HK1159681B - High lysine maize compositions and methods for detection thereof - Google Patents

Description

High-lysine corn composition and detection method thereof

This application is a divisional application of the invention patent application with application number "200480036889.3" and invention name "High lysine corn composition and detection method thereof".

This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application Serial No. filed December 11, 2003, which is hereby incorporated by reference in its entirety.

Field of the Invention

The present invention relates to the field of plant molecular biology. More particularly, the present invention relates to transgenic corn with increased lysine, and to assays and methods for identifying specific exogenous DNA that provides increased lysine.

Background of the Invention

Zea mays, commonly known as corn and maize, is a cereal widely used in animal feed. The cereal, i.e., grain, is a source of protein, starch, and oil for pigs, cattle, and poultry. Of the ten amino acids believed to be essential in mixed grain feed sources, corn is particularly limited in lysine, threonine, and methionine. Deficiencies in these amino acids, particularly lysine, require feed corn or corn meal to supplement these nutrients, often by adding soy meal. Increasing the lysine level in corn kernels as a method for making the seed or meal more nutritious as an animal feed would be beneficial in the art.

To increase lysine levels using molecular biology methods, a feedback-insensitive version of at least one lysine pathway enzyme, dihydrodipicolinate synthase (referred to herein as DHDPS), has been identified and used. A bacterial DHDPS gene isolated from Escherichia coli was shown in vitro to be approximately 200-fold less sensitive to inhibition by elevated lysine levels, and when introduced into transgenic tobacco, overexpression of the E. coli DHDPS gene resulted in elevated lysine levels in leaf tissue (Glassman et al., U.S. Patent No. 5,288,300). Falco et al. disclosed transgenic plants with elevated lysine levels in seeds and genes useful for producing these transgenic plants (U.S. Patent Nos. 5,773,691 and 6,459,019; U.S. Patent Application Publication No. 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 feedback-insensitive DHDPS from Escherichia coli and DHDPS from Corynebacterium (also known as cordapA) to generate transgenic rapeseed, tobacco, corn and soybean plants with increased levels of lysine in seeds. For corn, Falco et al. reported an increase of approximately 130% in free lysine in grain transformed with the cordapA gene relative to non-transformed grain.

It would be beneficial to be able to detect the presence or absence of a specific transgene in a plant or seed, or in the offspring of these plants or seeds, not only for the transgene itself, but also for its location in the host plant or seed genome. Identification of location further provides the identification of the transgenic event by which the genetic engineer inserts the transgene into the plant or seed ancestor plant.

SUMMARY OF THE INVENTION

The present invention provides constructs that can be used to generate transgenic events, as well as materials and methods that can be used to identify specific transgenic events, which result in transgenic plants that accumulate higher levels of lysine than closely related plants that do not contain the construct. Specifically, the present invention includes marker-free transgenic maize lines containing specific exogenous DNA introduced by standard maize transformation, referred to herein as "LY038 events" or "Event LY038." The present invention further provides methods for detecting the presence or absence of the LY038 event in DNA obtained from maize plants, seeds, or tissue samples. Maize plants of the present invention containing Event LY038 exhibit increased lysine in their grain relative to an ancestral or other substantially related plant. Furthermore, the present invention provides an exogenous DNA construct comprising a maize globulin 1 promoter, a rice actin 1 intron, a maize dihydrodipicolinate synthase chloroplast transport peptide-encoding DNA molecule, a coryneform bacterium dipicolinate synthase-encoding DNA molecule, and a maize globulin 1 3' untranslated region, which, when operably linked and expressed in transgenic plant cells and plants, results in increased lysine content in the grain, or a portion thereof, or a processed product derived therefrom. In another embodiment, the construct further comprises a lox site, which appears to be a component of a mechanism for removing a marker gene used to identify successful transformants.

With respect to identifying plants or seeds derived from a particular transgenic event, compositions and methods are provided for detecting the presence of the genomic insertion region, i.e., the site in the genome where the construct is located, in new corn plants comprising event LY038. DNA molecules are provided that comprise at least a portion of the exogenous DNA inserted into the genome and a portion of DNA from the corn genome flanking the insertion site (referred to herein as a "junction sequence").

In yet another embodiment of the present invention, novel DNA molecules are provided comprising at least about 20 base pairs of SEQ ID NO: 1 or 2, including base pairs 1781-1782 or 200-201, respectively. The present invention further provides DNA molecules having the sequence of an amplicon having the sequence of SEQ ID NO: 6, obtained by DNA amplification using primers of SEQ ID NOs: 3 and 4; and hybridization probes complementary to said amplicon having the sequence of SEQ ID NO: 5 and its complement. DNA molecules having the sequence of SEQ ID NO: 5 or 6 span the junction region between the exogenous DNA and the flanking maize genomic DNA and are diagnostic for event LY038 DNA when used in appropriate analytical tests. Other preferred DNA molecules of the present invention that span the junction region of the exogenous DNA/genomic insertion region of event LY038 are molecules having the sequences of SEQ ID NOs: 1, 2, and 11, and their complements. Another aspect of the present invention is a stably transformed maize plant or seed comprising these molecules.

A primer is said to be "sufficiently long" when its length allows it to function in a PCR reaction and specifically amplify the target sequence; a length of about 11 nucleotides or longer is sufficient; more preferably, about 18 nucleotides or longer, still more preferably about 24 nucleotides or longer, and even more preferably, about 30 nucleotides are sufficient to function and specifically amplify the target sequence. Those skilled in the art will appreciate that primers even longer than about 30 nucleotides can be usefully used in a PCR reaction and are therefore sufficiently long.

PCR primers that can be used to identify event LY038 include a sufficiently long transgenic portion of the DNA sequence of SEQ ID NO: 1 and a sufficiently long 5'-flanking maize DNA sequence of SEQ ID NO: 1, or a sufficiently long transgenic portion of the DNA sequence of SEQ ID NO: 2 and a sufficiently long 3'-flanking maize DNA sequence of SEQ ID NO: 2. These primers can be used in PCR methods to provide a DNA amplicon product that is diagnostic for event LY038 and its progeny. PCR primers homologous or complementary to any suitable length of SEQ ID NOs: 1 and 2 that can generate amplicons or probes diagnostic for event LY038 are another aspect of the invention. For example, without limitation, preferred primers diagnostic for event LY038 include those having at least about 18 contiguous nucleotides of either SEQ ID NO: 3 or 4. Amplicons produced using DNA primers that are diagnostic for event LY038 and its progeny are another aspect of the invention. A preferred amplicon diagnostic 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. These methods comprise obtaining DNA from a corn plant, seed, or tissue, contacting the sample DNA with a PCR primer set, performing PCR, and detecting the presence or absence of an amplicon. Preferred PCR primers diagnostic for event LY038 include oligonucleotide primers having the sequences of SEQ ID NOs: 3 and 4, which generate a LY038 event-specific amplicon having, for example, the sequence of SEQ ID NO: 6, which can be detected by a LY038 event-specific probe having, for example, the sequence of SEQ ID NO: 5.

Hybridization of a probe indicating the presence of event LY038 with an amplicon containing DNA specific for event LY038 can be detected by any suitable method known to those skilled in the art of nucleic acid manipulation, including TaqMan assays, Southern blot hybridization, and other methods known to those skilled in the art of molecular biology. Those skilled in the art will appreciate that detection of the amplicon can be performed using methods that do not involve hybridization of the probe to the amplicon, such as acrylamide gel or agarose gel analysis. Those skilled in the art will also appreciate that the length and sequence of the primers and probes can be varied from the exemplary sequences set forth in SEQ ID NOs: 3, 4, and 5 and still produce a PCR amplicon, or amplicon and probe set, that is diagnostic for event LY038.

In another aspect, the present invention provides methods for producing progeny plants comprising event LY038 DNA. The progeny plants can be inbred or hybrid plants. In yet another application, the present invention provides methods for marker-assisted propagation of event LY038. According to another aspect of the present invention, stably transformed corn plants comprising event LY038 DNA and further comprising increased lysine in the grain or portion thereof are provided.

The present invention further relates to a DNA detection kit comprising at least one DNA of sufficiently long consecutive nucleotides homologous or complementary to SEQ ID NO: 1 or 2, which functions as a DNA primer or probe specific for event LY038 or its progeny.

The present invention further relates to high-lysine corn (Zea maya) plants and seeds comprising event LY038, processed products thereof, and progeny thereof, including representative seeds deposited as ATCC Accession No. PTA-5623 (deposited on October 29, 2003). Furthermore, the present invention provides a corn plant or part thereof, including, for example, pollen or seeds produced by growing a plant comprising event LY038. Corn plants and seeds comprising event LY038 DNA are further aspects of the present invention, for which the DNA primer molecules of the present invention are useful for detecting event-specific sequences.

Processed products of the LY038 event include a portion of the corn kernel, such as the endosperm. Corn meal of the present invention can be made from kernels containing the LY038 transgenic DNA molecule, wherein the meal is high in lysine relative to other corn meal that does not contain the DNA molecule.

The above and other aspects of the present invention will become more apparent from the following detailed description, examples, and accompanying drawings. The following examples are included to demonstrate examples of certain preferred embodiments of the present invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent methods that the inventors have fully discovered to work in the practice of the present invention, and therefore can be considered to constitute examples of preferred modes for its practice. However, based on this description, it should be understood by those skilled in the art that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a plasmid map of pMON55221.

Figure 2A is a schematic diagram of the exogenous DNA insertion event LY038. The exogenous DNA and the relevant base pairs are indicated by italic fonts, and the maize genomic DNA and the relevant base pairs are indicated by normal fonts.

Figure 2B is the sequence at the 5' junction region comprising SEQ ID NO: 5 for event LY038.

Figure 2C is the sequence at the 3' junction region comprising SEQ ID NO: 11 for event LY038.

Sequence Description

The molecules with defined sequences used in the context of the present invention are given in the sequence listing submitted with this application. A summary of the sequence listing is as follows:

SEQ ID NO: 1 is a 1961 base pair (bp) polynucleotide sequence of the 5' DNA comprising the portion of the maize genome flanking the 5' side of the insertion site (bp 1-1781) and the transgenic insert portion of the LY038 event DNA (bp 1782-1961).

SEQ ID NO: 2 is an 867 bp polynucleotide sequence of the 3' DNA comprising the portion of the maize genome flanking the 3' side of the insertion site (bp 201-867) and the transgenic insert sequence of the LY038 event DNA (bp 1-200).

SEQ ID NOs: 3 and 4 are polynucleotide sequences of PCR primers that can be used to produce amplicons diagnostic for event LY038 DNA.

SEQ ID NO: 5 is the polynucleotide sequence of an oligonucleotide probe that can be used to hybridize to the amplicon to detect event LY038 DNA.

SEQ ID NO: 6 is the polynucleotide sequence of an amplicon diagnostic for event LY038 DNA.

SEQ ID NO:7 is the maize globulin 1 promoter (bp 48-1440; Kriz, Biochem. Genet., 27:239-251, 1989; Belanger and Kriz, Genetics, 129:863-872, 1991; U.S. Patent 6,329,574, the entire contents of which are incorporated herein by reference), the rice actin 1 intron (bp 1448-1928; McElroy et al., Plant Cell, 2:163-171, 1990), the maize DHDPS chloroplast transit peptide (bp 1930-2100; Frisch et al., Mol. Gen. Genet., 228:287-293, 1991), the Corynebacterium DHDPS gene (bp 2101-3003; Bonnassie et al., Nucleic Acids, 1996). Acids Research, 18:6421, 1990); Richaud et al., J. Bacteriol., 166:297-300, 1986), the polynucleotide sequence of the zein 1 3' untranslated region (bp 3080-4079; Belanger and Kriz, supra), and the loxP site (bp 4091-4124; Russell et al., Mol. Gen. Genet., 234:45-59, 1992).

SEQ ID NO: 8 is the polynucleotide sequence of the DHDPS gene of Corynebacterium (Bonnassie et al., Nucleic Acids Research, 18: 6421, 1990; Richaud et al., J. Bacteriol., 166: 297-300, 1986).

SEQ ID NO: 9 is a 1736 base pair polynucleotide sequence of additional maize genomic DNA flanking the 5' side of the LY038 event insertion site (see Figure 2A).

SEQ ID NO: 10 is a 359 base pair polynucleotide sequence of additional maize genomic DNA flanking the 3' side of the LY038 event insertion site (see Figure 2A).

SEQ ID NO: 11 is a 20 base pair polynucleotide sequence consisting of 10 consecutive nucleotides of the transgenic insert DNA and 10 consecutive nucleotides of the maize genomic DNA in the junction region shown in FIG2C .

Detailed Description of the Invention

As used herein, "exogenous" DNA refers to DNA that is not naturally derived from the specific construct, cell, or organism in which the DNA is found. Exogenous DNA can include DNA or RNA that is native to the genome but is located at a new position in the genome or is connected to other sequence elements that are not naturally associated with the exogenous DNA in their native state. The recombinant DNA construct used to transform plant cells contains exogenous DNA and often contains other elements discussed below. As used herein, "transgenic" means exogenous DNA that has been incorporated into the host genome or is capable of autonomous replication in the host cell and can trigger the expression of one or more cell products. Exemplary transgenics provide a host cell or plant regenerated therefrom that has a new phenotype relative to a corresponding non-transformed progenitor cell or plant, or a transformed progenitor cell or plant that contains other transgenes but does not contain the specific transgene in question. Transgenics can be introduced directly into a plant by genetic transformation, or can be inherited from any previous generation of plants transformed with exogenous DNA.

As used herein, "gene" or "coding sequence" means a DNA sequence from which an RNA molecule is transcribed. The RNA can be mRNA encoding a protein product, RNA that functions as an antisense molecule, or structural RNA such as tRNA, rRNA, snRNA, or other RNA. As used herein, "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 RNA molecule. An exemplary coding sequence is the Corynebacterium dihydrodipicolinate synthase gene (DHDPS; Bonnassie et al., Nucleic Acids Research, 18:6421, 1990; Richaud et al., J. Bacteriol., 166:297-300, 1986; bp 2101-3003 of SEQ ID NOs:7 and 8), which can be used to produce corn kernels with increased lysine. Corn plants transformed to contain and express the Corynebacterium DHDPS gene, which results in increased lysine in kernel tissue, are also referred to as high-lysine corn plants.

As used herein, "promoter" means a region of a DNA sequence necessary for the initiation of DNA transcription, resulting in the production of RNA complementary to the transcribed DNA; this region may also be referred to as a "5' regulatory region". A promoter is located upstream of a coding sequence to be transcribed and has a region that functions as a binding site for RNA polymerase and has regions that act with other factors to promote RNA transcription. Useful plant promoters include those that are constitutive, inducible, tissue-specific, temporally regulated, circadian, drought-induced, stress-induced, developmentally regulated, cold-induced, light-induced, and the like. Of particular importance for the present invention are embryo-specific promoters, such as, but not limited to, the maize globulin 1 promoter (Kriz, Biochem. Genet., 27:239-251, 1989; Belanger and Kriz, Genetics, 129:863-872, 1991; bp 48-1440 of SEQ ID NO:7).

As is well known in the art, in addition to the promoter, recombinant DNA constructs generally contain other regulatory elements, such as, but not limited to, a 3' untranslated region (e.g., a polyadenylation site or transcription termination signal), a transit or signal peptide, introns, and marker gene elements. A 3' untranslated region (3'UTR) that can be used in the practice of the present invention is the globin 1 3'UTR (Kriz, Biochem. Genet., 27:239-251, 1989; Belanger and Kriz, Genetics, 129:863-872, 1991; bp 3080-4079 of SEQ ID NO: 7). A particularly useful transit peptide is the maize DHDPS transit peptide (Frisch et al., Mol. Gen. Genet., 228:287-293, 1991; bp 1930-2100 of SEQ ID NO: 7). An intron that can be used in the context of the present invention is rice actin 1 intron 1 (McElroy et al., Plant Cell, 2: 163-171, 1990; bp 1448-1928 of SEQ ID NO: 7).

As used herein, the term "corn" means Zea mays, also known as maize, and includes all plant variants that can be bred with corn, including wild corn species. In the practice of the present invention, the methods and compositions for transforming plants by introducing exogenous DNA into the plant genome can include any known and proven methods. To date, microparticles and Agrobacterium-mediated gene delivery are the two most commonly used plant transformation methods. Microparticle-mediated transformation refers to the delivery of DNA coated on microparticles, which are driven into target tissues by several methods. Agrobacterium-mediated transformation is achieved by using genetically engineered soil bacteria belonging to the genus Agrobacterium. Several Agrobacterium species mediate the delivery of specific DNA, called "T-DNA," which can be genetically engineered to carry any desired DNA fragment into many plant species. Preferred methods for plant transformation are microprojectile bombardment as described in U.S. Patents 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861 and 6,403,865; and Agrobacterium-mediated transformation as described in U.S. Patents 5,635,055; 5,824,877; 5,591,616; 5,981,840; and 6,384,301, all of which are incorporated herein by reference.

As used herein, a "transgenic" organism is an organism whose genome has been altered by the incorporation of exogenous genetic material or other copies of native genetic material, such as by transformation or recombination. The transgenic organism can be a plant, mammal, fungus, bacteria, or virus. As used herein, a "transgenic plant" means a stably transformed plant, or any subsequent generation of progeny derived therefrom, wherein the DNA of the plant or its progeny comprises introduced exogenous DNA that was not originally present in a non-transgenic plant of the same line. The transgenic plant may additionally comprise sequences native to the transformed plant, but wherein the exogenous DNA has been altered so as to alter the expression level or pattern of the genes.

As used herein, a "stably" transformed plant is one in which the exogenous DNA is heritable. The exogenous DNA may be heritable as a DNA fragment that is retained in the plant cell and not inserted into the host genome. Preferably, the stably transformed plant comprises the exogenous DNA inserted into the chromosomal DNA of the nucleus, mitochondria, or chloroplasts, most preferably into the nuclear chromosomal DNA.

As used herein, a " _Ro transgenic plant" is a plant that has been directly transformed with exogenous DNA or regenerated from a cell or cluster of cells that has been transformed with exogenous DNA. As used herein, "progeny" means any subsequent generation, including seeds and plants thereof, derived from a particular parent plant or group of parent plants; the resulting progeny lines can be inbred or hybridized. Progeny of the transgenic plants of the present invention can be, for example, selfed, hybridized with transgenic plants, hybridized with non-transgenic plants, and/or backcrossed.

Seeds of the plants of the present invention can be collected from fertilized transgenic plants and used to grow subsequent generations of plants of the present invention, including hybrid plant lines containing the exogenous DNA of event LY038, which provide the benefit of increased lysine in corn kernels. The corn kernels can be processed into meal and oil products, or the kernels can be fed to animals without processing. The meal products specifically contain enhanced agronomic properties, including increased lysine. The present invention claims corn meal having increased lysine relative to other corn meal, wherein the corn meal contains the exogenous DNA of LY038.

The term "Event LY038 DNA" refers to a DNA fragment comprising the exogenous DNA of SEQ ID NO: 7 inserted into a specific location in the genome, flanked by bp 1-1781 of the maize genomic portion of SEQ ID NO: 1 on the 5' side of the insertion site, and bp 201-867 of the maize genomic portion of SEQ ID NO: 2 on the 3' side of the insertion site (as shown in FIG2A ), wherein the adjacent flanking genomic DNA is expected to be transmitted from the parent plant containing the exogenous DNA to progeny plants. More specifically, Event LY038 DNA also refers to each DNA region encompassing the interface between genomic DNA and the inserted exogenous DNA in the genome of the _Ro transformant, for example, the region surrounding the interface where the 5' end is in the genomic DNA and the 3' end is in the exogenous DNA, as described in SEQ ID NOs: 1, 2, 5, 6, and 11. Furthermore, the exogenous DNA sequence comprising an event DNA can be altered while the native sequence in a specific location of its host genome, e.g., portions of the sequence can be altered, deleted, or amplified and still comprise the event DNA, provided that the exogenous DNA continues to reside in the same location in the genome and provides increased lysine levels when expressed in the plant.

Transgenic "events" are produced by transforming plant cells with exogenous DNA constructs, regenerating plants resulting from the insertion of the exogenous DNA into the plant genome, and selecting specific plants characterized by the event DNA. Typically, a plurality of plant cells are transformed to produce a population of plants from which specific plants are selected. The term "event" refers to the starting _Ro transformant and the progeny of said transformant, which include the exogenous DNA, i.e., the event DNA, inserted into a specific and unique location in the genome. The term "event" also refers to progeny produced by sexual outcrossing, selfing, or repeated backcrossing, wherein at least one plant used for propagation is any generation of the starting _Ro transformant containing the event DNA.

Thus, a transgenic "event" is a plant that contains and is defined by "event DNA." In this manner, "event LY038" contains "LY038 event DNA." A plant can contain two or more different event DNAs and, therefore, two or more different events. Furthermore, a plant lacking a given transgenic event X does not contain the event DNA X in question. Event DNA can be passed from plant to plant, generation to generation, by any breeding scheme, protocol, or means known to those skilled in the art of corn breeding.

Plant transformation generally utilizes selectable markers and selection methods to distinguish cultured transformed cells from non-transformed cells. In some cases, the selectable marker gene is retained in the transgenic plant; in other cases, it is desirable to remove the selectable marker gene or other sequence introduced into the exogenous DNA. Homologous recombination is a method that can be used to delete marker genes located in transgenic plants (U.S. Patent No. 6,580,019, the entire contents of which are incorporated herein by reference). Another useful means of removing sequences from plants includes the use of site-specific recombinases and their respective site-specific target sites.

Many different site-specific recombinase systems can be used according to the present invention, including, but not limited to, the Cre/lox system of bacteriophage P1, and the FLP/FRT system of yeast. Phage P1 Cre/lox and yeast FLP/FRT systems constitute two particularly useful systems for site-specific integration or transgenic excision. In these systems, the recombinase (Cre or FLP) will specifically interact with its respective site-specific recombination sequence (lox or FRT, respectively) to reverse or excise the inserted sequence. The sequences of both systems are relatively short (lox is 34bp and FRT is 47bp) and are therefore convenient for use with transformation vectors. It has been demonstrated that the FLP/FRT and Cre/lox recombinase systems work effectively in plant cells. In a preferred embodiment, the Cre/lox recombinase system is used to remove selectable marker sequences, specifically the NPT II marker gene (see Figure 1) flanked by lox P recombination sites (bp 4091-4124 SEQ ID NO: 7; Russell et al., Mol. Gen. Genet., 234: 45-59, 1992).

Transgenic plants, seeds, or parts thereof that exhibit an enhanced desired characteristic, such as "increased lysine," are plants containing exogenous DNA that confers a desired, measurable change in the characteristic compared to a plant of substantially the same genotype lacking the desired exogenous DNA. Preferably, the enhanced desired characteristic is measured by comparing the characteristic in a transgenic plant having the exogenous DNA associated with the enhanced desired characteristic with the characteristic in a plant of substantially the same genotype but lacking the exogenous DNA. Such plants lacking the exogenous DNA can be naturally occurring wild-type plants or transgenic plants, preferably of the same species as the transgenic plant. Preferably, the plant lacking the exogenous DNA is a sibling of a plant containing the desired exogenous DNA that lacks the desired exogenous DNA. Such sibling plants may contain other exogenous DNAs. The increased lysine can be demonstrated by the plant through the accumulation of increased amounts of the amino acid in the grain and can be measured by any suitable method, such as mass spectrometry or high performance liquid chromatography of appropriately extracted tissue.

As used herein, a "probe" is an isolated oligonucleotide to which a detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent, dye, or enzyme, may be attached. Such a probe is complementary to a strand of a target nucleic acid. In the context of the present invention, such a probe is complementary to a strand of genomic DNA from event LY038, for example, from a corn plant or seed or other plant part from event LY038. Probes according to the present invention are substances including DNA, RNA, and polyamides that specifically bind to target DNA and can be used to detect the presence of the target DNA sequence.

A "primer" is an isolated oligonucleotide that can anneal to a complementary target DNA strand by nucleic acid hybridization and subsequently be extended along the target DNA by the action of a polymerase, e.g., a DNA polymerase. As used herein, the primers of the present invention are used to perform DNA amplification of a target nucleic acid sequence, e.g., by polymerase chain reaction (PCR) and may also be referred to as "PCR primers."

Probes and primers have enough nucleotide lengths so that they specifically bind to the target DNA sequence under the hybridization conditions or reaction conditions determined by the technician. This length can be any length that is enough to be used for the detection method of selection. Generally, a length of approximately 11 or more nucleotides is used, preferably approximately 18 or more nucleotides, more preferably approximately 24 or more nucleotides, and most preferably approximately 30 or more nucleotides. These probes and primers specifically hybridize to the target. Preferably, the probes and primers according to the present invention have continuous nucleotides of a DNA sequence completely similar to the target sequence, although probes that are different from the target DNA sequence and retain the ability to hybridize to the target DNA sequence can be designed by conventional methods. Use, for example, the schemes announced in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989, etc., and methods for preparing and using probes and primers are well known to those skilled in the art.

The evaluation of the flanking genomic DNA sequences around the transgenic event insertion site allows the design of detection method, and said detection method is specific for the given transgenic event that is inserted into the genome specific location.This detection method, it can be different between the identical or similar transgenics that are positioned at different insertion sites in the genome, is called the event-specific DNA detection method, for example, and the event-specific assay method.Described, for example, the event-specific assay method (U.S. Patent Application Publication No. 2002/0013960, incorporates its full content into this paper as a reference) of the corn event nk603 of glyphosate tolerance.

In preferred embodiments, nucleic acid probes of the present invention specifically hybridize to LY038 event-specific amplicons having the nucleic acid sequences of SEQ ID NOs: 3-6, or their complements, most preferably SEQ ID NO: 6, or its complement. In another aspect of the present invention, preferred nucleic acid probe molecules of the present invention share between approximately 80%, preferably approximately 90%, more preferably approximately 95%, even more preferably approximately 98%, and most preferably approximately 99% sequence identity with the nucleic acid sequences set forth in one or more of SEQ ID NOs: 3-6, or their complements. An exemplary probe diagnostic for event LY038 has the sequence of SEQ ID NO: 6. These probe molecules can be used by those skilled in the art as markers in plant propagation methods to identify progeny of genetic crosses. Hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art. These detection methods may include, but are not limited to, fluorescent tags, radioactive tags, antibody-based tags, and chemiluminescent tags.

As used herein, "homologous" refers to a nucleic acid sequence that will specifically hybridize under conditions of high stringency to the complement of the nucleic acid sequence to which it is being compared. Suitable stringency conditions, including time, temperature, and salt conditions that promote DNA hybridization, are known to those skilled in the art. Both temperature and salt can be varied, or the temperature or salt concentration can be held constant while the other variables are changed. In preferred embodiments, the polynucleic acids of the invention will specifically hybridize to one or more nucleic acid molecules set forth in SEQ ID NO: 1 or 2, or their complements, or fragments of either, under conditions of high to moderate stringency. In particularly preferred embodiments, the nucleic acids of the invention will specifically hybridize to one or more nucleic acid molecules set forth in SEQ ID NO: 1 or 2, or their complements, or fragments of either, under conditions of high stringency. Hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including, but not limited to, fluorescent tags, radioactive tags, antibody-based tags, and chemiluminescent tags.

As used herein, "amplicon" refers to the nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether corn plants obtained from a sexual cross contain genomic DNA from a transgenic event derived from a corn plant containing exogenous LY038 DNA, DNA extracted from a corn plant tissue sample can be subjected to a nucleic acid amplification method utilizing a DNA primer pair comprising a first primer derived from flanking sequences in the plant genome adjacent to the insertion site of the inserted heterologous DNA and a second primer derived from the inserted heterologous DNA to produce an amplicon diagnostic for the presence of the event DNA. The amplicon has a length and sequence that is also diagnostic for the event. Depending on the method used to detect the amplicon, the amplicon length can be the combined length of the primer pair plus one nucleotide base pair, preferably plus approximately 20 nucleotide base pairs, more preferably plus approximately 50 nucleotide base pairs, and even more preferably plus approximately 150 nucleotide base pairs or more. Alternatively, the primer pair can be derived from flanking sequences on either side of the inserted DNA to produce an amplicon that includes the entire inserted nucleotide sequence. One of the primer pairs derived from the plant genomic sequence can be located a certain distance from the inserted DNA. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or approximately 20,000 nucleotide bases.The term "use of an amplicon" specifically excludes primer dimers, which can form in DNA thermal amplification reactions.

Nucleic acid amplification can be accomplished by any of a variety of nucleic acid amplification methods known in the art, including polymerase chain reaction (PCR). The sequence of the heterologous DNA insert or the flanking DNA sequence from event LY038 can be confirmed (corrected if necessary) by amplifying DNA from seeds or plants extracted from ATCC deposit number PTA-5623 using DNA primers derived from the sequences provided herein, followed by standard DNA sequencing of the PCR amplicon or cloned DNA.

Primers and probes based on the flanking genomic DNA and insert sequences disclosed herein can be used to confirm (and correct, if necessary) the disclosed DNA sequences by conventional methods, for example, by recloning and sequencing these DNA molecules.

Amplicons produced by the amplification method are detected by a variety of techniques, including but not limited to gel-based analysis, genetic bit analysis (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, vol00, 4, p18-24), fluorescence polarization (Chen et al., Genome Res., 9:492-498, 1999), and molecular beacons (Tyangi et al., Nature Biotech., 14:303-308, 1996).

A specific object of the present invention is to detect by Taqman assay (available from Applied Biosystems, Foster City, California). The Taqman assay is a method for detecting and quantifying the presence of DNA sequences 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 hybridization to detect the amplified product using a specific FRET oligonucleotide probe. The FRET oligonucleotide probe is designed to have a 5' fluorescent reporter dye and a 3' quencher dye covalently attached to the probe' and 3' ends. The probe is designed to overlap the junction region of the genome and the inserted DNA. The FRET probe and PCR primers (one primer in the exogenous transgenic DNA sequence and one in the flanking genomic sequence) are cyclized in the presence of a thermostable polymer and dNTPs. Hybridization of the FRET probe causes the fluorescent moiety to split and release from the quencher moiety on the FRET probe. The fluorescent signal indicates the presence of the flanking/transgenic insert sequence due to successful amplification and hybridization.

PCR primers preferably used in Taqman assays are designed to (a) have a length of 18-25 bases and matching sequences in the flanking genomic DNA and transgene insert, (b) have a calculated melting temperature of about 57-60°C, e.g., corresponding to an optimal PCR annealing temperature of about 52-55°C, and (c) produce a product that includes the junction region between the flanking genomic DNA and the transgene insert and has a length of about 75-250 base pairs. The PCR primers are preferably located such that the junction sequence is at least one base away from the 3' end of each PCR primer. The PCR primers must not contain regions that are highly self- or mutually complementary.

FRET probes are designed to span the junction sequence. In preferred embodiments, FRET probes incorporate a chemical moiety at their 3' end that binds to the DNA secondary groove when the probe is annealed to the template DNA, thereby increasing the stability of the probe-template complex. The probes are preferably about 12 to about 17 bases in length and have a 3' secondary groove binding portion with a calculated melting temperature that is about 5 to about 7°C higher than that of the PCR primers. Primer design is disclosed in U.S. Patents 5,538,848; 6,084,102; and 6,127,121.

Another assay using the sequences of the present invention is a zygosity assay. A zygosity assay can be used to determine whether a plant containing an event is homozygous for the event DNA, i.e., contains the exogenous DNA at the same location on each chromosome of a chromosome pair, or is heterozygous for the event DNA, i.e., contains the exogenous DNA on only one chromosome of the chromosome pair. In one embodiment, a three-primer assay is employed, wherein Primer 1 specifically hybridizes and extends with the inserted exogenous DNA, Primer 2 specifically hybridizes and extends with the DNA flanking the 5' side of the inserted exogenous DNA, and Primer 3 specifically hybridizes and extends with the DNA flanking the 3' side of the inserted exogenous DNA. These three primers are diagnostic for the event. Typically, the exogenous DNA is of a size, e.g., about 3 to about 7 kilobases or larger, such that Primer 1 and Primer 3 no longer produce amplicons in a PCR reaction. When the three primers are mixed together in a PCR reaction with DNA extracted from a plant homozygous for a given event, a single amplicon is produced by Primer 1 and Primer 2, whose size and sequence will be indicative and diagnostic for the event DNA. When the three primers are mixed together in a PCR reaction with DNA extracted from a plant that does not contain the given event, a single amplicon is produced by Primer 1 and Primer 3, whose size and sequence will be indicative and diagnostic for maize genomic DNA lacking the exogenous DNA. When the three primers are mixed together in a PCR reaction with DNA extracted from a plant heterozygous for the given event, two amplicons are produced: 1) an amplicon produced by Primer 1 and Primer 3, whose size and sequence will be indicative and diagnostic for maize genomic DNA lacking the exogenous DNA, and 2) an amplicon produced by Primer 1 and Primer 2, whose size and sequence will be indicative and diagnostic for the event DNA. Methods for detecting various amplicons by zygosity assays are known to those of skill in the art and include, but are not limited to, gel electrophoresis, Taqman assays, Southern dot blots, Invader technology, sequencing, molecular beacons, pyrosequencing, and the like.

DNA detection kits can be developed using the compositions disclosed herein and DNA detection methods known in the art. The kits can be used to identify corn event LY038 DNA in a sample and can be applied to methods for propagating corn plants containing event LY038 DNA. The kits contain DNA sequences that can be used as primers or probes and are homologous or complementary to any portion of SEQ ID NO: 1 or 2, or to DNA contained in any transgenic genetic element of pMON55221 ( FIG. 1 ), which has been inserted into the genome of a corn plant to form event LY038 ( FIG. 2 ). These DNA sequences can be used in DNA amplification methods (PCR) or as probes in polynucleotide hybridization methods, i.e., Southern blot analysis or Northern blot analysis. The DNA molecule (SEQ ID NO:7) contained in the genome of event LY038 contains heterologous transgenic genetic elements, including the maize globulin 1 promoter (P-ZM glob1), the rice actin 1 intron (I-Os actin), a DNA molecule encoding the maize dihydrodipicolinate synthase chloroplast transit peptide (Zm DHDPS CTP), a DNA molecule encoding the coryneform dihydrodipicolinate synthase chloroplast transit peptide (DHDPS), the maize globulin 1 3' noncoding region (T-Zm glob1), 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 DNA primers or probes in DNA detection methods. The present invention contemplates that one skilled in the art of DNA detection can select one or more DNA molecules homologous or complementary to the transgenic DNA of SEQ ID NO:7 for use in methods for detecting transgenic DNA in the genome of LY038 and its progeny.

The following examples are included to illustrate examples of certain preferred embodiments of the present invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent methods discovered by the inventors to function well in the practice of the present invention, and thus can be considered to constitute examples of preferred modes for practicing the present invention. However, based on this disclosure, it should be understood by those skilled in the art that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention.

Example 1

Preparation of transgenic plants

Immature embryos of maize line H99 were isolated for transformation. Cassette DNA isolated from vector pMON55221 (see FIG. 1 ) containing the maize globulin 1 promoter (Kriz (1989), supra; Belanger and Kriz (1991), supra; U.S. Patent No. 6,329,574, the entire contents of which are incorporated herein by reference; bp 48-1440 of SEQ ID NO: 7), a rice actin 1 intron (McElroy et al. (1990), supra; bp 1448-1928 of SEQ ID NO: 7), a DNA molecule encoding the maize DHDPS chloroplast transit peptide (Frisch et al. (1991), DHDPS; bp 1930-2100 of SEQ ID NO: 7), a DNA molecule encoding the Corynebacterium dihydrodipicolinate synthase chloroplast transit peptide (Bonnassie et al. (1990), supra; Richaud et al. (1986), supra; bp 1448-1928 of SEQ ID NO: 7) were used. 2101-3003), the maize globulin 1 3' noncoding region (Belanger and Kriz (1991), supra; bp 3080-4079 of SEQ ID NO:7), a lox P site (U.S. Patent No. 5,658,772, specifically incorporated herein by reference in its entirety; bp 4091-4124 of SEQ ID NO:7), and the 35S promoter (Kay et al., Science, 236:1299-1302, 1987; U.S. Patent No. 5,164,316), a DNA molecule encoding the NPTII selectable marker (Potrykus et al. (1985), supra), the nos 3' UTR (Fraley et al., Proc. Natl. Acad. Sci. (USA), 80:4803-4807 (1983), supra), and the lox P site. The P site (U.S. Pat. No. 5,658,772, specifically incorporated herein by reference in its entirety) was attached to a gold particle. Exogenous DNA was introduced into immature maize embryos using microprojectile bombardment using methods known to those skilled in the art. Transformed cells were selected using a kanamycin selection scheme. Kanamycin-resistant calli were obtained and regenerated into several fertile _Ro transgenic plants using standard methods.

Example 2

Breeding protocols and lysine analysis

Table 1 summarizes the propagation plan and free lysine data used to develop corn event LY038, demonstrating high lysine in corn kernel tissue. Plants produced by the transformation method described in Example 1 were initially screened for the presence or absence of the Corynebacterium DHDPS sequence using PCR. The copy number of the transgenic insert was determined using TaqMan assay technology. Plants containing DNA molecules operably linked to the Corynebacterium DHDPS sequence as described in Example 1 and Figure 1 were allowed to reach maturity and produce _F1A seeds. For _F1A seed production, the initial transgenic _Ro plants were crossed with a non-transgenic elite corn inbred line.

_FiA plants were screened for the presence or absence of the NPTII sequence using a field-scorable kanamycin resistance test. Insensitivity to kanamycin indicated that the plants contained and were expressing the NPTII marker gene as shown in Figure 1 and described in Example 1; these plants are hereinafter referred to as NPTII+.

NPTII+ plants were crossed with transgenic maize lines expressing bacterial Cre recombinase to produce _F1B seeds. Free lysine levels were measured in samples of the resulting _F1B seeds collected from each ear. Sibling _F1B grains exhibiting greater than approximately 1000 ppm free lysine were promoted to field nurseries. _F1B progeny plants were assayed by PCR and/or DNA dot blot to determine the presence or absence of the DHDPS gene sequence, the sequence encoding the Cre recombinase, and the NPTII selectable marker gene sequence. The NPTII selectable marker gene was flanked by lox P sites (recombination sites), whereby Cre recombinase activity results in excision of the NPTII coding sequence. Plants containing the DHDPS and Cre recombinase sequences and lacking the NPTII sequence, hereinafter referred to as marker excision plants, were allowed to self-pollinate to produce _F2A seeds. Free lysine levels in positive and negative _F2A seeds were measured.

Having obtained _F2A seeds containing the exogenous DHDPS gene of interest and lacking the NPTII selectable marker gene, it is now necessary to propagate them to remove the Cre recombinase sequence. _F2A seeds from marker-excised plants are planted in the field and re-assayed using PCR and/or Southern dot blot analysis to determine the presence or absence of the DHDPS gene sequence, the sequence encoding the Cre recombinase, and the NPTII selectable marker gene sequence. Plants containing the DHDPS sequence and lacking the Cre recombinase and NPTII sequences are selected as "positive" plants. Sibling plants lacking the DHDPS, Cre recombinase, and NPTII sequences are selected as "negative" plants to serve as negative controls. Plants containing the DHDPS sequence are self-pollinated to produce _F3 seeds and promoted to the next generation in the field. Similarly, negative plants lacking the DHDPS, Cre recombinase, and NPTII sequences are self-pollinated to produce _F3 seeds. Free lysine is measured in both positive and negative _F3 seeds.

Positive plants were grown in the field from _F3 seeds. A single plant, homozygous by Taqman assay, was selected and designated LY038. The _F3 plants were either A) self-pollinated to produce _F4-38 ears or B) crossed with inbred lines to produce _F4-38A ears for positive and negative selection. Free lysine was measured in the positive and negative _F4-38 seeds.

_F1-38 seeds from event LY038 were grown in the field and self-pollinated to produce _F2B seeds, which were assayed for free lysine.

_F4-38 seeds of event LY038 were grown in the field to produce _F4-38 plants and either A) self-pollinated to produce _F5-38 seeds or B) crossed with inbred lines to produce hybrid _F1-38B seeds for agronomic evaluation. _F5-38 seeds of event LY038 were grown in the field and either A) self-pollinated to produce _F6-38 seeds or B) crossed with inbred corn varieties to produce additional hybrid _F2C seeds for additional agronomic evaluation.

The above-disclosed Monsanto Event LY038 corn seed deposit was generated by self-pollinating _F5-38 plants to produce _F6-38 seeds and self-pollinating _F6-38 plants to produce _F7-38 seeds. The American Type Culture Collection (Manassas, VA) accession number for Event LY038 is PTA-5623.

Free lysine accumulation was monitored during development of event LY038 maize lines.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 can be used to assess the lysine content of mature grain containing Event LY038. The inventors of the present invention contemplate that other methods known in the art can be used to detect and quantify lysine to provide similar findings of increased lysine content in seeds of LY038.

Free lysine in corn kernels from event LY038 was analyzed using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Individual mature corn kernel samples from event LY038 were weighed, ground into a fine, homogeneous powder, and extracted with an extraction solvent containing methanol, water, and formic acid. In the case of large kernels, approximately 30 mg of the ground powder was used. Lysine in the sample extracts was separated using liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry. Following separation, lysine was quantified using its mass spectrometry peak area relative to a corresponding standard curve prepared using a deuterated _d4 -lysine internal standard (IS).

In another method, the lysine content of corn kernels was assessed based on free lysine by high-performance liquid chromatography (HPLC). Individual corn kernels or aggregates of kernels from event LY038 were ground into a fine, homogeneous powder as described, and in this case, approximately 30 mg of powder was used for analysis. Amino acids were extracted with 5% trichloroacetic acid and detected by pre-column primary amine derivatization with o-phthalaldehyde (OPA). The resulting amino acid adducts, isoindole, are hydrophobic and have excellent fluorescent properties, which can then be detected on a fluorescence detector. Using reversed-phase chromatography, separation was achieved by the hydrophobicity of the R groups on each amino acid. To help stabilize the fluorophore, a thiol group such as 2- _{mercaptoethanol} ( _SHCH2CH2OH ) or 3- _{mercaptopropionic} acid ( _SHCH2CH2COOH ) was added.

Table 1. Breeding plan and lysine analysis used to identify high-lysine maize event LY038.

^* represents ppm free lysine in mature grain containing event LY038

ND = Not Determined

- means less than about 400 ppm free lysine

+ indicates about 1000 to about 1200 ppm free lysine

++ indicates about 1200 to about 1400 ppm free lysine

+++ indicates greater than about 1400 ppm free lysine

i = inbred kernel data

Based on the experiments described here, free lysine in corn kernels containing the LY038 construct increased by between approximately 200% (e.g., F1B, etc.) and almost 300% (e.g., FiA). Intermediate increases in free lysine were also observed (e.g., _F1-38A ).

Example 3

Determination of flanking sequences

Genomic DNA was isolated from a maize plant designated LY038 and used in experiments to determine the maize genomic sequence flanking the transgenic DNA insert. The sequence of the flanking sequences and the junction region between the genomic flanking sequence and the transgenic insert were determined using three different methods: tail PCR and the Genome Walker ^™ kit from ClonTech (Cat. No. K1807-1, ClonTech Laboratories, Palo Alto, California), as well as inverse PCR.

Tail PCR is a method for isolating genomic DNA sequences flanking a known insertion sequence using denaturing primers and a biotin capture step. Primers complementary to the exogenous DNA are used in the initial PCR reaction along with a variety of variant primers. Typically, primers specific for the exogenous DNA and denaturing primers are used in pairs, rather than as a set. The denaturing primers hybridize to a certain extent to the maize genomic sequence flanking the inserted DNA, allowing for the generation of PCR amplicons. The initial PCR amplicon is mixed with a biotin-labeled primer complementary to the transgenic portion of the amplicon and allowed to anneal. Amplicons annealed to the biotin primers are captured using streptavidin, and unbound amplicons are washed away. The annealed amplicons are subjected to a second PCR reaction using nested primers complementary to the exogenous DNA portion of the amplicon and a variety of denaturing primers. The PCR amplicons from the second PCR are subjected to agarose gel electrophoresis, and the bands are excised from the gel and separated. The separated PCR amplicons are sequenced. Tail PCR and sequencing were used to identify the 3' flanking genomic DNA sequence of event LY038.

Flanking DNA was isolated using the Genome Walker method according to the manufacturer's recommended conditions. The 5' flanking genomic DNA sequence of event LY038 was identified using tail PCR and the Genome Walker kit. The Genome Walker method amplified the product of the restriction enzyme ScaI to generate an amplicon that could be used to identify the 5' flanking genomic DNA sequence of event LY038.

Using tail PCR and the Genome Walker method, several hundred base pairs or more of DNA flanking the insertion site of the DNA construct in event LY038 were generated. Additional genomic DNA flanking these events was obtained using inverse PCR, bioinformatics analysis, and comparison with a maize genomic DNA sequence database. Using a combined approach, flanking sequences were identified in the sequences of SEQ ID NOs: 1, 2, 9, and 10. A maize plant is an aspect of the present invention when it contains a DNA molecule within its genome that can be used as a template for DNA amplification to provide an amplicon comprising a linked DNA molecule as described herein, wherein the linked DNA molecule is diagnostic for maize event LY038 DNA in a DNA sample extracted from a maize tissue sample.

Example 4

Event-specific primer and probe assay information

For each event, PCR primers and probes were designed for Taqman assays, namely SEQ ID NOs: 3 and 4. PCR primers with the sequences of SEQ ID NOs: 3 and 4 were used in the Taqman assay to generate an amplicon diagnostic for event LY038; the amplicon had the sequence of SEQ ID NO: 6, and the probe for detecting this amplicon had the sequence of SEQ ID NO: 5. When the primers and probe were subjected to the PCR conditions listed in Table 2, a fluorescent signal indicated the production of the amplicon detected by the probe. Appropriate control samples, such as various negative and positive DNA controls, were used to demonstrate that the PCR primers and probes were specific for the event of interest.

In addition to the primer and probe sets described, any primer and probe set derived from SEQ ID NO: 1 or 2 that is specific for event LY038 DNA when used in a PCR amplification reaction to generate a DNA amplicon diagnostic for event LY038 DNA is an aspect of the present invention and can be readily prepared by one skilled in the art. PCR conditions for generating a Taqman assay diagnostic for event LY038 DNA are included in Table 2.

When performing the PCR or Taqman assays described herein, one skilled in the art will include appropriate control samples. Including a positive control DNA sample, a negative control DNA sample, and other controls is appropriate and aids in the interpretation of the results. Furthermore, one skilled in the art will appreciate how to prepare internal control primers and probes for Taqman PCR reactions using published standard methods (e.g., as disclosed by Applied Biosystems, Foster City, California). One skilled in the art will also appreciate that the specific primer sequences, probes, and reaction conditions specified herein can be modified to produce an assay diagnostic for event LY038 DNA. Furthermore, one skilled in the art will appreciate that the products of the PCR reaction can be analyzed by analytical gel electrophoresis.

Table 2. PCR reaction mixture and conditions diagnostic for event LY038 DNA.

^* Primer mix resuspended in 18 M HO at a concentration of 20 μM each.

Example: 100 μl of 100 μM first primer, 100 μl of 100 μM second primer, 300 μl of 18 MΩ water.

^** Probe resuspended in 18 MΩ water at a concentration of 10 μM.

^*** May include but not be limited to:

Negative DNA control (e.g., non-transgenic DNA)

Negative water control (no template DNA)

Positive control (event LY038)

Sample DNA (from leaves, seeds, other plant parts)

Internal control primers can be made for a wide range of genes or genomic regions, and their design is known to those skilled in the art.

A sample of Monsanto corn seed of Event LY038 disclosed above was deposited under the Budapest Treaty on October 29, 2003, with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110. Event LY038 is assigned the ATCC designation PTA-5623. The deposit will remain with the depositary for a period of 30 years, or five years after the last request, or for the life of the patent, whichever is longer, and may be replaced as needed 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 arrangement and detail without departing from these principles. We claim protection for all modifications that come within the spirit and scope of the appended claims.

Sequence Listing

<110> Dizigan, Mark

Malvar, Thomas

Kelly, Rebecca

Voyles, Dale

Luethy, Michael

Malloy, Kathleen

<120> High-lysine corn composition and its detection method

<130> 06009.0030.00PC00

<150> US 60/529,182

<151> 2003-12-11

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ggatccggcc cggatctcgc ggggaatggg gctctcggat gtagatctgc gatccgccgt 1680

tgttggggga gatgatgggg ggtttaaaat ttccgccatg ctaaacaaga tcaggaagag 1740

gggaaaaggg cactatggtt tatattttta tatatttctg ctgcttcgtc aggcttagat 1800

gtgctagatc tttctttctt ctttttgtgg gtagaatttg aatccctcag ctttgttcat 1860

cggtagtttt tcttttcatg atttgtgaca aatgcagcct cgtgcggagc ttttttgtag 1920

gtagccacca tggtttcgcc gacgaatctc ctcccggcgc ggaagatcac ccctgtctca 1980

aatggcggcg cagcgacggc gagcccctct tctccctcgg tggccgcacg gccacggcga 2040

ctcccttcag gcctccaatc tgtgactggt agagggaagg tttccttggc agccatcact 2100

agtacaggtt taacagctaa gaccggagta gagcacttcg gcaccgttgg agtagcaatg 2160

gttactccat tcacggaatc cggagacatc gatatcgctg ctggccgcga agtcgcggct 2220

tatttggttg ataagggctt ggattctttg gttctcgcgg gcaccactgg tgaatcccca 2280

acgacaaccg ccgctgaaaa actagaactg ctcaaggccg ttcgtgagga agttggggat 2340

cgggcgaagc tcatcgccgg tgtcggaacc aacaacacgc ggacatctgt ggaacttgcg 2400

gaagctgctg cttctgctgg cgcagacggc cttttagttg taactcctta ttactccaag 2460

ccgagccaag agggattgct ggcgcacttc ggtgcaattg ctgcagcaac agaggttcca 2520

atttgtctct atgacattcc tggtcggtca ggtattccaa ttgagtctga taccatgaga 2580

cgcctgagtg aattacctac gattttggcg gtcaaggacg ccaagggtga cctcgttgca 2640

gccacgtcat tgatcaaaga aacgggactt gcctggtatt caggcgatga cccactaaac 2700

cttgtttggc ttgctttggg cggatcaggt ttcatttccg taattggaca tgcagccccc 2760

acagcattac gtgagttgta cacaagcttc gaggaaggcg acctcgtccg tgcgcgggaa 2820

atcaacgcca aactatcacc gctggtagct gcccaaggtc gcttgggtgg agtcagcttg 2880

gcaaaagctg ctctgcgtct gcagggcatc aacgtaggag atcctcgact tccaattatg 2940

gctccaaatg agcaggaact tgaggctctc cgagaagaca tgaaaaaagc tggagttcta 3000

taatgatcta gagcaagccg aattcctgca gcccggggga tccactagtt ctagagcggc 3060

cgccaccgcg gtggagctcg ccaaaacgag caggaagcaa cgagagggtg gcgcgcgacc 3120

gacgtgcgta cgtagcatga gcctgagtgg agacgttgga cgtgtatgta tatacctctc 3180

tgcgtgttaa ctatgtacgt aagcggcagg cagtgcaata agtgtggctc tgtagtatgt 3240

acgtgcgggt acgatgctgt aagctactga ggcaagtcca taaataaata atgacacgtg 3300

cgtgttctat aatctcttcg cttcttcatt tgtccccttg cggagtttgg catccattga 3360

tgccgttacg ctgagaacag acacagcaga cgaaccaaaa gtgagttctt gtatgaaact 3420

atgacccttc atcgctaggc tcaaacagca ccccgtacga acacagcaaa ttagtcatct 3480

aactattagc ccctacatgt ttcagacgat acataaatat agcccatcct tagcaattag 3540

ctattggccc tgcccatccc aagcaatgat ctcgaagtat ttttaatata tagtattttt 3600

aatatgtagc ttttaaaatt agaagataat tttgagacaa aaatctccaa gtattttttt 3660

gggtattttt tactgcctcc gtttttcttt atttctcgtc acctagttta attttgtgct 3720

aatcggctat aaacgaaaca gagagaaaag ttactctaaa agcaactcca acagattaga 3780

tataaatctt atatcctgcc tagagctgtt aaaaagatag acaactttag tggattagtg 3840

tatgcaacaa actctccaaa tttaagtatc ccaactaccc aacgcatatc gttccctttt 3900

cattggcgca cgaactttca cctgctatag ccgacgtaca tgttcgtttt ttttgggcgg 3960

cgcttacttt cttccccgtt cgttctcagc atcgcaactc aatttgttat ggcggagaag 4020

cccttgtatc ccaggtagta atgcacagat atgcattatt attattcata aaagaattcg 4080

agggggatcc ataacttcgt atagcataca ttatacgaag ttatcttaga gcggccgcca 4140

ccgcggtgga gctcggatcc actagtaacg gccgcc 4176

<210> 8

<211> 903

<212> DNA

<213> Corynebacterium glutamicum

<400> 8

agtacaggtt taacagctaa gaccggagta gagcacttcg gcaccgttgg agtagcaatg 60

gttactccat tcacggaatc cggagacatc gatatcgctg ctggccgcga agtcgcggct 120

tatttggttg ataagggctt ggattctttg gttctcgcgg gcaccactgg tgaatcccca 180

acgacaaccg ccgctgaaaa actagaactg ctcaaggccg ttcgtgagga agttggggat 240

cgggcgaagc tcatcgccgg tgtcggaacc aacaacacgc ggacatctgt ggaacttgcg 300

gaagctgctg cttctgctgg cgcagacggc cttttagttg taactcctta ttactccaag 360

ccgagccaag agggattgct ggcgcacttc ggtgcaattg ctgcagcaac agaggttcca 420

atttgtctct atgacattcc tggtcggtca ggtattccaa ttgagtctga taccatgaga 480

cgcctgagtg aattacctac gattttggcg gtcaaggacg ccaagggtga cctcgttgca 540

gccacgtcat tgatcaaaga aacgggactt gcctggtatt caggcgatga cccactaaac 600

cttgtttggc ttgctttggg cggatcaggt ttcatttccg taattggaca tgcagccccc 660

acagcattac gtgagttgta cacaagcttc gaggaaggcg acctcgtccg tgcgcgggaa 720

atcaacgcca aactatcacc gctggtagct gcccaaggtc gcttgggtgg agtcagcttg 780

gcaaaagctg ctctgcgtct gcagggcatc aacgtaggag atcctcgact tccaattatg 840

gctccaaatg agcaggaact tgaggctctc cgagaagaca tgaaaaaagc tggagttcta 900

taa 903

<210> 9

<211> 1736

<212> DNA

<213> Corn

<400> 9

gaatccctca gctttgttca tcggtagttt ttcttttcat gatttgtgac aaatgcagcc 60

tcgtgcggag cttttttgta ggtagccacc atggagtgca caccggtctc tactctagtc 120

gcaacatttg caccctcacg acgctgcgac tgcgggggga tttcaacccg tcggctccta 180

tcttcggctt ctactctagt ctcatcgtgt gtggtgcccc gttgcaactg cggggggatg 240

ttagactgtg tgtgggctgg caccactgtt gggctgccga cccattaggg ttagggttaa 300

gtctctctat gtatgtaccc cttctctatg caatagatta agcattaggg tttcctacac 360

acaccaggat tttaagtgaa caaatgtgcc cctttacaaa ctaacaatga ccatgaaatg 420

cctaagatta ccaatgatcc aagaccagga atttttatta ttttgtggtg ttgcttttgt 480

aagctaatta ggttgtttat ttttggtgct ttatagttgt ccaacctttat tgccttttcc 540

accaattggg tacactcatt cttcaacaca ttcattaact atttcatgtt tttttaactt 600

gaactgcgct tatctctgtt cttttatttt atgacttgga tgaaataagt cagttagtcg 660

tattaaccat ttggctgcca ttttacatgt gatagattat ttatttcttg attcctgata 720

gtttttttta ttcgtggctt ctactgttct aatatgaact atggactaaa tattttattt 780

tgatttcctg ctacttgcag attgaaatgt cggagatacg tgctttagtc agccgagcta 840

ctgctaggag tcttgttctg attgatgaaa tatgtagagg cacagaaact gcaaaaggaa 900

catgtatagc tggtagcatc attgaaagac ttgataatgt tggctgccta ggcatcatat 960

caactcacct gcatgggatt ttcgacctgc ctctctcact tagcaacact gatttcaaag 1020

ctatgggaac tgaagtggtc gatggatgca ttcatccaac atggaaactg attgatggca 1080

tatgtagaga aagccttgct tttcaaacag caaggaggga aggcatgcct gacttgataa 1140

tcaccagggc tgaggagcta tatttgagta tgagtacaaa taacaagcag ggagcatcag 1200

tggcgcacaa tgagcctcct aatggcagcc ccagtgtaaa tggcttggtt gaggagcctg 1260

aatctctgaa gaacagactg gaaatgctgc ctggtacctt tgagccgctg cggaaggaag 1320

ttgagagtgc tgttactacg atgtgtaaga aaatactgtc ggacctttac aacaaaagta 1380

gcatcccaga actggtcgag gtggtctgcg ttgctgtagg tgctagagag caaccaccgc 1440

cttccactgt tggcagatct agcatctacg tgattatcag aagcgacaac aggctctatg 1500

ttggacaggt aaattcatga tcgcattttt ttttctgcgt tgttcattat ctcaggtgat 1560

acagccccat aaaatgccga cactgacgac agatctcctt tttttctgcc tgcagacgga 1620

cgatcttctg gggcgcttga acgccccaca gatcgaagga aggcatgcgg gacgctacgg 1680

tattatacgt cttggtccct ggcaagagcg ttgcctgcca gctggaaacc cttctc 1736

<210> 10

<211> 359

<212> DNA

<213> Corn

<400> 10

aatgaagata aaccccctgc ttataatgaa tcaggcttat aacaacaaat tataatatga 60

aggatttaat taactttagt tttcttcaaa cttgtagctg accatatatt tatatatctt 120

ttagttgagt aaaacatgca atcatttctt taggcttctc ctttctagta cttataattt 180

caccttctct ataatagtat tcagaagtaa atgtatcctt taaaatagta ctcagattta 240

tttttaattt ttttcatttt tgttagccag tttcttttct ttgtgttat ctacgattca 300

ggttcaacat taattaaatc tgaaacgcac aaacacattt aaaggaaagt gggaagctt 359

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 11

aacggccgcc ataattattg 20

Claims (10)

1. A method for producing transgenic maize plants containing event LY038, the method comprising the step of crossing a first transgenic parent maize plant containing event LY308 DNA in its genome with a second parent maize plant, wherein the progeny plant contains event LY308 DNA in its genome, wherein a representative sample of maize event LY038 has been deposited with the American Type Culture Collection (ATCC) accession number PTA-5623. 2. The method of claim 1, further comprising the following steps: (a) Crossing offspring plants with themselves or with a third maize plant to produce seeds of a second offspring plant in subsequent generations; (b) The seeds are used to grow a second generation of offspring plants, and the second generation of offspring plants is crossed with itself or a fourth maize plant to produce seeds of a third generation of offspring plants; and (c) Repeat steps (a) and (b) for at least one more generation to produce inbred maize plants with increased lysine levels and containing SEQ ID NO:5 and SEQ ID NO:11. 3. The method of claim 1, further comprising: (a) DNA samples obtained from maize plants of any generation; (b) Contacting a DNA sample with a labeled nucleic acid molecule, said labeled nucleic acid molecule comprising SEQ ID NO:5 or SEQ ID NO:11; and (c) Implementing marker-assisted propagation methods, wherein plants genetically linked to a marker nucleic acid molecule complement are selected; and (d) Select the plants with high lysine content. 4. A method for producing transgenic maize plants with a high lysine content, specifically LY038, comprising: (a) Planting maize seeds, said maize seeds having a DNA molecule containing SEQ ID NO: 7 in their genome; (b) A plant grown from said maize seeds, wherein said plant has a higher lysine content compared to non-transgenic maize plants, and said event LY038DNA refers to a DNA fragment containing exogenous DNA of SEQ ID NO:7 inserted at a specific location in the genome, bp 1-1781 of the maize genome portion of SEQ ID NO:1 flanking the insertion site at the 5' side, and bp 201-867 of the maize genome portion of SEQ ID NO:2 flanking the insertion site at the 3' side. The representative sample of the maize event LY038 has been deposited with American Type Culture Collection (ATCC) accession number PTA-5623. 5. The method of claim 4, wherein the genome comprises DNA having a sequence selected from SEQ ID NO: 1, 2, 5, 6 and 11. 6. A corn meal containing maize event LY038, derived from tissue of maize event LY038, wherein the corn meal generates an amplicon in a DNA amplification method, the amplicon comprising sequences selected from the following: SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 11, and the event LY038 DNA refers to a DNA fragment containing exogenous DNA of SEQ ID NO: 7 inserted into a specific location in the genome, bp 1-1781 of the maize genome portion of SEQ ID NO: 1 flanking the insertion site at the 5' side, and bp 201-867 of the maize genome portion of SEQ ID NO: 2 flanking the insertion site at the 3' side, wherein a representative sample of the maize event LY038 has been deposited with the American Type Culture Collection (ATCC) accession number PTA-5623. 7. A maize endosperm comprising maize event LY038, derived from tissue of maize event LY038, wherein the endosperm generates an amplicon in a DNA amplification method, the amplicon comprising sequences selected from the following: SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 11, and the event LY038 DNA refers to a DNA fragment comprising exogenous DNA of SEQ ID NO: 7 inserted into a specific location in the genome, bp 1-1781 of the maize genome portion of SEQ ID NO: 1 flanking the insertion site at the 5' side, and bp 201-867 of the maize genome portion of SEQ ID NO: 2 flanking the insertion site at the 3' side, wherein a representative sample of the maize event LY038 has been deposited with the American Type Culture Collection (ATCC) accession number PTA-5623. 8. A method for producing maize plants with increased lysine content compared to non-GMO maize plants, the method comprising the following steps: (a) Crossing a maize plant with another maize plant grown from ATCC seed deposit PTA-5623 or its offspring; (b) Obtaining at least one progeny plant; and (c) Select progeny plants containing the LY308 DNA and exhibiting increased lysine content compared to non-transgenic maize plants. The representative sample of the maize event LY038 has been deposited with American Type Culture Collection (ATCC) accession number PTA-5623. 9. Maize meal containing event LY038, wherein the genome of the meal contains DNA molecules selected from SEQ ID NO: 1, 2, 5, 6 and 11, and the event LY038 DNA refers to a DNA fragment containing exogenous DNA of SEQ ID NO: 7 inserted into a specific location in the genome, bp 1-1781 of the maize genome portion of SEQ ID NO: 1 flanking the insertion site at the 5' side, and bp 201-867 of the maize genome portion of SEQ ID NO: 2 flanking the insertion site at the 3' side, wherein a representative sample of the maize event LY038 has been deposited with the American Type Culture Collection (ATCC) accession number PTA-5623. 10. Maize endosperm containing event LY038, wherein the genome of the endosperm contains DNA molecules selected from SEQ ID NO: 1, 2, 5, 6 and 11, and the event LY038 DNA refers to a DNA fragment containing exogenous DNA of SEQ ID NO: 7 inserted into a specific location in the genome, bp 1-1781 of the maize genome portion of SEQ ID NO: 1 flanking the insertion site at the 5' side, and bp 201-867 of the maize genome portion of SEQ ID NO: 2 flanking the insertion site at the 3' side, wherein a representative sample of the maize event LY038 has been deposited with the American Type Culture Collection (ATCC) accession number PTA-5623.