from each ear. F -? Co-descendant grains exhibiting more than about 1000 ppm free lysine were exposed to a field nursery. Progeny plants F-? _ Were tested by PCR and / or Southern blot to determine the presence or absence of the DHDPS gene sequence, the Cre recombinase coding sequence and the selectable marker gene sequence of NPTII. The NPTII selectable marker gene was flanked by lox P sites (recombination sites) andAs such, the activity of Cre recombinase resulted in the excision of the coding sequence of NPTII. Plants comprising the DHDPS and Cre recombinase sequences and lacking the NPTII sequences, referred to below as plants with suppressed marker, were allowed to self-pollinate to produce F2A seed. The free lysine content in the positive and negative F2A seed was determined. Having obtained F2A seed comprising the exogenous DHDPS gene of interest and lacking the NPTII selectable marker gene, it was now necessary to select the Cre recombinase sequences. The F2A seed of plants with suppressed marker was seeded in the field, and once again tested by PCR and / or Southern blot to determine the presence or absence of the DHDPS gene sequence, the Cre recombinase and the sequence of the NPTII selectable marker gene. Plants comprising the DHDPS sequence and lacking sequences for Cre recombinase and NPTII were selected as "positive" plants. Co-descendant plants lacking the sequences of DHDPS, Cre recombinase and NPTII were selected as "negative" plants, to serve as negative controls. Plants that comprised the DHDPS sequence were self-pollinated to create F3 seed and exposed to the next generation in the field. Also, negative plants lacking the DHDPS, Cre recombinase and NPTII sequences were self-pollinated to create F3 seed. The free lysine content in the positive and negative F3 seed was determined. Positive plants were developed in the field from seed F3 An individual plant, determined by Taqman® test as homozygous, was selected and designated as LY038. Plants F3 were, A) self-pollinated to produce cobs F-38, or B) crossed with an inbred line to produce cobs FI-3SA of positive and negative selections. The free lysine content in the positive and negative F4-38 seed was determined. Seed F ^ SA of event LY038 was developed in the field, and allowed to self-pollinate to produce seed F2_ for which the free lysine content was determined. Seed F4-38 of event LY038 was developed in the field to produce F4-38 plants, and A) was allowed to self-pollinate to produce F5-38 seed, or B) crossed with an inbred line to produce hybrid F1-38B seed used for agronomic evaluation. Seed F-38 of event LY038 was developed in the field, and A) self-pollinated to produce seed F6-38, or B) crossed with a variety of inbred line maize to produce additional hybrid F2C seed used for further agronomic evaluation. Monsanto Company corn seed deposits were generated from the LY038 event described above, through the self-pollination of F5-38 plants to produce F6-38 seed and the self-pollination of Fd-38 plants to produce seed F7-38- The number of access of the American Type Culture Collection (Manassas, VA) for the event LY038, is PTA-5623. The accumulation of free lysine was monitored during the development of corn lines of the LY038 event. The lysine accumulation values are summarized in Table 1, and represent the amount of free lysine present in the mature grain on a dry weight basis, in parts per million. Different methods are useful for evaluating the lysine content of mature grains that comprise event LY038. Other methods known in the art that are useful for detecting and quantifying the lysine content are contemplated by the inventors of the present invention, to provide similar findings of an increase in the seed lysine content of the LY038 event. Liquid chromatography-mass spectrometry / mass spectrophotometry (LC-MS / MS) was used to analyze the free lysine content in the corn kernels of event LY038. Individual mature corn grain samples from event LY038 were weighed first, ground to a fine homogeneous powder, and extracted with an extraction solvent comprising methanol, water and formic acid. In situations where the grains were piled, approximately 30 mg of ground powder was used. Multiple reaction monitoring mass spectrometric (MRM) and liquid chromatography techniques were used to separate the lysine in the sample extract. After separation, lysine was quantified using its maximum area of mass spectrometry against its corresponding standard calibration curve that was prepared using an internal standard (IS) of deuterized d-lysine. In another method, the lysine content of corn kernels was based on the evaluation of free amino acids by high performance liquid chromatography (HPLC). Individual corn grains or groups of grains of event LY038 were milled to a fine homogeneous powder as described and, in this case, approximately 30 mg of powder was used for analysis. Amino acids were extracted with trichloroacetic acid at 5%, and amino acid detection was achieved through pre-derivatization of primary amines in a column with o-phthalaldehyde (OPA). The resulting amino acid adduct, an isoindol, is hydrophobic and has excellent fluorescence characteristics, which can then be detected in a fluorescence detector. Through the use of inverted phase chromatography, separation is achieved through the hydrophobic nature of the R groups located in each amino acid. To help stabilize the fluorophore, a thiol such as 2-mercaptoethanol (SHCH2CH2OH) or 3-mercaptopropionic acid (SHCH2CH2COOH) is added.

TABLE 1 Genetic improvement scheme and lysine analysis used to identify high lysine corn, event LY038 Represents the ppm of free lysine in mature grains comprising event LY038 ND = not determined - indicates less than about 400 ppm of free lysine + indicates approximately 1000 to approximately 1200 ppm of free lysine ++ indicates approximately 1200 to approximately 1400 ppm of free lysine +++ indicates more than approximately 1400 ppm of free lysine i = Inbred grain data.

Based on the experiments described herein, the free lysine content in corn grains containing the construction LY038, increased between approximately 200% (for example, FIB, among others) and almost 300% (for example, F-? A). Intermediate increases in free lysine content were also observed (e.g., F? -38A).

EXAMPLE 3 Determination of the flanking sequence Genomic DNA was isolated from maize plants designated as LY038, and was used in experiments to determine the genomic sequence of the corn flanking the insertion of transgenic DNA. Three different methods were used to determine the flanking sequences and the sequence of the junction between the genomic flanking sequence and the transgenic insert: final PCR and the CloneTech Genome Walker ™ kit (catalog number K1807-1, ClonTech Laboratories, Palo Alto, California), and inverted PCR. The final PCR is a method for isolating genomic DNA sequences flanking a known insert sequence, using degenerate primers and a biotin capture step. A primer complementary to exogenous DNA is used in the primary PCR reactions together with a variety of degenerate primers. Typically, specific primers were used for the exogenous DNA and degenerate primers in pairs and not in groups. The degenerate primers hybridize to a certain degree with the genomic sequence of the corn flanking the inserted DNA that allows the generation of PCR amplicons. The primary PCR amplicons are mixed with a biotin-labeled primer complementary to the transgene portion of the amplicon, and allowed to bind. The amplicons attached to the biotin primers were captured using streptavidin, and unbound amplicons were entrained. The bound amplicons were subjected to secondary PCR reactions using a nested primer that was complementary to the exogenous DNA portion of the amplicon, and a variety of degenerate primers. The PCR amplicons from the secondary PCR reaction were subjected to agarose gel electrophoresis, and bands were cut from the gel and isolated. The isolated PCR amplicons were sequenced. The 3 'flanking genomic DNA sequence of event LY038 was identified using final PCR and sequencing. The Genome Walker method for flanking DNA isolation was carried out according to the conditions suggested by the manufacturer. Final PCR and the Genome Walker team were used to identify the 5 'flanking DNA sequence of event LY038. For Genome Walker, products of the restriction enzyme Seal were amplified to produce amplicons useful for the identification of the 5 'flanking genomic DNA sequence of the LY038 event. The use of the final PCR and the Genome Walker methods generated several hundred base pairs or more of DNA sequence flanking the insertion site of the DNA construct in the LY038 event. Inverted PCR and bioinformatics analysis and comparison with maize genomic DNA sequence databases were used to obtain additional genomic DNA flanking these events. Through the use of the combined methods, the flanking sequences in the sequence of SEQ ID NOs: 1, 2, 9 and 10 are identified. A corn plant is an aspect of the present invention, when that corn plant contains within its genome a DNA molecule that can be used as a template in a DNA amplification reaction to provide an amplicon comprising a DNA binding molecule described in the present invention, wherein the DNA binding molecule serves as a diagnostic for DNA of event LY038 of the corn in a sample of DNA extracted from a sample of corn tissue.

EXAMPLE 4 Specific event initiator and probe test information For each event, useful PCR primers and probes were designed in a Taqman® test, namely, SEQ ID NOs: 3 and 4. The use of PRC primers having the sequence of SEQ ID NOs: 3 and 4 in one Taqman® test, resulted in an amplicon that serves as a diagnostic for the event LY038; the amplicon has the sequence of SEQ ID NO: 6, and the probe useful for the detection of this amplicon has the sequence of SEQ ID NO: 5. When the primers and the probes were subjected to the PCR conditions outlined in Table 2 , a fluorescent signal indicated that amplicons were produced that were not detected by the probe. By including the appropriate control samples, eg, several positive and negative DNA controls, it was shown that the PCR primers and the probes were specific for the desired event. In addition to the initiator and probe kit, any initiator and probe equipment derived from SEQ ID NO: 1 or 2, specific for DNA from event LY038 that when used in a PCR amplification reaction produces a DNA amplicon that serves as a diagnostic for DNA of event LY038, is an aspect of the present invention, and is readily prepared by those skilled in the art. PCR conditions that produce a Taqman® test that serves as diagnostics for DNA from event LY038 are included in Table 2. The skilled artisan would include the appropriate control samples when carrying out the PCR or Taqman® assays described herein. invention. The inclusion of positive control DNA samples, negative control DNA samples and other controls is adequate, and helps in the interpretation of results. In addition, the person skilled in the art would know how to prepare internal control primers and probes for the Taqman® PCR reaction using standard methods published, for example, by Applied Biosystems, Foster City, California. The person skilled in the art would also realize that the particular initiator sequences, probes and reaction conditions specified herein, can be modified and produce a diagnostic test for DNA from event LY038. In addition, the person skilled in the art would know that the products of the PCR reaction can be analyzed by gel electrophoresis for analysis.

TABLE 2 Mixture of PCR reaction and diagnostic conditions for DNA of event LY038 mixed primers resuspended in water at 18 megaohms at a concentration of 20 μM of each primer Example: 100 μl of the first primer at a concentration of 100 μM, 100 μl of the second primer at a concentration of 100 μM, 300 μl of water at 18 megaohms ** Probe resuspended in water at 18 megaohms, at a concentration of 10 μM *** May include, but is not limited to, negative DNA control (eg, non-transgenic DNA) negative water control (no template DNA) control positive (event LY038) Sample DNA (from leaf, seed and other parts of the plant samples) Combinations of internal control probe and primer can be made with a wide variety of genes or genomic regions, the design of which is known to experts in the art.

Deposits were made from the corn seed of Monsanto Company representative of the event LY038 described above, under the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110. The ATCC access number for event LY038 is PTA-5623. The deposit will be kept in the depository for a period of 30 years, or 5 years after the last application, or during the effective life of the patent, which lasts longer, and will be replaced as necessary during that period. Having illustrated and described the principles of the present invention, it should be apparent to those skilled in the art, that the present invention can be modified in detail and arrangement without departing from said principles. All modifications that are within the spirit and scope of the appended claims are claimed.

Claims (43)

NOVELTY OF THE INVENTION CLAIMS

1. - A DNA construct comprising, in an operably linked array: (a) a first DNA segment comprising a corn globulin 1 promoter; (b) a second DNA segment comprising an intron of rice actin 1; (c) a third DNA segment comprising a DNA molecule encoding DHDPS transit peptide in corn chloroplasts; (d) a fourth DNA segment comprising a DNA molecule encoding Corynebacterium DHDPS; and (e) a fifth DNA segment comprising a 3 'untranslated region of corn globulin 1.

2. The DNA construction according to claim 1, further characterized in that the third DNA segment is SEQ ID NO: 8.

3. The construction of DNA in accordance with the claim 1, further characterized in that it also comprises a recombination site.

4. The DNA construct according to claim 3, further characterized in that said recombination site is a lox P site.

5. The DNA construct according to claim 4, further characterized in that it has SEQ ID NO: 7

6. A stably transformed monocotyledonous plant comprising the DNA construct according to any of claims 1 to 5 in its genome.

7. The monocot plant stably transformed according to claim 6, further characterized in that said plant is a variety of corn.

8. The monocot plant stably transformed according to claim 6, further characterized in that the plant generates more lysine in comparison with a second plant having a substantially similar genotype, in which the second plant does not include the construction of DNA.

9. The monocot plant stably transformed according to claim 6, further characterized in that the genome of said transgenic maize plant comprises SEQ ID NO: 1 or 2.

10. The monocot plant stably transformed according to claim 9, further characterized in that the genome of said transgenic maize plant comprises DNA from event LY038, and wherein the DNA of event LY038 comprises SEQ ID NO: 7 flanked by the pb (base pairs) 1 to 1781 of SEQ ID NO: 1 and bp 201 to 867 of SEQ ID NO: 2.

11. Seed of progeny of a stably transformed maize plant according to claim 7, wherein said progeny comprises the DNA construct according to any of claims 1 to 5 in its genome.

12. - A method for producing a progeny plant comprising DNA from event LY038 in its genome, comprising the step of crossing a first progenitor maize plant comprising DNA of event LY038 in its genome, with a second progenitor maize plant, in wherein said progeny plant comprises DNA of event LY038 in its genome, and wherein the DNA of event LY038 comprises SEQ ID NO: 7 flanked by bp 1 to 1781 of SEQ ID NO: 1 and bp 201 to 867 of SEQ ID NO: 2. The method according to claim 12, further characterized in that it further comprises the steps of: (a) crossing the first progeny plant with itself or with a third maize plant to produce a seed of a second progeny plant of a subsequent generation; (b) developing a second progeny plant of a subsequent generation from said seed, and crossing the second progeny plant of a subsequent generation with itself or a fourth maize plant, to produce a seed of a third progeny plant of a subsequent generation; and (c) repeating steps (a) and (b) for at least one additional generation, to produce an inbred maize plant derived from the maize plant comprising event LY038. 14. The method according to claim 12, further characterized in that it comprises: (a) obtaining a DNA sample from a progeny corn plant of any generation; (b) contacting the DNA sample with a labeling nucleic acid molecule homologous or complementary to a DNA molecule selected from the group consisting of SEQ ID NOs: 1-6; and (c) carrying out a markers-assisted breeding method for event LY038, wherein plants are selected that are genetically linked to a complement of the marking nucleic acid molecule; and (d) selecting said plants for high lysine content. 15. The method according to claim 12, further characterized in that said second maize plant does not comprise an exogenous DNA identified as event LY038, and wherein said second maize plant is of a genotype different from that of said maize plant than it comprises an exogenous DNA identified as event LY038, thus producing a hybrid corn plant. 16. A pair of DNA primer molecules suitable for detecting DNA from event LY038, comprising: a first DNA molecule and a second DNA molecule, wherein each of the molecules comprises at least 12 contiguous nucleotides that share at least about 90% sequence identity with a portion of SEQ ID NO: 1, and wherein said first DNA molecule is homologous or complementary to the maize genome portion of SEQ ID NO: 1, and said The second DNA molecule is homologous or complementary to the insertion portion of the transgene of SEQ ID NO: 1. 17. The pair of DNA primer molecules according to claim 16, further characterized in that the primers are SEQ ID NO: 3 and SEQ ID NO: 4. 18.- A pair of DNA primer molecules suitable for detecting DNA from event LY038, comprising: a first DNA molecule and a second DNA molecule, wherein each of the molecules comprises at least 12 contiguous nucleotides that share at least about 90% sequence identity with a portion of SEQ ID NO: 2, and wherein said first DNA molecule is homologous or complementary to the maize genome portion of SEQ. ID NO: 2, and said second DNA molecule is homologous or complementary to the insertion portion of the transgene of SEQ ID NO: 2. 19.- A method for determining the presence of an exogenous DNA of LY038 in a tissue sample of corn, which comprises the steps of: (a) obtaining DNA from the corn tissue sample; (b) contacting said DNA with a pair of PCR primers, wherein a first primer of said pair of hybrid PCR primers with a sequence within base pairs 1 to 1781 of SEQ ID NO: 1 or its complement , and wherein a second primer of said pair of hybrid PCR primers with a sequence within the base pairs 1782 to 1961 of SEQ ID NO: 1 or its complement; (c) amplifying DNA with said pair of PCR primers, thereby producing a DNA amplicon molecule comprising at least base pairs 1781 to 1782 of SEQ ID NO: 1 or its complement; and (d) detecting said DNA amplicon molecule. 20. A stably transformed corn plant, which when analyzed by a method according to claim 19, produces a DNA amplicon comprising SEQ ID NO: 5. 21. The method according to claim 19, characterized in addition because said corn tissue sample is derived from a seed or maize plant developed from said seed, wherein a representative seed is a seed of ATCC deposit PTA-5623 or progeny thereof, comprising DNA from event LY038 in its genome. 22. A method for determining the presence of an exogenous DNA of LY038 in a maize tissue sample, comprising the steps of: (a) obtaining DNA from the maize tissue sample; (b) contacting said DNA with a pair of primers, wherein a first primer of said hybrid primer pair with a sequence within base pairs 1 to 200 of SEQ ID NO: 2 or its complement, and a second initiator of said hybrid primer pair with a sequence within base pairs 201 to 867 of SEQ ID NO: 2 or its complement; (c) carrying out a DNA amplification method of said DNA with said pair of primers, thereby producing a DNA amplicon molecule comprising at least base pairs 200 to 201 of SEQ ID NO: 2 or its complement; and (d) detecting said DNA amplicon molecule. 23. A stably transformed corn plant, which when analyzed by a method according to claim 22, produces a DNA amplicon comprising SEQ ID NO: 11. 24.- The method according to claim 22, characterized in addition because said corn tissue sample is derived from a seed or maize plant developed from said seed, wherein a representative seed is a seed of ATCC deposit PTA-5623 or progeny thereof, comprising DNA from event LY038 in its genome. 25. A DNA detection equipment, comprising: at least one DNA molecule, wherein the molecule comprises at least 12 or more contiguous nucleotides that share at least approximately 90% to approximately 100% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, which functions as an initiator or DNA probe in a method specifically designed to detect DNA from the LY038 event of maize, and progeny thereof, in a DNA sample. 26.- A method for producing a maize seed with a higher lysine content, comprising: (a) planting maize seed having in its genome a DNA molecule comprising the DNA construct according to claim 1; (b) developing plants from said corn seed, whereby said DNA molecule is expressed in corn cells to produce maize seed with a higher lysine content than corn seed lacking said DNA molecule; and (c) harvesting said seed. 27. The method according to claim 26, further characterized in that the genome comprises DNA having the sequence selected from the group consisting of SEQ ID NO: 1, 2, 5, 6 and 11. 28.- An integral flour of corn comprising exogenous DNA of LY038. 29. The corn flour in accordance with claim 28, further characterized in that it contains a molecule of Exogenous DNA of LY038, wherein said molecule is detected in a sample containing the DNA of the wholemeal flour by the method according to claim 19. 30.- The cornmeal integral meal according to claim 28, further characterized by contains a molecule of Exogenous DNA of LY038, wherein said molecule is detected in a sample that contains the DNA of the integral flour by the method according to claim 22. 31.- The corn integral flour according to claim 28, further characterized because comprises LY038 DNA selected from the group consisting of SEQ ID NO: 1, 2, 5, 6 and 11. 32.- The corn integral meal according to claim 28, further characterized in that it comprises a DNA molecule of LY038, wherein said molecule is a template in a DNA amplification method, wherein said method provides an amplicon comprising SEQ ID NO: 5 or 11. 33.- A corn endosperm with increased lysine content with respect to other corn endosperms , wherein the corn endosperm comprises exogenous DNA of LY038. 34. The corn endosperm according to claim 33, further characterized in that it contains an exogenous DNA molecule of LY038, wherein said molecule is detected in a sample containing the Endosperm DNA by the method according to claim 19. 35.- The corn endosperm according to the claim 33, further characterized in that it contains an exogenous DNA molecule of LY038, wherein said molecule is detected in a sample containing the Endosperm DNA by the method according to claim 22. 36.- The corn endosperm according to the claim 33, further characterized in that it comprises LY038 DNA selected from the group consisting of SEQ ID NO: 1, 2, 5, 6 and 11. 37.- The maize endosperm according to the claim 33, further characterized in that it comprises a DNA molecule of LY038, wherein said molecule is a template in a DNA amplification method, wherein said method provides an amplicon comprising SEQ ID. NO: 5 or 11. 38.- A transgenic corn plant, where the representative seed of said plant was deposited with the seed deposit of ATCC accession number PTA-5623, identified as event LY038. 39.- A corn plant or parts thereof produced by developing the seed of claim 38, comprising the DNA construct according to any of claims 1 to 5 in its genome. 40.- The corn plant or parts thereof according to claim 38, further characterized in that it comprises pollen, ovule, seed, roots or leaves, comprising the DNA construction according to any of claims 1 to 5 in its genome 41. The corn plant according to claim 38, further characterized in that it also comprises progeny thereof. 42. The corn plant according to claim 38, further characterized in that the genome of said plant or progeny thereof comprises a DNA molecule selected from the group consisting of SEQ ID NO: 1, 2, 5, 6 and 11. 43.- An isolated DNA molecule comprising SEQ ID NO: 1, 2, 9 or 10, wherein said molecule is linked to a high lysine trait in corn.