CN112280743B

CN112280743B - Corn event 2A-7 and identification method thereof

Info

Publication number: CN112280743B
Application number: CN202011220206.7A
Authority: CN
Inventors: 赖锦盛; 赵海铭; 宋伟彬
Original assignee: Beijing Liangyuan Biotechnology Co ltd
Current assignee: Beijing Liangyuan Biotechnology Co ltd
Priority date: 2020-11-04
Filing date: 2020-11-04
Publication date: 2023-06-02
Anticipated expiration: 2040-11-04
Also published as: CN112280743A

Abstract

The invention provides transgenic maize events 2A-7, and maize plants or parts, seeds, cells, or progeny thereof containing a nucleic acid molecule diagnostic for the maize event. This event shows resistance to lepidopteran insect infestation. The invention also provides methods of detecting the presence of a nucleic acid molecule unique to the corn event in a sample, as well as probes and primers for detecting the presence of the corn event in a sample. The invention further relates to methods of producing maize plants and seeds comprising nucleic acid molecules diagnostic for such maize events.

Description

Corn event 2A-7 and identification method thereof

Technical Field

The present invention relates to transgenic maize events (transgenic corn event) 2A-7, and maize plants or parts, seeds, cells or progeny thereof containing a nucleic acid molecule diagnostic for the maize event. The invention also provides methods of detecting the presence of a nucleic acid molecule unique to the corn event in a sample, as well as probes and primers for detecting the presence of the corn event in a sample. The invention further relates to methods of producing insect resistant maize plants and seeds.

Background

Corn is the grain crop with the widest global planting range and the largest yield, and occupies the first three grains (corn, wheat and rice). However, current corn pests are serious, and the corn pests in the world are about 350 species, wherein the corn borers are the most widely distributed and most harmful, which are important corn pests in the world, the occurrence of which seriously affects the yield and quality of corn, and causes great economic loss to corn production. Therefore, effective measures are adopted to control the harm of the corn, which is a urgent problem to be solved for improving the corn yield and increasing the income of farmers.

Since there is no suitable pest-resistant variety, the main method of pest control has been to spray chemical pesticides during growth. However, chemical pesticides not only kill pests, but also kill natural enemies of the pests, causing ecological balance destruction and environmental pollution. Thus, there is an urgent need for corn plants that are resistant to corn pests, particularly lepidopteran pests.

The insect-resistant gene can be introduced into the corn variety by the transgenic technology, so that the insect resistance of transgenic corn is improved, and meanwhile, each plant of the transgenic corn has a considerable resistance, so that the insect-resistant effect is obviously better and more stable than the pesticide spraying control effect, the investment of manpower and material resources can be saved, and the social resources can be effectively saved.

However, integration of foreign genes in the host plant genome is random. If the foreign gene happens to insert into a very important host gene, and thus mutates, the random insertion event can be fatal to the recipient plant. In addition, even if random insertion events do not impair the function of host cell genes, the expression of inserted foreign genes can be affected by "positional effects" caused by surrounding genomic DNA. In some cases, the gene is inserted into sites where the positional effect is strong enough to prevent synthesis of an effective amount of product from the introduced gene. Therefore, it is often necessary to screen a large number of events to identify superior events that characterize the introduced target gene in optimal expression. It is necessary to identify one or more unique sequences that are associated only with the elite event in order to diagnose the presence of the transgenic event.

Disclosure of Invention

The present invention relates to a maize event designated 2A-7 comprising a nucleic acid molecule encoding Cry1Ab and Cry2Ab proteins stably integrated in its genome that confers insect resistance to 2A-7 maize plants. The seeds of the corn event 2A-7 are preserved in China general microbiological culture collection center (CGMCC) and have a preservation number of CGMCC NO.17848. The transgenic corn plants have agronomic performance substantially equivalent to non-transgenic isogenic lines in the absence of insect stress. Under insect pressure, the transgenic corn plant has obviously improved resistance to insect attack by corn borer, armyworm, cotton bollworm, carpopodium borer and the like compared with non-transgenic corn plants.

Maize transformant 2A-7 and products thereof

Accordingly, in a first aspect, the present invention provides a maize plant or part, seed, cell or progeny thereof having incorporated into its genome an exogenous nucleic acid molecule comprising a Cry1Ab and a Cry2Ab gene, wherein said exogenous nucleic acid molecule is flanked by 5 'flanking regions having the nucleotide sequence of SEQ ID NO 5 at positions 1-432 or 300-432 and said exogenous nucleic acid molecule is flanked by 3' flanking regions having the nucleotide sequence of SEQ ID NO 5 at positions 8532-9031 or 8532-8800.

The present invention also provides a maize plant or part, seed, cell or progeny thereof having incorporated into its genome an exogenous nucleic acid molecule comprising Cry1Ab and Cry2Ab genes, wherein said exogenous nucleic acid molecule is flanked by a 5 'flanking region and a 3' flanking region, the sequence of the part comprising the 5 'flanking region and the exogenous nucleic acid molecule adjacent thereto is as shown in SEQ ID No. 1, and the sequence of the part comprising the 3' flanking region and the exogenous nucleic acid molecule adjacent thereto is as shown in SEQ ID No. 2.

The maize plants of the invention, or parts, seeds, cells or progeny thereof, are resistant to insect attack. In certain embodiments, the insect is selected from lepidopteran insects. In certain embodiments, the insect is selected from one or more of the lepidopteran pests, such as armyworm, corn borer (e.g., asian corn borer), cotton bollworm, peach borer, spodoptera frugiperda, and the like. In some embodiments, the maize plants of the invention, or parts, seeds, cells, or progeny thereof, also have resistance to herbicides (e.g., glyphosate, glufosinate, etc.).

In certain embodiments, the position of the exogenous nucleic acid molecule in the genome corresponds to between Chr3:179141694bp-179141724bp of the B73 reference genomic sequence (version V4). In certain embodiments, integration of the exogenous nucleic acid molecule results in deletion of a portion of the sequence in the original genome, which is Chr3:179141695bp-179141723bp (B73 reference genome V4 version), for a total of 29bp, the deleted region being the maize endogenous gene Zm00001d042767, the function of which is predicted to be glucoan endo-13-beta-glucosidase 14, insertion of the fragment of interest and deletion of the Chr3:179141695bp-179141723bp sequence will inactivate the function of the gene. The gene is double-copy in corn, and the gene number of another homologous gene is Zm00001d012292.

In certain embodiments, the nucleotide sequences of the Cry1Ab and Cry2Ab genes are codon optimized for expression in monocot (e.g., maize) cells. In certain embodiments, the Cry1Ab gene is as set forth in SEQ ID NO. 12. In certain embodiments, the Cry2Ab gene is as set forth in SEQ ID NO. 13.

In certain embodiments, the exogenous nucleic acid molecule further comprises a Bar gene. In certain embodiments, the Bar gene is set forth in SEQ ID NO. 14.

In certain embodiments, the exogenous nucleic acid molecule comprises a 35S polyA terminator, a Bar gene, a CAMV 35S promoter, a nos polyA terminator, a Cry1Ab gene, a Gly promoter, a CAMV 35S promoter, an adh1 enhancer, a Cry2Ab gene, and a nos polyA terminator.

In certain embodiments, the exogenous nucleic acid molecule comprises the sequence of nucleotides 483-8524 of SEQ ID NO. 5 or a complement thereof.

It will be readily appreciated that during integration of the exogenous nucleic acid into the genome, unintended integration sequences may be generated, both for non-genomic sequences and for inserts of interest (e.g., T-border sequences), such as those resulting from genomic repair during integration of the insert. Thus, the exogenous nucleic acid molecule incorporated into the genome of a maize plant or part, seed, cell or progeny thereof of the present invention comprised therein may comprise such an unintended integration sequence. Thus, in certain exemplary embodiments, the exogenous nucleic acid molecule comprises the sequence of nucleotides 433-8531 of SEQ ID NO. 5 or a complement thereof.

In certain embodiments, the genome of the maize plant or part, seed, cell or progeny thereof comprises the sequence set forth in any one of SEQ ID NOs 1-5 or a complement thereof. In certain embodiments, the genome of the maize plant or part, seed, cell, or progeny thereof comprises the sequence set forth in SEQ ID NO. 5 or a complement thereof.

In certain embodiments, when genomic DNA of the maize plant or part, seed, cell or progeny thereof is amplified using SEQ ID NO. 6 and SEQ ID NO. 7 as forward and reverse primers, respectively, an amplicon having a length of about 200-300bp (e.g., about 258 bp) is produced. In certain embodiments, when the genomic DNA is amplified using SEQ ID NO. 8 and SEQ ID NO. 9 as forward and reverse primers, respectively, an amplicon having a length of about 250-350bp (e.g., about 310 bp) is produced.

In certain embodiments, the corn seed from which the corn plant or portion, seed, cell or progeny thereof is produced is deposited with the China general microbiological culture Collection center (CGMCC) and has a accession number of CGMCC No.17848. In certain embodiments, the maize plant or part, seed, cell or progeny thereof is propagated and/or bred from a maize plant grown from the maize seed.

In a second aspect, the invention provides a corn seed which is preserved in China general microbiological culture collection center (CGMCC) and has a preservation number of CGMCC NO.17848. The invention also provides a maize plant or part, seed, cell or progeny thereof produced from the maize seed of the second aspect. In certain embodiments, the maize plant or part, seed, cell or progeny thereof is propagated and/or bred from a maize plant grown from a maize seed as described in the second aspect.

In a third aspect, the invention provides a preparation comprising a maize plant or part, seed, cell or progeny thereof of the first aspect, or a maize seed of the second aspect, or a maize plant or part, seed, cell or progeny thereof produced from a maize seed of the second aspect.

In certain embodiments, the article is a food, agricultural product (e.g., feed), cosmetic, pharmaceutical, or industrial product.

In certain embodiments, the preparation comprises genomic DNA of a maize plant or part, seed, cell or progeny thereof of the first aspect, or a maize seed or maize plant produced therefrom of the second aspect.

In certain embodiments, a detectable amount of a polynucleotide is present in the preparation that is unique to maize event 2A-7.

In certain embodiments, the article of manufacture comprises a sequence selected from the group consisting of the sequences set forth in any one of SEQ ID NOs:1-5 or the complement thereof.

In certain embodiments, when the nucleic acids contained in the preparation are amplified using SEQ ID NO. 6 and SEQ ID NO. 7 as forward and reverse primers, respectively, amplicons of about 200-300bp (e.g., about 258 bp) in length are produced. In certain embodiments, when the nucleic acids contained in the preparation are amplified using SEQ ID NO. 8 and SEQ ID NO. 9 as forward and reverse primers, respectively, an amplicon having a length of about 250-350bp (e.g., about 310 bp) is produced.

In certain embodiments, the article is selected from the group consisting of corn ear, bract-removed corn, corn silk, corn pollen, corn grits, corn flour, crushed corn, corn flour, corn oil, corn starch, corn slurry, corn malt, corn sugar, corn syrup, margarine produced from corn oil, unsaturated corn oil, saturated corn oil, corn flakes, popcorn, ethanol and/or juice produced from corn (liquor), distillers dried grains produced from corn fermentation (DDGS), animal feed from corn, cosmetics, and bulking agents.

In certain embodiments, the portion of the maize plant of any of the preceding aspects is selected from the group consisting of kernels, pollen, ovules, flowers, shoots (shoots), roots, stems (stalks), silks, inflorescences (tassels), ears and leaves.

The invention also relates to the use of a maize plant or part, seed, cell or progeny thereof as described in any of the preceding aspects for the production of food, agricultural products (e.g. feed), cosmetics, pharmaceuticals or industrial products.

In certain embodiments, the corn plant or part, seed, cell or progeny thereof is used to produce a food, feed, starch or for brewing.

In certain embodiments, the portion of the maize plant is selected from the group consisting of kernels, pollen, ovules, flowers, shoots, roots, stems, silks, inflorescences, ears and leaves.

In certain embodiments, the maize plant or part, seed, cell, or progeny thereof is used to produce a preparation selected from the group consisting of: corn ear, bract-removed corn, corncob, corn pollen, corn grits, corn flour, crushed corn, corn flour, corn oil, corn starch, corn slurry, corn malt, corn sugar, corn syrup, margarine produced from corn oil, unsaturated corn oil, saturated corn oil, corn flakes, popcorn, ethanol and/or juice (liquor) produced from corn, distillers dried grains produced from fermentation of corn (DDGS), animal feed from corn, cosmetics, and bulking agents.

Method for identifying event 2A-7

In view of the randomness of integration of exogenous fragments in the plant genome in transgenic events, the insertion sites of exogenous fragments in the genome are different for different transgenic events. The flanking sequences are specific for the particular transgenic event. Thus, specific detection of transgenic events can be achieved using flanking sequences of the insert. Such as hybridization with probes comprising a portion of the flanking sequences and a portion of the exogenous insert sequence, or design of specific primers comprising a portion of the flanking sequences and a portion of the exogenous insert sequence for PCR amplification, etc.

Thus, in a fourth aspect, the invention provides a primer pair comprising a first primer comprising a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g.at least 18; e.g.15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 1-432 (e.g.nucleotides 200-432) of SEQ ID NO. 5 or the complement thereof or a sequence having at least 80% (e.g.80% to 100%, or 90% to 100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to said nucleotide sequence, and a second primer comprising a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g.15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 483-432 (e.g.483-1000) of SEQ ID NO. 5 or the complement thereof or a nucleotide sequence having at least 80% (e.g.80% to 100%, or at least 85%, at least 90% to 95%, at least 99% or at least 100% sequence identity to said nucleotide sequence.

In certain embodiments, when the sequence shown in SEQ ID NO. 5 is amplified using the first and second primers, an amplicon having a length of 100-500bp is produced. In certain embodiments, the amplicon is 200-400bp, e.g., 200-300bp, e.g., about 258bp, in length.

In certain embodiments, the first primer comprises a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g., at least 18; e.g., 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 300-400 of SEQ ID NO. 5 or the complement thereof or a sequence having at least 80% (e.g., 80% -100%, or 90% -100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the nucleotide sequence, and the second primer comprises a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g., at least 18; e.g., 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 500-600 of SEQ ID NO. 5 or the complement thereof or a sequence having at least 80% (e.g., 80% -100%, or 90% -100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the nucleotide sequence.

In certain embodiments, the first primer comprises a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g., at least 18; e.g., 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 300-350 of SEQ ID NO. 5 or the complement thereof or a sequence having at least 80% (e.g., 80% to 100%, or 90% to 100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the nucleotide sequence.

In certain embodiments, the second primer comprises a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g., at least 18; e.g., 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 520-600 (e.g., nucleotides 520-570) of SEQ ID NO. 5 or a complement thereof or a sequence having at least 80% (e.g., 80% -100%, or 90% -100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the nucleotide sequence.

In certain embodiments, the first primer comprises the sequence set forth in SEQ ID NO. 6 or a sequence having at least 80% (e.g., 80% to 100%, or 90% to 100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity thereto, and the second primer comprises the sequence set forth in SEQ ID NO. 7 or a sequence having at least 80% (e.g., 80% to 100%, or 90% to 100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity thereto.

In a fifth aspect, the invention provides a primer pair comprising a first primer comprising a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g. at least 18; e.g. 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 483-8524 of SEQ ID NO:5 (e.g. nucleotides 8000-8524) or the complement thereof or a nucleotide sequence having at least 80% (e.g. 80% to 100%, or 90% to 100%, or at least 85%, at least 90%, at least 95%, at least 99% or 100%) compared to said nucleotide sequence, and a second primer comprising a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g. at least 18; e.g. 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 8532-9031 of SEQ ID NO:5 (e.g. nucleotides 8532-8800) or the complement thereof or a nucleotide sequence having at least 80% (e.g. 80% to 100%, or at least 85% to 90%, at least 95% or at least 99% compared to said nucleotide sequence.

In certain embodiments, when the sequence shown in SEQ ID NO. 5 is amplified using the first and second primers, an amplicon having a length of 100-500bp is produced. In certain embodiments, the amplicon is 200-400bp, e.g., 250-350bp, e.g., about 310bp in length.

In certain embodiments, the first primer comprises a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g., at least 18; e.g., 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 8400-8500 of SEQ ID NO. 5 or the complement thereof or a sequence having at least 80% (e.g., 80% -100%, or 90% -100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the nucleotide sequence, and the second primer comprises a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g., at least 18; e.g., 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 8700-8800 of SEQ ID NO. 5 or the complement thereof or a sequence having at least 80% (e.g., 80% -100%, or 90% -100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the nucleotide sequence.

In certain embodiments, the first primer comprises a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g., at least 18; e.g., 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 8400-8600 (e.g., nucleotides 8400-8500, nucleotides 8450-8550, or nucleotides 8450-8500) of SEQ ID NO. 5 or a complement thereof or a sequence having at least 80% (e.g., 80% -100%, or 90% -100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the nucleotide sequence.

In certain embodiments, the second primer comprises a nucleotide sequence consisting of at least 15 consecutive nucleotides (e.g., at least 18; e.g., 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of the sequence of nucleotides 8500-9000 (e.g., nucleotides 8500-8800, nucleotides 8700-8800, or nucleotides 8750-8800) of SEQ ID NO. 5 or a sequence complementary thereto or having at least 80% (e.g., 80% -100%, or 90% -100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the nucleotide sequence.

In certain embodiments, the first primer comprises the sequence set forth in SEQ ID NO. 8 or a sequence having at least 80% (e.g., 80% to 100%, or 90% to 100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity thereto, and the second primer comprises the sequence set forth in SEQ ID NO. 9 or a sequence having at least 80% (e.g., 80% to 100%, or 90% to 100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity thereto.

In a sixth aspect, the invention provides a method of detecting a nucleic acid molecule unique to maize event 2A-7 in a sample comprising maize nucleic acid, comprising:

(1) Contacting the primer pair of the fourth or fifth aspect with the sample;

(2) Performing a nucleic acid amplification reaction; and

(3) Detecting the product of step (2) by gel electrophoresis (e.g., agarose gel electrophoresis);

wherein the maize event 2A-7 is a maize plant or part, seed, cell, or progeny thereof as described in any of the above aspects.

In certain embodiments, the maize event 2A-7 comprises an exogenous nucleic acid molecule encoding mcry1Ab and mcry2Ab incorporated into its genome that flanks the 5 'flanking region and the 3' flanking region, the sequence of the portion of the exogenous nucleic acid molecule comprising the 5 'flanking region and adjacent thereto is shown in SEQ ID NO. 1, and the sequence of the portion of the exogenous nucleic acid molecule comprising the 3' flanking region and adjacent thereto is shown in SEQ ID NO. 2.

In certain embodiments, the maize event 2A-7 comprises in its genome the sequence set forth in any one of SEQ ID NOs 1-5 or a complement thereof.

In certain embodiments, in step (3), the sample is indicated to comprise a nucleic acid molecule unique to maize event 2A-7 when the desired amplicon, that is, the amplicon generated when the primer pair is used to amplify a nucleic acid of genomic DNA of maize event 2A-7, is detected.

In certain embodiments, the method comprises:

(1) Contacting the primer pair of the fourth aspect with the sample;

(2) Performing a nucleic acid amplification reaction; and

wherein when an amplicon having a length of about 250-260bp (e.g., about 258 bp) is detected, it is indicative of the presence of a nucleic acid molecule in the sample that is unique to maize event 2A-7.

In certain embodiments, the primer pair comprises: a first primer comprising a sequence shown in SEQ ID NO. 6 or a sequence having at least 80% (e.g., 80% to 100%, or 90% to 100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity thereto and a second primer comprising a sequence shown in SEQ ID NO. 7 or a sequence having at least 80% (e.g., 80% to 100%, or 90% to 100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity thereto.

In certain embodiments, the method comprises:

(1) Contacting the primer pair of the fifth aspect with the sample;

(2) Performing a nucleic acid amplification reaction; and

Wherein when an amplicon having a length of about 305-315bp (e.g., about 310 bp) is detected, it indicates the presence of a nucleic acid molecule in the sample that is unique to maize event 2A-7.

In certain embodiments, the primer pair comprises: a first primer comprising a sequence shown in SEQ ID NO. 8 or a sequence having at least 80% (e.g., 80% to 100%, or 90% to 100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity thereto and a second primer comprising a sequence shown in SEQ ID NO. 9 or a sequence having at least 80% (e.g., 80% to 100%, or 90% to 100%, or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity thereto.

In a seventh aspect, the invention provides a method of detecting a nucleic acid molecule unique to maize event 2A-7 in a sample comprising maize nucleic acid, comprising:

(1) Contacting a primer pair with the sample; wherein when the primer pair is used to amplify a nucleic acid of genomic DNA of maize event 2A-7, an amplicon comprising a nucleotide sequence selected from the group consisting of: the sequence shown in any one of SEQ ID NOs 1-5 or a complementary sequence thereof;

(2) Performing a nucleic acid amplification reaction, thereby producing the amplicon; and

(3) Detecting the amplicon;

In certain embodiments, in step (3), the detecting comprises DNA sequencing.

In certain embodiments, in step (3), the amplicon is detected using a nucleic acid probe specific for the amplicon. In certain embodiments, the detecting comprises the steps of:

(a) Contacting the nucleic acid probe with the amplification product of step (2);

(b) Subjecting the amplification product and the nucleic acid probe to stringent hybridization conditions; and

(c) Detecting hybridization of the nucleic acid probe to the amplification product;

wherein when hybridization is detected, the presence of a nucleic acid molecule unique to maize event 2A-7 in the sample is indicated.

In an eighth aspect, the invention provides a method of detecting the presence of a nucleic acid molecule unique to maize event 2A-7 in a sample comprising maize nucleic acid, comprising:

(1) Contacting a nucleic acid probe specific for a target sequence with the sample, the target sequence comprising a nucleotide sequence selected from the group consisting of: the sequence shown in any one of SEQ ID NOs 1-5 or a complementary sequence thereof;

(2) Subjecting the sample and nucleic acid probe to stringent hybridization conditions; and

(3) Detecting hybridization of the nucleic acid probe to a sample;

In certain embodiments, when hybridization is detected, the presence of a nucleic acid molecule in the sample that is unique to maize event 2A-7 is indicated.

In certain embodiments, the nucleic acid probe comprises: the sequence of the 5' flanking region and the portion of the exogenous nucleic acid molecule adjacent thereto, or a sequence having at least 80% (e.g., 80% -100% or 90% -100%, e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the sequence.

In certain embodiments, the nucleic acid probe hybridizes to SEQ ID NO:1 or SEQ id no:2 or a complement thereof or a fragment of either has at least 80% (e.g., 80% -100% or 90% -100%, e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity.

In certain embodiments, the nucleic acid probe comprises the sequence set forth in SEQ ID NO. 1 or 2 or a complement thereof.

In certain embodiments, the nucleic acid probe is provided with a fluorescent label, such as a Taqman probe.

Herein, the term "primer" is an isolated nucleic acid that is annealed to a complementary target DNA strand by nucleic acid hybridization to form hybridization between the primer and the target DNA strand, and then extended along the target DNA strand by a polymerase (e.g., a DNA polymerase). Multiple pairs or sets of primers may be used for amplification of nucleic acid molecules, for example by Polymerase Chain Reaction (PCR) or other conventional nucleic acid amplification methods.

The term "nucleic acid probe" is an isolated nucleic acid linked to a conventionally detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent, or enzyme. Such probes are complementary to one strand of the target nucleic acid, and in the case of the present invention, hybridize to one strand of genomic DNA from maize events 2A-7. Probes of the present invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.

Methods of making and using probes and primers are described, for example, in molecular cloning: a Laboratory Manual, version 2, volumes 1-3, editions by Sambrook et al, coldSpring Harbor Laboratory Press, cold Spring Ha r bor, NY,1 989 (hereinafter, "Sam brook et al, 1 989"); current Protocols in Molecular Biology, ausubel et al, greene Publishing and Wiley-Interscience, new York,1992 (periodic updates) (hereinafter, "Ausubel et al, 1992"); and Innis et al, PCR Protocols: a Guide to Methods and Applications, academic Press: san Diego,1990. The PCR Primer pairs can be obtained from known sequences, for example, by using a computer program intended for this purpose, such as Primer (version 0.5, 1991,Whitehead Institute for Biomedical Research,Cambridge,MA).

The nucleic acid probes and primers of the invention hybridize under stringent conditions to a target DNA sequence. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA at a transgenic event in a sample. In this context, two nucleic acid molecules are said to be capable of specifically hybridizing to each other if they are capable of forming antiparallel double stranded nucleic acid structures. If the nucleic acid molecules exhibit complete complementarity, one nucleic acid molecule is referred to as the "complement (complementary sequence)" of the other nucleic acid molecule. In this context, a molecule is said to exhibit "complete complementarity" when each nucleotide of the molecule is complementary to a nucleotide of another molecule. Two molecules are said to be "minimally complementary" if they hybridize to each other with sufficient stability that they remain annealed to each other under at least conventional "low stringency" conditions. Similarly, molecules are said to be "complementary" if they hybridize to each other with sufficient stability that they remain annealed to each other under conventional "high stringency" conditions. Conventional stringent conditions are described by Sambrook et al, 1989 and Haymes et al: nucleic AcidHybridization, A Practical Approach IRL Press, washington, DC (1985). Thus, deviations from complete complementarity are allowed, provided that these deviations do not completely preclude the ability of the molecule to form a double-stranded structure. In order for a nucleic acid molecule to function as a primer or probe, it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure at the particular solvent and salt concentration used.

Suitable stringent conditions for promoting DNA hybridization, such as 6.0 x sodium chloride/sodium citrate (SSC) at about 45 ℃, followed by washing with 2.0 x SSC at 50 ℃, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, john Wiley & Sons, n.y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the washing step may be selected from the group consisting of 50℃with low stringency, about 2.0 XSSC to 50℃with high stringency, about 0.2 XSSC. In addition, the temperature of the washing step may also be increased from low stringency conditions at room temperature of about 22 ℃ to high stringency conditions at about 65 ℃. Both the temperature and the salt concentration are variable, or either the temperature or the salt concentration may be kept constant while the other variable is changed. In certain embodiments, the probes or primers of the invention specifically hybridize to a target sequence under moderately stringent conditions (e.g., about 2.0 XSSC and about 65 ℃). In certain embodiments, the probes or primers of the invention specifically hybridize to a target sequence under high stringency conditions.

In certain embodiments, the probes of the invention hybridize to SEQ ID NO:1 or SEQ id no:2 or a complement thereof or a fragment of any one thereof has a sequence identity of at least 80% (e.g., 80% -100% or 90% -100%, e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%). Hybridization of the probe to the target DNA molecule can be detected by any of a number of methods known to those skilled in the art, including, but not limited to, fluorescent labeling, radio-labeling, antibody-based labeling, and chemiluminescent labeling.

In reference to amplification of a target nucleotide sequence (e.g., by PCR) using a particular amplification primer pair, "stringent conditions" refer to conditions that are: the primer pair is hybridized only to the target nucleic acid sequence to which the primer having the corresponding wild-type sequence (or its complement) should bind in a DNA thermal amplification reaction and preferably yields a unique amplification product (amplicon).

The term "specific for (target sequence)" means that the probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample containing the target sequence.

The term "amplicon" refers to a nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a maize plant results from a sexual hybridization of genomic DNA containing a transgenic event of a maize plant of the invention, a nucleic acid amplification method can be performed on DNA extracted from a maize plant tissue sample using a primer pair comprising a flanking sequence in the genome of the plant derived from an insertion site immediately adjacent to the insertion site of the heterologous DNA and a second primer derived from the insertion of the heterologous DNA to produce an amplicon diagnostic for the presence of the event DNA. The amplicon has a length and has a sequence that is also diagnostic for the event. The length of the amplicon may be within a combined length of the primer pair plus one nucleotide base pair, preferably about 50 nucleotide base pairs, more preferably about 250 nucleotide base pairs, and even more preferably about 450 nucleotide base pairs. Alternatively, the primer pair may be derived from flanking sequences on either side of the inserted DNA so as to generate an amplicon comprising the entire inserted nucleotide sequence. The members of the primer pair from the plant genomic sequence may be located at a distance from the inserted DNA molecule, which may range from one nucleotide base pair up to about twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers that may form in a DNA thermal amplification reaction.

Nucleic acid amplification can be achieved by any of a variety of nucleic acid amplification methods known in the art, including Polymerase Chain Reaction (PCR). Various methods of amplification are known in the art, and are described in, inter alia, U.S. Pat. nos. 4,683,195 and 4,683,202 and PCR Protocols: a Guide to Methods and Applications, innis et al, academic Press, san Diego,1990.PCR amplification methods have been developed to amplify genomic DNA up to 22kb and phage DNA up to 42kb (Cheng et al, proc. Natl. Acad. Sci. USA91:5695-5699, 1994). These methods, as well as other methods known in the art of DNA amplification, may be used in the practice of the present invention. Heterologous DNA insert or flanking sequences from maize events 2A-7 can be tested as follows: primers derived from the sequences provided herein are used to amplify these sequences from the event followed by standard DNA sequencing of PCR amplicons or cloned DNA.

Amplicons produced by these methods can be detected by a variety of techniques. One such method is genetic bit analysis (Genetic Bit Analysis) (Nikiforov et al, nucleic Acid Res.22:4167-4175, 1994) in which DNA oligonucleotides are designed that overlap with both adjacent flanking genomic DNA sequences and insert DNA sequences. Oligonucleotides were immobilized in wells of a microtiter plate. After PCR of the region of interest (using one primer located in the insert sequence and one primer located in the adjacent flanking genomic sequence), the single-stranded PCR product can be hybridized to the immobilized oligonucleotide and used as a template for a single base extension reaction using a DNA polymerase and a labeled ddNTP specific for the next desired base. The reader may be fluorescent or ELISA-based. The signal indicates the presence of the inserted/flanking sequences due to successful amplification, hybridization and single base extension.

Another method is the technique of pyrosequencing (pyrosequencing technique) described by wind (Innov.Pharma.Tech.00:18-24, 2000). In this method, oligonucleotides are designed that overlap adjacent genomic DNA and insert DNA junction regions. The oligonucleotides were hybridized to single stranded PCR products from the region of interest (one primer located in the insert and the other primer located in the flanking genomic sequence) and incubated in the presence of DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5' phosphate sulfate, and luciferin. dNTPs were added alone and the optical signal generated by incorporation was measured. The light signal indicates the presence of the transgene insert/flanking sequences due to successful amplification, hybridization, and single base or multiple base extension.

Fluorescence polarization (Genome Res.9:492-498, 1999) as described by Chen et al is one method that may be used to detect the amplicons of the invention. This method was used to design oligonucleotides that overlap with genomic flanking and insert DNA junction regions. The oligonucleotides were hybridized to single stranded PCR products from the region of interest (one primer located in the insert DNA sequence and the other primer located in the flanking genomic DNA sequence) and incubated in the presence of DNA polymerase and fluorescent labeled ddNTPs. Single base extension results in incorporation of ddNTP. Incorporation can be measured from polarization change using a fluorometer. The polarization change indicates the presence of the transgene insert/flanking sequences due to successful amplification, hybridization, and single base extension.

Taqman probe method (PE Applied Biosystems, foster City, calif.) is described as a method for detecting and quantifying the presence of a DNA sequence and is well understood from the instructions provided by the manufacturer. Briefly, FRET oligonucleotide probes are designed that overlap with genomic flanking and insert DNA junction regions. The FRET probe and PCR primers (one primer located in the insert DNA sequence and one primer located in the flanking genomic sequence) are cycled in the presence of thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage of the fluorescent moiety on the FRET probe from the quenching moiety and release of the fluorescent moiety. The fluorescent signal indicates the presence of flanking/transgene insert sequences due to successful amplification and hybridization.

Molecular beacons have been described for sequence detection as described by Tyangi et al (Nature Biotech.14:303-308, 1996). Briefly, FRET oligonucleotide probes are designed that overlap with flanking genomic and insert DNA junction regions. The unique structure of the FRET probe allows it to contain a secondary structure that holds the fluorescent moiety and the quenching moiety in close proximity. The FRET probe and PCR primers (one primer located in the insert DNA sequence and one primer located in the flanking genomic sequence) are cycled in the presence of thermostable polymerase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe to the target sequence results in removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties, which results in the generation of a fluorescent signal. The fluorescent signal indicates the presence of flanking/transgene insert sequences due to successful amplification and hybridization.

In the method of any of the above aspects, the sample can be any sample comprising corn event 2A-7DNA, so long as the sample comprises at least a detectable amount of a polynucleotide that diagnoses the presence of event 2A-7 in the sample (i.e., a polynucleotide that is unique to corn event 2A-7). In certain embodiments, the sample may be selected from the group consisting of corn ear, bract-removed corn, corn silk, corn pollen, corn grits, corn flour, crushed corn, corn flour, corn oil, corn starch, corn slurry, corn malt, corn sugar, corn syrup, margarine produced from corn oil, unsaturated corn oil, saturated corn oil, corn flakes, popcorn, ethanol and/or juice produced from corn (liquor), distillers dried grains produced from corn fermentation (DDGS), animal feed from corn, cosmetics, and bulking agents.

The invention also provides a kit for identifying maize event 2A-7 comprising the nucleic acid probes and/or primers described above which hybridise specifically under high stringency conditions to the target sequences set out in any one of SEQ ID NOs 1 to 5. In certain embodiments, the kit further comprises additional materials necessary to enable the nucleic acid hybridization or amplification method to be completed.

The invention also relates to the use of a primer, primer pair, nucleic acid probe or kit as described in any of the above aspects for detecting a nucleic acid molecule unique to maize event 2A-7 in a sample comprising maize nucleic acid or for detecting the presence of maize event 2A-7.

The invention also relates to an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOs: any of the sequences shown in SEQ ID NOs 1 to 5 or a complement thereof.

Method for producing insect-resistant maize plants

In a ninth aspect, the present invention provides a method of producing an insect-resistant corn plant comprising:

(1) Crossing a first parent corn plant with a second parent corn plant; wherein the first or second parent corn plant is as defined in the first or second aspects;

(2) Obtaining a first generation progeny plant from the crossing of (1); and

(3) Selecting from among these first generation progeny plants a progeny plant that is resistant to the insect, wherein the progeny plant is indicated to be an insect-resistant maize plant when the progeny plant satisfies at least one of the following (3 a) - (3 c):

(3a) The progeny plant contains in its genome the sequence set forth in any one of SEQ ID NOs 1-5 or the complement thereof;

(3b) When the genome DNA of the progeny plant is amplified by using SEQ ID NO. 6 and SEQ ID NO. 7 as forward primer and reverse primer, respectively, an amplicon having a length of about 200-300 (e.g., about 258 bp) bp is produced; and/or the number of the groups of groups,

(3c) When the genomic DNA of the progeny plant is amplified using SEQ ID NO. 8 and SEQ ID NO. 9 as forward and reverse primers, respectively, an amplicon having a length of about 250-350bp (e.g., about 310 bp) is produced; or alternatively, the first and second heat exchangers may be,

(3d) Hybridization can be detected when genomic DNA of a progeny plant is detected using a nucleic acid probe specific for the sequence set forth in any one of SEQ ID NOs 1-5 or its complement (e.g., a nucleic acid probe comprising the sequence set forth in SEQ ID NO:1 or 2 or its complement).

In certain embodiments, the first or second parent maize plants comprise incorporated into their genome an exogenous nucleic acid molecule encoding mcry1Ab and mcry2Ab flanking a 5 'flanking region and a 3' flanking region, the sequence of the portion comprising the 5 'flanking region and the exogenous nucleic acid molecule adjacent thereto is as shown in SEQ ID No. 1, and the sequence of the portion comprising the 3' flanking region and the exogenous nucleic acid molecule adjacent thereto is as shown in SEQ ID No. 2.

In certain embodiments, the first or second parent maize plant comprises in its genome the sequence set forth in any one of SEQ ID NOs 1-5 or a complement thereof.

In certain embodiments, the method further comprises the steps of:

(4) Selfing the progeny plant obtained in step (3), thereby producing a plurality of second generation progeny plants;

(5) Selecting from these second generation progeny plants a plant that is resistant to insects, wherein said plant is indicated to be an insect-resistant maize plant when said plant meets at least one of said (3 a) - (3 c).

In a tenth aspect, the present invention provides a method of producing hybrid corn seed that can be grown into an insect-resistant corn plant comprising: crossing a first parent corn plant with a second parent corn plant and harvesting the resulting hybrid seed, wherein the first parent corn plant and/or the second parent corn plant is as defined in the first aspect or the second aspect.

In certain embodiments, the first and/or second parent corn plants comprise incorporated into their genome an exogenous nucleic acid molecule encoding mcry1Ab and mcry2Ab flanking the 5 'flanking region and the 3' flanking region, the sequence of the portion comprising the 5 'flanking region and the exogenous nucleic acid molecule adjacent thereto is shown in SEQ ID No. 1, and the sequence of the portion comprising the 3' flanking region and the exogenous nucleic acid molecule adjacent thereto is shown in SEQ ID No. 2.

In certain embodiments, the first parent corn plant and/or the second parent corn plant comprises in its genome the sequence set forth in any one of SEQ ID NOs 1-5 or a complement thereof.

In certain embodiments, the method comprises:

(1) Planting seeds of a first inbred maize line, which is a maize plant as defined in the first or second aspects; and planting seeds of a second inbred line having a different genotype;

(2) Cultivating a period of time resulting from said planted corn plant until flowering;

(3) Emasculating flowers of plants of one of the maize inbred lines;

(4) Sexual crossing the two different inbred lines with each other; and

(5) Harvesting the hybrid seed thus produced.

In certain embodiments, the first inbred maize line comprises an exogenous nucleic acid molecule encoding mcry1Ab and mcry2Ab incorporated into its genome, flanked by a 5 'flanking region and a 3' flanking region, the sequence of the portion of the exogenous nucleic acid molecule comprising the 5 'flanking region and adjacent thereto is shown in SEQ ID No. 1, and the sequence of the portion of the exogenous nucleic acid molecule comprising the 3' flanking region and adjacent thereto is shown in SEQ ID No. 2.

In certain embodiments, the first inbred maize line comprises in its genome the sequence set forth in any one of SEQ ID NOs 1-5 or a complement thereof.

In certain embodiments, the first inbred maize line provides the female parent. In certain embodiments, the first inbred maize line provides the male parent.

In the method of the ninth or tenth aspect, the insect may be selected from lepidopteran insects, for example one or more of the lepidopteran pests, such as armyworms, corn borers (e.g. asian corn borers), cotton bollworms, carpopodium borers, spodoptera frugiperda, and the like.

Those of ordinary skill in the art will recognize that the transgenic genotypes of 2A-7 can be introgressed by breeding into other maize lines containing different transgenic genotypes, thereby achieving characteristic accumulation in the resulting seed and progeny plants and imparting an increased activity profile to these plants.

The transgenic genotypes of the present invention can be introgressed into any maize inbred or hybrid using art-recognized breeding techniques. The goal of plant breeding is to combine different desirable traits into a single variety or hybrid. For field crops, these characteristics may include resistance to insects and diseases, tolerance to herbicides, tolerance to heat and drought, reduced time to crop maturation, greater yield, and better agronomic quality. With the mechanical harvesting of many crops, uniformity of plant characteristics (e.g., germination and clump establishment, growth rate, maturity, and height of the plant and ear) is important.

Field crops are cultivated by techniques that utilize plant pollination methods. A plant is self-pollinated if pollen from one flower is transferred to the same or another flower of the same plant. If the pollen is from a flower of a different plant, the plant is cross pollinated.

Corn (maize) can be cultivated by both self-pollen and cross-pollination techniques. Corn has separate male and female flowers on the same plant, located on the tassel and ear, respectively. Natural pollination of corn occurs when wind blows pollen from the tassel to the filaments protruding from the top of the ear.

Reliable methods for controlling male fertility in plants provide opportunities for improved plant breeding. This is especially true for the development of maize hybrids that rely on certain male sterility systems. There are several options available to breeders to control male fertility, such as manual or mechanical emasculation (or emasculation of the tassel), cytoplasmic male sterility, genetic male sterility, gametocides, and the like.

Hybrid maize seeds are typically produced by incorporating a male sterility system that either manually or mechanically de-tassels.

Two maize inbreds are planted on a block of land in interleaved strips and pollen bearing tassel is removed from one inbred (female). If sufficient isolation of the foreign maize pollen source is possible, the ear of the emasculated inbred line will only fertilize from the other inbred line (male) and the resulting seed is a hybrid and will form a hybrid plant.

The use of male sterile inbreds is only one factor in maize hybrid production. Plant breeding techniques known in the art and used in maize plant breeding programs include, but are not limited to, regression selection, backcrossing, pedigree breeding, selection for limited length polymorphism enhancement, selection for genetic marker enhancement, and transformation.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "corn" refers to maize or corn and includes all plant varieties that can be cultivated with corn, including wild maize varieties.

As used herein, the term "Cry2Ab gene" has a meaning well known to those skilled in the art, which is an endotoxin gene found in the entomopathogenic bacterium bacillus thuringiensis (Bacillus thuringiensis, bt for short), and can be found in various public databases (e.g., genBank: M23724). The inventors have optimized the codon of Cry2Ab for the sequence characteristics of monocotyledonous plants to obtain a sequence (mCry 2 Ab) particularly suitable for expression in maize cells, the sequence of the mCry2Ab being shown in SEQ ID NO. 13 and see Chinese patent application CN201410483143.2.

As used herein, the term "Cry1Ab gene" has a meaning well known to those skilled in the art, which is an endotoxin gene found in the entomopathogenic bacterium bacillus thuringiensis (Bacillus thuringiensis, bt for short), and can be found in various public databases (e.g., genBank: M13898). The inventors have optimized the codon of Cry1Ab for the sequence characteristics of monocotyledonous plants to obtain a sequence (mCry 1 Ab) particularly suitable for expression in maize cells, the sequence of the mCry1Ab being shown in SEQ ID NO. 12 and see Chinese patent application CN201710701976.5.

As used herein, the term "Bar gene" has a meaning well known to those skilled in the art, which is a herbicide resistant gene derived from streptomyces hygroscopicus. Bar gene is often used as a selection marker gene, which can generate phosphoryl acetyl transferase, can acetylate free amino of glufosinate herbicide, and achieves the purpose of detoxification, thereby facilitating the screening of positive callus in genetic transformation. Bar gene sequences are known in the art, see, for example, thompson CJ et al, EMBO J.1987Sep;6 (9):2519-23.. In certain exemplary embodiments, the Bar gene has a sequence as set forth in SEQ ID NO. 14.

As used herein, the terms "corn event", "event" and "transgenic event" are used interchangeably and refer to a recombinant plant produced by transforming and regenerating a plant cell or tissue with heterologous DNA (e.g., an expression cassette comprising a gene of interest). The term "event" encompasses the original transformant of the heterologous DNA and/or the progeny of the transformant. The term "event" also encompasses progeny produced by a sexual outcrossing between a transformant and another maize line. Even after repeated backcrossing with the recurrent parent, the insert DNA and flanking DNA from the transformed parent are present at the same chromosomal location in the progeny of the hybrid. The term "event" also includes DNA from the original transformant, comprising the inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA, which is expected to be transferred into progeny that received the inserted DNA comprising the relevant transgene as a result of a sexual cross of the parental line comprising the inserted DNA (e.g., the original transformant and progeny resulting from selfing) with the parental line not comprising the inserted DNA. Typically, transformation of plant tissue produces a plurality of events, each event representing insertion of a DNA construct into a different location of the genome of a plant cell. Based on the expression of the transgene or other desired characteristics, a particular event is selected. Thus, "event 2A-7", "2A-7" or "2A-7 event" may be used interchangeably.

As used herein, the term "transformation" is the process of introducing a heterologous nucleic acid into a host cell or organism. In particular, "transformation" refers to the stable integration of a DNA molecule into the genome of a related organism.

As used herein, the term "transformed/transgenic/recombinant" refers to a host organism, such as a bacterium or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also exist as an extrachromosomal molecule. Such extrachromosomal molecules are capable of automatic replication. Transformed cells, tissues or plants are understood to include not only the end product of the transformation process but also the progeny of their transgenes. "non-transformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism, i.e., a bacterium or plant, which does not contain a heterologous nucleic acid molecule. As used herein, "transgenic" refers to a plant, plant cell, or multiple, structured or unstructured plant cells that have integrated nucleic acid sequences representing the relevant genes into the plant genome by well known genetic manipulation and gene insertion techniques, and typically into the chromosome, mitochondria, or other chromosome-containing organelle of the nucleus, at a site different from, or more than, the site normally found in a natural plant or plant cell. Transgenic plants result from manipulation and insertion (as opposed to naturally occurring mutations) of such nucleic acid sequences to produce non-naturally occurring plants or plants having a non-naturally occurring genotype. Transformation techniques for plants and plant cells are well known in the art and may include, for example, electroporation, microinjection, agrobacterium-mediated transformation, and biolistic transformation (ballistict ransformation).

As used herein, the expression "unique to corn event 2A-7" refers to a characteristic of corn event 2A-7. Thus, nucleic acids unique to event 2A-7 were not found in other maize plants other than 2A-7. In certain embodiments, the nucleic acid unique to event 2A-7 may be selected from SEQ ID NOs:1-5.

As used herein, the term "genotype" is the genetic material inherited by a parent corn plant, and not all genetic material is necessarily expressed in the progeny corn plant. Thus, the expression "2A-7 genotype" refers to the exogenous genetic material transformed into the genome of a plant in maize event 2A-7 as well as genetic material flanking the inserted sequence.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum 62 matrix or PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

Advantageous effects of the invention

The event 2A-7 provided by the invention can obviously resist the invasion of lepidoptera insects and has good herbicide resistance. Meanwhile, 2A-7 has excellent genetic stability and safety, and is equivalent to the agronomic performance of a commercially available variety (such as Zheng 58) under the condition of no insect pressure. The event 2A-7 has wide application prospect and realizes positive social and ecological benefits.

Drawings

FIG. 1 shows the pCAMBIA3301+mcry1Ab+mcry2Ab vector map.

FIG. 2 shows the result of electrophoresis of the PCR product of the reference gene in example 2. Wherein, lane 1: industrialized transgenic corn; lane 2: industrialized transgenic soybean; lane 3: industrialized transgenic rape; lane 4: industrialized transgenic cotton; lane 5: industrialized transgenic rice; lane 6: transgenic corn 2A-5; lane 7: transgenic corn 2A-7; lane 8: transgenic corn 2A-7; lane 9: transgenic corn 2A-7; lane 10: transgenic corn 2A-7; lane 11: a non-transgenic receptor control; lane 12: blank control; lane 13: positive control t+zssiib (Zm 00001d 052263) plasmid (10 pg); lane M: molecular weight marker DL2000 plus.

FIG. 3 shows the result of PCR product electrophoresis of the 5' -flanking region-specific reaction system in example 2. Wherein, lane 1: industrialized transgenic corn; lane 2: industrialized transgenic soybean; lane 3: industrialized transgenic rape; lane 4: industrialized transgenic cotton; lane 5: industrialized transgenic rice; lane 6: transgenic corn 2A-5; lane 7: transgenic corn 2A-7; lane 8: transgenic corn 2A-7; lane 9: transgenic corn 2A-7; lane 10: transgenic corn 2A-7; lane 11: a non-transgenic receptor control; lane 12: blank control; lane 13: the pre-cloned T+2A-7 5 'plasmid (10 pg) containing 5' flanking sequences and insert sequences; lane M: molecular weight marker DL2000 plus.

FIG. 4 shows the result of PCR product electrophoresis of the 3' -flanking region-specific reaction system in example 2. Lane 1: industrialized transgenic corn; lane 2: industrialized transgenic soybean; lane 3: industrialized transgenic rape; lane 4: industrialized transgenic cotton; lane 5: industrialized transgenic rice; lane 6: transgenic corn 2A-5; lane 7: transgenic corn 2A-7; lane 8: transgenic corn 2A-7; lane 9: transgenic corn 2A-7; lane 10: transgenic corn 2A-7; lane 11: a non-transgenic receptor control; lane 12: blank control; lane 13: T+2A-7 3 'plasmid (10 pg) containing 3' -terminal flanking sequences and insertion sequences; lane M: molecular weight marker DL2000 plus.

FIG. 5 shows the results of the identification of the resistance to armyworm by 2A-7 in example 3.

FIG. 6A shows the identification of the resistance of 2A-7 ears to corn borers in example 3. Wherein, from top to bottom, the formula is 2A-7, zheng 58 and Zhengdan 958.

FIG. 6B shows the results of the identification of the resistance of 2A-7 stalks to corn borers in example 3. Wherein, 2A-7, zheng 58 and Zhengdan 958 are sequentially arranged from left to right.

FIG. 7 shows the results of the identification of the resistance of 2A-7 ears to cotton bollworms in example 3. Wherein, from top to bottom, the formula is 2A-7, zheng 58 and Zhengdan 958.

FIG. 8 shows the results of in-house measurements of Spodoptera frugiperda on the 2A-7 leaves of example 3. Wherein the upper part is a contrast Zheng 58 blade, and the lower part is a 2A-7 blade.

FIG. 9 shows the results of the in-house measurements of Spodoptera frugiperda with the 2A-7 filaments of example 3. Wherein the upper part is the control Zheng 58 filament, and the lower part is the 2A-7 filament.

FIG. 10 shows the results of 2A-7 pair Spodoptera frugiperda field measurements in example 3. Wherein, the left side is 2A-7, and the right side is control Zheng 58.

Sequence information

The information of the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequence

Description of preservation of biological Material

The invention relates to the following biological materials which have been preserved in China general microbiological culture collection center (CGMCC) (No. 1, 3 of North West Lu, chaoyang district, beijing city):

Seeds of maize (Zea mays) 2A-7 having a collection number CGMCC No.17848 and a collection date of 2019, 10 months and 28 days.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1 transformation and selection of maize event 2A-7

The selection marker gene bar (SEQ ID NO: 14), the target genes mCry1Ab (SEQ ID NO: 12) and mCry2Ab (SEQ ID NO: 13) and their matched regulatory elements are inserted into T-DNA of pCAMBIA3301 (purchased from Hunan Fenghui biotechnology Co., ltd.) to construct a plant expression vector according to a conventional molecular biology method, named pCAMBIA3301+mCry1Ab+mCry2Ab, the matched regulatory elements of each inserted gene are shown in the following table, the map is shown in FIG. 1, and the promoter Gly can be seen in Chinese patent application CN201710702435.4. Positive clones were identified for maize transformation by enzymatic cleavage and two-way sequencing.

Table 2: regulatory elements in transformation vectors

Regulatory element	Function of	Source
			Gly	Promoter of target gene mcry1Ab	Corn
nos	Terminator of target gene mcry1Ab	Agrobacterium tumefaciens
			CaMV35s	Promoter of target gene mcry2Ab	Cauliflower mosaic virus
adh1	Enhancing expression of mCry2Ab	Corn
			nos	Terminator of target gene mcry2Ab	Agrobacterium tumefaciens
CaMV35s	Promoter of selectable marker gene bar	Cauliflower mosaic virus
			35S ployA	Terminator of selectable marker gene bar	Cauliflower flowerMosaic virus

The vector is transformed into agrobacterium EHA105 (purchased from Beijing Hua Vietnam biotechnology Co., ltd.) and then is used for infecting maize immature embryo with agrobacterium containing target gene, and the specific transgenic method is as follows:

the recipients used in the transgenic procedure were the cross F1-generations of the inbred lines HiIIA and HiIIB (publicly available from the "Maize genetic resource stock center" (Maize GDB, 2010)). Firstly, planting inbred lines HiIIA and HiIIB in the field, and bagging the inbred lines from the inbred line to the powder scattering; then preparing pollination, wherein two pollination modes are adopted: hiIIA is used as female parent and HiIIB is used as male parent; hiIIA is used as male parent, hiIIB is used as female parent, immature embryo on pollinated ear grain is taken 9-11 days after pollination, then agrobacterium infection is carried out indoors, the young embryo which is invaded by agrobacterium is placed on a selection medium for multiple screening, resistant callus is obtained, the resistant callus is regenerated into seedling, and transgenic T0 generation plant is obtained. After the T0 generation of the transgene is obtained, pollen of the T0 generation transgenic plant is used for crossing some female parent for seed production, such as Zheng 58 and the like. The agrobacterium infection method is adopted to introduce the insertion sequence into the embryo of the receptor plant, and the transgenic plant is obtained after screening by herbicide dipropyl amine phosphorus. 2000T 0 generation transformants are obtained through multiple times of transformation, and corn transformants 2A-7 with high expression, single copy and genetic stability of both mcry1Ab and mcry2Ab are obtained through detection of the protein content of mcry1Ab and mcry2Ab, screening of copy numbers of inserted fragments, detection of stability of continuous multiple generations and identification of agronomic characters at different places.

Further, the integration of exogenous DNA in the genome of maize transformant 2A-7 was identified as follows: fresh corn leaves are taken to extract corn genomic DNA. TAIL-PCR was performed involving a total of 5 degenerate primers: LAD1-1, LAD1-2, LAD1-3, LAD1-4, AC1,6 specific primers: RB-0a, RB-1a, RB-2a, LB-0a, LB-1a, LB-2a, and the sequences are shown in the following table.

Table 3: primers used in TAIL-PCR

Note that: n=a/T/C/G, b=g/T/C, v=a/G/C, d=a/G

The PCR product was ligated with B vector (Beijing full gold Biotechnology Co., ltd. CB 101-01) and the ligation product was transformed into E.coli competent cells. The single clone was selected for PCR identification and the amplified product was sent to Beijing Oncomelania biosciences, inc. for sequencing. The sequencing results were aligned with the T-border sequence and the maize genome and the exogenous fragment integration was analyzed.

It was determined that integration of the foreign DNA resulted in deletion of a portion of the sequence in the acceptor genome, which was Chr3:179141695bp-179141723bp (B73 reference genome version V4), the deleted region being 29bp in total, of the maize endogenous gene Zm00001d042767, the function of which was predicted to be glucoan endo-13-beta-glucosidase 14, and insertion of the target fragment and deletion of the Chr3:179141695bp-179141723bp sequence would inactivate the function of the gene. The gene is double-copy in corn, and the gene number of another homologous gene is Zm00001d012292. The 2A-7 transformant was inserted between Chr3:179141694bp-179141724bp (version V4 of the B73 reference genome), and the sequence of integration of the T-border into the genome was shown as nucleotides 483-8524 of SEQ ID NO. 5, the composition of SEQ ID NO. 5 being shown in the following table. In the 2A-7 genome, the exogenous sequence flanks a 5 'flanking region having the nucleotides 1-432 of SEQ ID NO. 5 and a 3' flanking region having the nucleotides 8532-9031 of SEQ ID NO. 5. The 5 'junction sequence encompassing part of the 5' flanking region and part of the non-genomic sequence is shown as SEQ ID NO. 1 (corresponding to 423-442 of SEQ ID NO. 5) and the 3 'junction sequence encompassing part of the non-genomic sequence and part of the 3' flanking region is shown as SEQ ID NO. 2 (corresponding to 8522-8541 of SEQ ID NO. 5).

Table 4: integration of maize transformants 2A-7 into genomic sequence information

Note that: (1) Unexpected integration sequences, i.e., non-genomic sequences and non-T-border sequences, may be sequences resulting from genomic repair when the insert is integrated; (2) The unlabeled positions in the table are the sequences between the genes in the T-border region.

Preparation of 2A-7 seeds

The 2A-7T0 generation plants are obtained by genetic transformation of 3301+mcry1Ab+mcry2Ab vectors, homozygous 2A-7 seeds are obtained by continuous 2 generation selfing, and the seeds are preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.17848 and the preservation date of 2019, 10 and 28 days.

Example 2 identification method of maize event 2A-7

1. Materials and methods

1.1, method for extracting corn genome DNA

(1) Taking CTAB solution, and carrying out water bath at 65 ℃ in advance;

(2) About 0.1g of fresh corn leaves are cut into pieces, placed in a precooled mortar, rapidly ground into powder in liquid nitrogen and immediately transferred into a precooled 2mL EP tube (typically no more than 1/2 tube volume);

(3) Rapidly adding 0.8mL CTAB buffer solution of 65 ℃ in an EP tube, gently shaking uniformly, and carrying out water bath for 30min at 65 ℃ and gently shaking from time to time;

(4) Placing in a fume hood for about 15min, and cooling to room temperature;

(5) An equal volume of chloroform was added: isoamyl alcohol (24:1), mixing, and slightly oscillating for 15min;

(6) Centrifuging at 12000rpm for 8min at room temperature;

(7) Aspirate supernatant to a new 1.5mL EP tube;

(8) Adding an equal volume of pre-chilled isopropyl alcohol (pre-chilled at 4 ℃);

(9) Centrifuging at 12000rpm for 8min at room temperature;

(10) Discarding the supernatant, adding 1mL of 75% ethanol, mixing well, discarding the supernatant (ethanol precipitation);

(11) Placing in a fume hood until the ethanol is completely volatilized (1-2 h);

(12) The DNA was dissolved in 300. Mu.L of TE Buffer and kept at 4℃overnight.

1.2, transformation event-specific PCR method

The 5 'and 3' flanking sequences of events 2A-7 are shown as nucleotide numbers 1-432 and nucleotide numbers 8532-9031 of SEQ ID NO. 5, respectively. The sequences of the 5 'and 3' insertion sites of maize transformants 2A-7 involved forward and reverse primers (tables 5-6), respectively, and PCR reactions were performed under the reaction conditions and reaction systems shown in tables 7-8, respectively.

Table 5:5' terminal primer information

Table 6:3' terminal primer information

Table 7: PCR reaction conditions

Table 8: PCR reaction conditions

Components of the reaction System	Final concentration	Volume of each reaction (. Mu.L)
			1. Nuclease-free water		12.6
2. Reaction buffer	1×	2
			3.dNTP’s	0.25mM	2
4. Forward primer	0.25	0.5
			5. Reverse primer	0.25	0.5
DNA polymerase (5U/. Mu.L)	0.1U/μL	0.4
			DNA sample (50 ng,25 ng/. Mu.L)	2.5ng/μL	2
Total volume of		20

Meanwhile, the maize endogenous gene zSSIIb (Zm 00001d 052263) is used as an internal reference gene, the forward primer zSSIIb-F is shown as SEQ ID NO. 10, and the reverse primer zSSIIb-R is shown as SEQ ID NO. 11.

PCR amplified products were detected by electrophoresis on ethidium bromide stained agarose gel (3%). Appropriate molecular mass standards are added during electrophoresis to determine the size of the amplified product, which is visualized using a gel imaging system.

2. Method authentication data

About 1000ng of genomic DNA extracted from the following samples was used as a template for PCR amplification of a corn transformant 2A-7 specific system at the 5 'end and the 3' end and an internal standard system to determine the specificity of the method: 4 different corn single plants containing the transformant 2A-7, corn transformant 2A-5, industrialized transgenic corn, industrialized transgenic soybean, industrialized transgenic cotton and industrialized transgenic rice. Wherein:

1) Industrialized transgenic corn (Bt-11, bt-176, MON863, MON810, GA21, NK603, T25, TC1507, MON89034, MON88017, 59122, MIR604, 3272, MON 87460) is mixed to prepare 1 sample, the content is 1%

2) Industrialized transgenic soybean (MON 87769, 356043, 305523, CV127, MON89788, A5547-127, A2704-12) is mixed to prepare 1 sample, and the content is 1%)

3) Industrialized transgenic rape (MS 1, MS8, RF1, RF2, RF3, T45, oxy235, topas19/2 are mixed to prepare 1 sample, the content is 1%)

4) Industrialized transgenic cotton (MON 1445, MON531, MON15985, LLCOTTON25, MON 88913) is mixed to prepare 1 sample, each 1%)

5) Industrialized transgenic rice (KF-6, KMD-1, M12, KF-2, KF-8 are mixed to prepare 1 sample, the content is 1 percent respectively).

The results of the detection of the reference gene are shown in FIG. 2, which shows that all corn samples showed the expected amplification products and all non-corn samples did not show the amplification products.

The results of the amplification using the 5' primer set shown in Table 5 are shown in FIG. 3, which shows that clear single bands consistent with the expected size fragments were observed for all samples containing transformants 2A-7, without non-specific amplification; for all other corn and non-corn samples, no expected amplification products were observed.

The results of the amplification using the 3' primer set shown in Table 6 are shown in FIG. 4, which shows that clear single bands consistent with the expected size fragments were observed for all samples containing transformants 2A-7, without non-specific amplification; for all other corn and non-corn samples, no expected amplification products were observed.

Since the two primers of the specific PCR bind to the specific regions of the T-DNA and the receptor genome, respectively, the PCR amplification process can be completed only when the two binding regions are contiguous. In the case of the transgenic process, however, the integration of the T-DNA is random, and the binding regions of other transformants are hardly adjacent to each other except for the 2A-7 transformant, and even if they are adjacent to each other, the product will be different from the expected size. Thus, the 5 'primer pair and the 3' primer set described above can be used to detect maize transformants 2A-7.

In addition, the presence of the 5 'or 3' binding sequence can also be detected by DNA probes specifically hybridizing thereto in view of the uniqueness of the 5 'and 3' binding sequences to identify 2A-7 maize events.

Example 3 field pest control trait identification for corn event 2A-7

1. Test basis

The basis for this test reference is: bulletin-10.1-2007, department of agriculture 953.

2. Test materials

2.1 test corn

(1) The breeding process of the transgenic insect-resistant corn 2A-7T5 generation comprises the steps of infecting F1 generation immature embryo hybridized by HiIIA and HiIIB by using agrobacterium containing a target vector, obtaining T0 generation transgenic plant, carrying out hybridization and backcross by using Zheng Wei round parents, obtaining T3 generation, and then carrying out continuous selfing for 2 generation to obtain 2A-7 of homozygous Zheng 58 background;

(2) Receptor control zheng 58; and

(3) The conventional maize variety Zhengdan 958 for local production and use.

The quality of the materials meets the requirement of GB4404.1 that the quality of the materials is not lower than that of the second-stage corn seeds.

2.2 test insects

Armyworm Mythimna separata: an indoor artificial feed for the larvae (hatching time is 12-24 h) of the armyworms or an armyworms population fed by corn seedlings;

asian corn borer Ostrinia furnacalis: the corn borer initially hatched larvae (hatching time is 2-12 h) are indoor artificially fed Asian corn borer populations;

cotton bollworm Helicoverpa armigera: the first hatched larva (incubation time is 12-24 h) is the indoor artificial breeding cotton bollworm population.

Spodoptera frugiperda Spodoptera frugiperda: the spodoptera frugiperda initially hatched larva (hatching time is 12-24 h) is a population of indoor artificially-raised spodoptera frugiperda.

3. Isolation measure

The experimental isolation was performed by using a 300 meter period of flowering phase isolation for more than 25 days.

4. Experimental method

Insect resistance artificial inoculation identification: according to the standard of the Ministry of agriculture 953 bulletin-10.1-2007.

The field test design adopts a random block design, and is repeated three times, and the cell area is 30m ² (5 m is multiplied by 6 m), the row spacing is 60cm, the plant spacing is 25cm, the soil fertility level and cultivation management are the same as those of field production, and no pesticide is sprayed in the whole growth period. The interval of 2m is arranged between different pest-receiving test cells, so that the pest is prevented from spreading among different cells. Before and after the insect inoculation, certain humidity in the field should be ensured, and if drought occurs, watering should be performed in time.

4.1 Clay

The insect-catching method comprises the following steps: the identification of the resistance to the armyworm is carried out in the heart leaf stage, the corn plant development is carried out until the 4-6 leaf stage is reached, and the artificial insect grafting amount in each cell is not less than 40 plants. And (3) inoculating 30-40 of the newly hatched larvae of each plant which are artificially fed, inoculating the larvae into corn leaves, and carrying out second insect inoculation after 3 days of insect inoculation, wherein the number and method of insect inoculation are the same as those of the first insect inoculation, and the insect inoculation selection is carried out in the evening.

Survey recording: 14 days after insect inoculation, corn leaves were investigated for pest readiness and larval survival by the myxoworms.

And (3) expressing the result: based on the pest damage degree of corn leaf, the average value of pest damage grade (leaf feeding grade) of corn leaf of each cell is calculated, the judgment standard is shown in table 9, and then the pest resistance level of transgenic pest-resistant corn is judged according to the regulation of table 10.

Table 9: grading standard for pest damage degree of corn leaves

Table 10: evaluation criterion for resistance of corn to armyworm

Mean value of leaf grade in heart leaf stage	Type of resistance
		1.0～2.0	High resistance to HR
2.1～4.0	anti-R
		4.1～6.0	Medium resistance MR
6.1～8.0	Sense S
		8.1～9.0	High sense HS

4.2 Asian corn borer

The insect-catching method comprises the following steps: the resistance identification of target insect Asiatic corn borer includes artificial inoculation of corn borer in the heart leaf period (8-10 leaf period for corn plant to develop) and silking period, each period is inoculated with 2 times, each period has artificial inoculation of not less than 40 plants in each district, and each plant is inoculated with 60-80 first larva of Asiatic corn borer. The insect-catching selection is carried out in the evening, and if the insect-catching is carried out in the weather above medium rain, the insect-catching is carried out for 1 time.

Recording of the heart leaf period survey: the feeding condition of the upper leaf blade by corn borers is investigated by each plant after 2-3 weeks of the leaf-stage insect grafting. Each identification material randomly selects 15-20 strains/row, each strain records the leaf feeding grade of the corn borer according to the description in table 11 (the leaf feeding grade is divided according to the diameter size and the number of leaf worm holes formed after the corn borer larvae feed on the heart leaves).

Table 11: grading standard for damage degree of corn borers to heart leaves

The average of the extent of leaf damage (leaf feeding grade) to the identified material population was calculated for corn borers. The calculation method comprises the following steps:

average leaf rating = Σ (leaf rating x number of plants planted at this rating)/total number of plants investigated

And (3) expressing results in the heart leaf stage: the insect pest level of each identified material was divided according to the average of the leaf levels, as shown in table 12.

Table 12: resistance evaluation criterion of corn to corn borer

Insect pest level	Mean value of leaf grade in heart leaf stage	Resistance to
			1	1.0～2.9	High resistance to HR
3	3.0～4.9	anti-R
			5	5.0～6.9	Medium resistance MR
7	7.0～8.9	Sense S
			9	9.0	High sense HS

Laying period survey records: and (5) investigating the maize female ear damage condition, the number of wormholes, the length (cm) of wormhole tunnels and the age and survival number of surviving larvae, and evaluating the female ear damage degree and the plant damage condition.

And (3) expressing the result of the spinning period: evaluation of insect resistance in corn ear stage according to the damage condition of female ear, the number of boreholes, the tunnel length (cm) of the boreholes, the age of surviving larvae and the number of surviving larvae, calculating the average value of the damage level of the corn borer in each cell ear stage on the female ear, judging the standard as shown in table 13, and distinguishing the resistance level of the corn borer in the corn ear stage according to the regulation of table 14.

Table 13: grading standard for damage degree of corn ear period to Asian corn borer

Grade of female ear damage	Description of symptoms
		1	The female ear is not damaged
2	The damage of the filament is less than 50 percent
		3	Most of the filaments are not less than 50% of the damage; the larvae survive, and the age is less than or equal to 2 years
4	The damage of spike tip is less than or equal to 1cm, and the larvae survive, and the age is less than or equal to 3 years
		5	The damage of the spike tip is less than or equal to 2cm; or the larvae survive, and the age is less than or equal to 4 years; the tunnel length is less than or equal to 2cm
6	The damage of the spike tip is less than or equal to 3cm; or the larvae survive, and the age is more than or equal to 4 years; tunnel length is less than or equal to 4cm
		7	The damage of the spike tip is less than or equal to 4cm, and the tunnel length is less than or equal to 6cm
8	The damage of the spike tip is less than or equal to 5cm, and the tunnel length is less than or equal to 8cm
		9	The damage of the spike tip is more than 5cm, and the tunnel length is more than 8cm

Table 14: resistance evaluation standard of corn female ear to Asian corn borer

Average value of the grade of damaged leaves of female spike	Type of resistance
		1.0～2.0	High resistance to HR
2.1～3.0	anti-R
		3.1～5.0	Medium resistance MR
5.1～7.0	Sense S
		≥7.1	High sense HS

4.3 Cotton bollworm

The insect-catching method comprises the following steps: the resistance identification of the cotton bollworms is carried out in the period of spinning and powdering, and each plant is respectively inoculated with 20-30 first hatched larvae. The artificial insect-grafting amount in each district is not less than 40 plants, the artificial insect-grafting amount is equal to the first insect-grafting amount after 3 days of insect-grafting on maize filaments. The insect-catching selection is carried out in the evening, and if the insect-catching is carried out in the weather above medium rain, the insect-catching is carried out for 1 time.

Investigation of hazard levels: the artificial inoculation is carried out on the 14 th to 21 th days, the pest rate of female ears is investigated plant by plant, and the number of the survival larvae of each female ear and the pest length of the female ear are investigated.

And (3) expressing the result: evaluation of insect resistance in corn ear period according to the insect damage rate of female ear, the number of surviving larvae and the insect damage length (cm) of female ear, the average value of the insect damage level of cotton bollworms in corn ear period in each cell is calculated, the judgment standard is shown in table 15, and the insect resistance level of corn ear period to cotton bollworms is judged according to the regulation in table 16.

Table 15: grading standard for damage degree of corn ear period by cotton bollworm

Grade of female ear damage	Description of symptoms
		0	The female ear is not damaged
1	Only the filament is damaged
		2	The head of the ear is damaged by 1cm
3+	Every 1cm of pest under the top of the ear, the corresponding pest level is increased by 1 level
		…N

Table 16: evaluation criterion for resistance of female corn ear to cotton bollworm

Average value of the grade of damaged leaves of female spike	Type of resistance
		0～1.0	High resistance to HR
1.1～3.0	anti-R
		3.1～5.0	Medium resistance MR
5.1～7.0	Sense S
		≥7.1	High sense HS

4.4 spodoptera frugiperda

The insect-catching method comprises the following steps: the resistance identification of target pest spodoptera littoralis in the center leaf stage and in the ear stage is carried out by artificial inoculation in the center leaf stage (the small horn mouth stage, the corn plant development to 8-10 leaf stage) and the laying stage, wherein each period is inoculated with 2 times, each district is inoculated with not less than 40 plants, and each plant is inoculated with 20-30 spodoptera littoralis initially hatched larvae. The insect-catching selection is carried out in the evening, and if the insect-catching is carried out in the weather above medium rain, the insect-catching is carried out for 1 time.

Survey recording: after 14 days of insect inoculation, the extent to which the corn leaf is damaged by spodoptera frugiperda was investigated.

And (3) expressing the result: based on the extent to which the corn leaf is damaged by spodoptera frugiperda, an average value of the damage level (leaf feeding level) of spodoptera frugiperda to the corn leaf in each cell is calculated, the judgment criteria are shown in the following table, and then the resistance level of the transgenic insect-resistant corn to spodoptera frugiperda is judged according to the regulations of tables 17 and 18.

Table 17: grading standard for pest degree of spodoptera littoralis on corn leaves

Leaf grade	Description of symptoms
		1	The blade is harmless, or only has needle-like insect holes (less than or equal to 1 mm)
2	Only a small number of wormholes with the size of the bullet holes (less than or equal to 5 mm) are formed on the individual blades
		3	The small number of blades are provided with wormholes with the sizes of bullet holes (less than or equal to 5 mm)
4	The individual blade is notched (less than or equal to 10 mm)
		5	Few leaves are provided with nicks (less than or equal to 10 mm)
6	Part of the blades are provided with notches (less than or equal to 10 mm)
		7	Individual leaf portions are fed, and few leaves are provided with large cuts (less than or equal to 10 mm)
8	A few leaves are eaten, and a part of the leaves are provided with large-piece nicks (less than or equal to 10 mm)
		9	Most of the leaves are fed

Table 18: evaluation criterion for resistance of corn to spodoptera frugiperda

And (3) expressing the result of the spinning period: evaluation of the insect resistance in the ear period of corn the average value of the insect resistance grade of spodoptera frugiperda in each cell to the ear was calculated based on the damage condition of the female ear, the number of boreholes, the tunnel length (cm) of the boreholes, and the number of surviving larvae, the judgment standard is shown in table 19, and the level of the insect resistance of the ear period of corn to spodoptera frugiperda was discriminated according to the regulation in table 20.

Table 19: grading standard for pest extent of spodoptera littoralis in corncob stage

Table 20: evaluation criterion for resistance of corn female ear to spodoptera frugiperda

5. Analysis of results

5.1 analysis of the Clay resistance identification results

The results of the insect-receiving identification were investigated 2-3 weeks after the insect-receiving. As shown in fig. 5 and table 21, analysis of parameters such as leaf hazard rate, wormhole or nick size, etc. showed that the leaf hazard rate, wormhole or nick size of transgenic corn 2A-7 was significantly lower than that of its corresponding non-transgenic corn variety and local normal cultivar corn variety in the 5% significant level range, and reached significant difference levels. Experimental results show that the transgenic corn 2A-7 has better control effect on target pest armyworms.

Table 21: leaf damage level investigation result

5.2 analysis of the results of the identification of the resistance of corn borers

5.2.1 blade hazard class investigation

The leaf stage (V6-V8) was inoculated with insects, and leaf feeding levels were investigated plant by plant 14 days after the inoculation. Leaf hazard level investigation results show that transgenic 2A-7 corn has good control effect on target insect corn borer, the 2A-7 leaf hazard level is obviously lower than that of the corresponding non-transgenic corn variety and local common cultivated corn variety within a 5% obvious level range, and the obvious difference level is achieved (table 22). The results show that the resistance of transgenic corn 2A-7 Asian corn borer is superior to that of the corresponding non-transgenic corn variety and the local common cultivated corn variety.

Table 22: leaf-stage leaf damage level investigation result of corn borers

5.2.2 female ear hazard level investigation

The spiny period is inoculated with insects, and the identification result of the insects is investigated before harvest. As shown in fig. 6A-6B and tables 23-24, analysis of five parameters, such as the female ear hazard rate, the number of individual plant boreholes, the individual plant tunnel length, the number of individual plant living worms and the age of surviving larvae, shows that the female ear hazard rate, the number of individual plant boreholes, the individual plant tunnel length, the number of individual plant living worms and the age of surviving larvae of the transgenic corn 2A-7 are significantly lower than the corresponding non-transgenic corn varieties and local common cultivated corn varieties within a significant level range of 5%, and reach significant difference levels. Experimental results show that the transgenic corn 2A-7 has better control effect on target insect Asiatic corn borer.

Table 23: anti-corn borer effect in spinning period

Table 24: corn borer hazard level investigation result of female ear

As can be seen from the above results, the average damage levels of the female ears of 2A-7, control and main cultivar Zhengdan 958 were 1.00, 7.57 and 7.40, respectively. By combining the data, the resistance effects of the 2A-7 insect-resistant corn and the two control corn on corn borers are obviously different and are expressed as high resistance level.

5.2.3 ear stalk hazard level investigation

The ear handles are used as the only channels for nutrient transportation in corn grouting, play an important role in seed development, and meanwhile, the ear handles play a supporting role on the ears in the harvesting period, so that an important guarantee is provided for mechanized harvesting. For this purpose, we investigated parameters such as the ear stem damage rate, the number of surviving larvae of the ear stem, the ear stem damage length (cm), etc., and analysis showed that the ear stem damage rate, the number of surviving larvae of the ear stem, the ear stem damage length (cm) of transgenic corn 2A-7 were significantly lower than those of the corresponding non-transgenic corn varieties and the local normal cultivated corn varieties within a significant level of 5% (table 25). Experimental results show that the transgenic corn 2A-7 has better control effect on the corn borer which is a target pest.

Table 25: corn borer resisting effect of ear stem

5.3 analysis of the results of the identification of the resistance to bollworms

The spiny period is inoculated with insects, and the identification result of the insects is investigated 2-3 weeks after the insects are inoculated. As shown in fig. 7 and table 26, analysis of the parameters of the female ear pest rate, the number of surviving larvae, and the female ear pest length (cm) showed that the female ear pest rate, the number of surviving larvae, and the female ear pest length (cm) of transgenic corn 2A-7 were significantly lower than those of the corresponding non-transgenic corn varieties and the local normal cultivar corn varieties within the 5% significant level range, and reached significant difference levels. Experimental results show that the transgenic corn 2A-7 has better control effect on target pests, namely cotton bollworms.

Table 26: anti-cotton bollworm effect in spinning period

The results fully demonstrate that 2A-7 has obvious resistance to attack by lepidopteran pests such as corn borer, armyworm, cotton bollworm and the like.

5.4 results of identification of Spodoptera frugiperda resistance

5.4.1 blade hazard class investigation

The heart leaf stage (the small horn mouth stage, the corn plant grows to 8-10 leaves stage) is inoculated, and the inoculation identification result is investigated 2-3 weeks after inoculation. The results are shown in Table 27, and analysis of parameters such as leaf hazard rate, wormhole or nick size show that the leaf hazard rate, wormhole or nick size of the 2A-7 transformant is significantly lower than that of the corresponding non-transgenic corn control variety within a 5% significant level range, and a significant difference level is achieved. Experimental results show that the transformant leaves have good control effect on target pest spodoptera littoralis and reach high resistance level.

Table 27: leaf stage leaf blade hazard level investigation result of spodoptera frugiperda

5.4.2 female ear hazard level investigation

The spiny period is inoculated with insects, and the identification result of the insects is investigated before harvest. As shown in the results tables 28-29, analysis of five parameters including female ear hazard rate, individual plant hole count, individual plant tunnel length, individual plant live worm count and survival larva age shows that the female ear hazard rate, individual plant hole count, individual plant tunnel length, individual plant live worm count and survival larva age of the transgenic corn 2A-7 are significantly lower than those of the corresponding non-transgenic corn varieties and local common cultivation corn varieties within a 5% significant level range, and reach significant difference levels. Experimental results show that the transgenic corn 2A-7 has better control effect on target pest spodoptera littoralis.

Table 28: anti-spodoptera littoralis effect during laying period

Table 29: investigation result of hazard level of spodoptera frugiperda on female ears

In addition, FIGS. 8-9 show the results of indoor bioassays of 2A-7 leaf or filament resistance to Spodoptera frugiperda, respectively. FIG. 10 shows the results of field tests for resistance of 2A-7 to Spodoptera frugiperda.

The results show that the transgenic corn 2A-7 has good resistance to spodoptera frugiperda.

Example 4 identification of glufosinate resistance of maize events 2A-7

1. Test protocol

1.1 test materials

Baosta (18% glufosinate-ammonium soluble), manufactured by Bayer corporation.

2A-7: transformant T5 generation material was identical to example 3.

1.2 design of experiments

(1) Test design

Random block design, 3-4 replicates. The cell is provided with a 1.0m wide isolation zone, and the cell area is not less than 24m ² The processing comprises the following steps: the transgenic corn is not sprayed with herbicide; spraying a target herbicide to transgenic corn; the corresponding non-transgenic corn is not sprayed with herbicide; the corresponding non-transgenic corn is sprayed with the target herbicide.

(2) Glufosinate administration dosage

The applied dosages of the herbicide used are divided into: medium dose of pesticide registration label (600 g active ingredient/hectare), medium dose of 2 times amount (1200 g active ingredient/hectare), medium dose of 4 times amount (2400 g active ingredient/hectare). For water amounts of 450L/hectare.

1.3 period of application

Is applied according to the recommended time of glufosinate-resistant corn.

Post-emergence stems and leaves are used for treating glufosinate, and general resistance identification is used in the 3-5 leaf stage of corn.

1.4 spray apparatus requirements

(1) Selection of nebulizers

Manual knapsack sprayer or CO requiring uniform pressure, wide spray width and stable flow rate ₂ Compressing the atomizer. And spraying evenly.

(2) Spray head

A fan-shaped spray head is selected.

(3) Spraying method

Each treatment needs to be sprayed at one time. The dosage is calculated according to the actual area of the spraying district. The experiment should be repeated with rainfall within 12 hours after the drug.

1.5 resistance identification investigation

Maize seedling rates, plant heights, and phytotoxicity symptoms were investigated and recorded at 1 week, 2 weeks, and 4 weeks, respectively, after dosing. 15 corn plants are taken from each cell.

After harvesting the corns, taking 2 rows of corns in the middle of each cell for measuring yield.

1.6 analysis and presentation of results

Herbicide damage rate was calculated as follows.

Wherein:

x-percent victims in percent (%);

n-peer number of victims;

s-the number of grades;

t-total plant number;

m-highest level.

The classification of the symptoms of phytotoxicity is according to GB/T17980.42-2000.

Stage 1: the corn grows normally without any harm symptoms;

2 stages: the corn is slightly harmful to the human body, and the harm is less than 10%;

3 stages: the corn has the same pesticide injury, can be recovered later, and does not influence the yield;

4 stages: the corn has heavy phytotoxicity, is difficult to recover, and causes yield reduction;

5 stages: the corn has serious phytotoxicity and cannot be recovered, so that the yield is obviously reduced or prevented.

2. Experimental results

2.1 Rate of victims

The damage rate of the transformant was examined at 1 week, 2 weeks and four weeks of spraying, respectively, and the results are shown in the following table.

Table 30: failure rate of glufosinate treatment

From the above table, it can be seen that there is a slight phytotoxicity after 2 times of medium dose glufosinate, the phytotoxicity is less than 10%, and the phytotoxicity is greater than 10% after 4 times of medium dose glufosinate, the transgenic corn 2A-7.

2.2 2A-7 plant height investigation

The strain heights of the transformants were investigated at 1 week, 2 weeks and four weeks of spraying, respectively, and the results are shown in the following table.

Table 31: influence of glufosinate-ammonium treatment on plant height

From the above table, transgenic corn 2A-7 showed little but no significant difference from the non-sprayed control plant height after spraying glufosinate at different concentrations.

2.3 2A-7 yield investigation

The single spike kernel yield and moisture content were investigated at harvest and the acre yield (14% moisture) was calculated as the results are given in the table below.

Table 32: effect of glufosinate-ammonium treatment on yield

From the above table, transgenic corn 2A-7 showed reduced but no significant difference in yield after spraying glufosinate at different concentrations than the control without spraying.

Based on the above results, the classification results of the phytotoxicity symptoms according to GB/T17980.42-2000 are shown in the following table. The transgenic corn 2A-7 has normal growth and no harm symptoms after the glufosinate is dosed in the spraying process, the resistance grade is 1 grade, the corn is slightly phytotoxicity after the glufosinate is dosed in 2 times of the spraying process, the phytotoxicity is less than 10%, the resistance grade is 2 grade, the corn is slightly phytotoxicity after the glufosinate is dosed in 4 times of the spraying process, the plant height can be recovered, the yield is reduced compared with the micro spraying process, and the resistance grade is 4 grade. The results show that 2A-7 has excellent resistance to herbicides such as glufosinate.

Table 33: glufosinate resistance ranking

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> Chinese university of agriculture

<120> corn event 2A-7 and methods of identifying same

<130> IDC190193

<160> 25

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> 5' junction sequence

<400> 1

ggcagtgggt tgcccacggt 20

<210> 2

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> 3' junction sequence

<400> 2

caatccagat ccacgcgcat 20

<210> 3

<211> 442

<212> DNA

<213> artificial sequence

<220>

<223> 5 'junction sequence+5' flanking sequence

<400> 3

ttcaaactgg ccctccttaa agagaagata aaaatcggag acctcatatc actagtgacc 60

atagaaaaaa tgagaatttc gtgggtctgt ctgaatatga aattcaattc cacctaggga 120

agggagacac ttccagttcc agtgcgggat tttaccgaac gctaacaatg agcgcctggc 180

gtgcgttaat ttggcagtgt cggcgcagca gaagttcggg atcaactacg ggcagatcgc 240

gaacaatctc ccggacccga cgcaggtggc gaccctcctc cgatcgatga acgtgaacaa 300

ggtgaagctc tacgacgcgg acccccgggt gctgacggcg ttcgcgaaca cgggcgtgga 360

gttcatcatc gccgtgggca acgagaacct gcagaccatg gccggcagcc ccgccgcggc 420

gcggcagtgg gttgcccacg gt 442

<210> 4

<211> 511

<212> DNA

<213> artificial sequence

<220>

<223> 3 'junction sequence+3' flanking sequence

<400> 4

gcaatccaga tccacgcgca tcacctgcgt caccgtgggc aacgaggtct tctccggcaa 60

cgacacggcc acgatggcca gcctcctccc cgctatgaag gccgtccacg cggcgctcgc 120

ggacctgggc ctcggcggcc aggccaccgt ctcgtcggcg cactccgtga acgtgctcgc 180

cgccagcttc ccgccgtcgt cgggcgcgtt ccgggaggac ctggcggagt acatgaagcc 240

catcctggac ttccacgccc agacggggtc gccgttcctc atcaacgcct accccttctt 300

cgcgtacaag gcgagcccgg gcagcgtgtc gctgccgtac gtgctgttcg agcccaaccc 360

gggggtgcgc gaccccagca ccggcctcag ctacgacaac atgctgtacg cccagatcga 420

cgccgtgtac gccgccatga aggccatggg gcacacggac gtgggcgtcc ggatctcgga 480

gaccgggtgg ccgtccaggg gcgacgagga c 511

<210> 5

<211> 9031

<212> DNA

<213> artificial sequence

<220>

<223> exogenous nucleic acid molecule+flanking sequences at both ends

<400> 5

ttcaaactgg ccctccttaa agagaagata aaaatcggag acctcatatc actagtgacc 60

atagaaaaaa tgagaatttc gtgggtctgt ctgaatatga aattcaattc cacctaggga 120

agggagacac ttccagttcc agtgcgggat tttaccgaac gctaacaatg agcgcctggc 180

gtgcgttaat ttggcagtgt cggcgcagca gaagttcggg atcaactacg ggcagatcgc 240

gaacaatctc ccggacccga cgcaggtggc gaccctcctc cgatcgatga acgtgaacaa 300

ggtgaagctc tacgacgcgg acccccgggt gctgacggcg ttcgcgaaca cgggcgtgga 360

gttcatcatc gccgtgggca acgagaacct gcagaccatg gccggcagcc ccgccgcggc 420

gcggcagtgg gttgcccacg gtgacgcagg tgacgcaggt gatgcgcgtg gacggccttc 480

ataacaaatt gacgcttaga caacttaata acacattgcg gacgttttta atgtactgaa 540

ttaacgccga attaattcgg gggatctgga ttttagtact ggattttggt tttaggaatt 600

agaaatttta ttgatagaag tattttacaa atacaaatac atactaaggg tttcttatat 660

gctcaacaca tgagcgaaac cctataggaa ccctaattcc cttatctggg aactactcac 720

acattattat ggagaaactc gagtcaaatc tcggtgacgg gcaggaccgg acggggcggt 780

accggcaggc tgaagtccag ctgccagaaa cccacgtcat gccagttccc gtgcttgaag 840

ccggccgccc gcagcatgcc gcggggggca tatccgagcg cctcgtgcat gcgcacgctc 900

gggtcgttgg gcagcccgat gacagcgacc acgctcttga agccctgtgc ctccagggac 960

ttcagcaggt gggtgtagag cgtggagccc agtcccgtcc gctggtggcg gggggagacg 1020

tacacggtcg actcggccgt ccagtcgtag gcgttgcgtg ccttccaggg gcccgcgtag 1080

gcgatgccgg cgacctcgcc gtccacctcg gcgacgagcc agggatagcg ctcccgcaga 1140

cggacgaggt cgtccgtcca ctcctgcggt tcctgcggct cggtacggaa gttgaccgtg 1200

cttgtctcga tgtagtggtt gacgatggtg cagaccgccg gcatgtccgc ctcggtggca 1260

cggcggatgt cggccgggcg tcgttctggg ctcatggtag actcgagaga gatagatttg 1320

tagagagaga ctggtgattt cagcgtgtcc tctccaaatg aaatgaactt ccttatatag 1380

aggaagggtc ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca gtggagatat 1440

cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc 1500

tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct 1560

ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga 1620

tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt 1680

gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga 1740

cgagagtgtc gtgctccacc atgttcacat caatccactt gctttgaaga cgtggttgga 1800

acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg accactgtcg 1860

gcagaggcat cttgaacgat agcctttcct ttatcgcaat gatggcattt gtaggtgcca 1920

ccttcctttt ctactgtcct tttgatgaag tgacagatag ctgggcaatg gaatccgagg 1980

aggtttcccg atattaccct ttgttgaaaa gtctcaatag ccctttggtc ttctgagact 2040

gtatctttga tattcttgga gtagacgaga gtgtcgtgct ccaccatgtt ggcaagctgc 2100

tctagccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 2160

acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 2220

tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 2280

ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gaattcgccc 2340

gatctagtaa catagatgac accgcgcgcg ataatttatc ctagtttgcg cgctatattt 2400

tgttttctat cgcgtattaa atgtataatt gcgggactct aatcataaaa acccatctca 2460

taaataacgt catgcattac atgttaatta ttacatgctt aacgtaattc aacagaaatt 2520

atatgataat catcgcaaga ccggcaacag gattcaatct taagaaactt tattgccaaa 2580

tgtttgaacg ggtgacctca cacggccttc tgagccctct ccaggtcgta ctcggcctcg 2640

aaggtcacct cggcgggcac gaactcgatg cggtcgatgt acacctcgtt gccgctgttg 2700

aacacgtggg cgctcagggt gaacacgctg ctgccgttgc tgaagttgaa gggggtggtg 2760

aagcccacgg tgcggaagct gccgctctgc aggttgctgc cgctgctcat ggtggcgctg 2820

aagttgccct ggttgatggg gcggccgtcg atgctggtgt ggaactgcag gttggtggtg 2880

ctggcgtagc ggatgcggac gcggtagcgc tggctcaggg gggcggtgat gttcacgcgc 2940

agggtgctga tctggccggg gctggtgcgg cgcaggatgt cgccgccggt gaagccgggg 3000

cccttcacca cgctggtgcc gctgcccagg ttggtgctct tggtcagggg gatctgggtg 3060

atctggctgc tggggatgat gttgttgaac tcggcggacc tgtggatcca ggagaacatt 3120

ggagccctga tgatggacac ggaggagttg gagaaaccgg acctgaacat ggacacgtgg 3180

gaaagcctgt gggagaaacc ctgccttggt ggcacgttgt tgttctgtgg tggaatctcg 3240

tcaagggagt ccacggtacc ggacttcctg tacacggcgg agggaaggtt ggaggaggta 3300

ccgtaggcga actcggtacc gtcaagcacg gaaagctgct ggttgttgat accgatgttg 3360

aagggcctcc tgtaaagggt ggaggaaagg gtcctgtaca caccctgacc aagctgggcc 3420

acgatcctct gctgtggagc ggcgttaccc atggtaccgt aaagtgggaa ggtgaactcg 3480

ggaccggaga aacccactgg gctggccatg atctggtggc cggaccagta gtactcaccg 3540

cggtgggcgt cggtgtagat ggtgatggag ttaaggatgt ccataaggtg tgggctcctg 3600

atggacccct cgataccctg ggcggaaccg cggaaggaac cgtcgaagtt ctcaagcact 3660

gggttggtgt agatctccct ggtaagctgg gacacggtcc tgatagggta cgtcctggag 3720

tcgtagttag ggaacaggga cacgatgtca agcacggtaa gggtaagctc cctcctgaac 3780

tggttgtacc tgatccagtc cctggagtcg ggaccccaca ccctctcaag accggtgttg 3840

taccacctca cggcgtggtc ggtgtagtta ccgataagcc tggtaaggtc gttgtacctg 3900

gagttgatgg tggcggcgtc gaaaccccac ctctgaccga acacggacac gtccctaagc 3960

acggaaaggt gaaggttggc ggcctgcacg tacacggaca gcagtggcac ctggtagttc 4020

tgcacggcga acagtgggat ggcggtggta agggcggagt tcatgtcgtt gaactggatc 4080

ctcatctcct ccctaagggc gggattggtg ggatcggcct cccactccct gaaggactcg 4140

gcgtagatct ggtaaaggtt ggaaagaccc tcaagcctgg agatggcctg gttcctggcg 4200

aactcctcga tcctctggtt gataagctgc tcgatctgca caaggaaggc gtcccactgg 4260

gagggaccga agatgcccca gatgatgtcc acaagaccaa gcacgaaacc ggcaccgggc 4320

acgaactcgg aaagaaggaa ctgggtaagg gaaagggaga tgtcgatggg agtgtaaccg 4380

gtctcgatcc tctcaccacc aagcacctcc acctcgggat tggaaaggca gttgtaggga 4440

atgcactcgt tgatgttggg attgttgtcc atctcgatcc gctcacccac gaacccgaac 4500

cccggagaac ctcacgaacc cgaacccgaa ccggaggggg aggagaagcc aacccacctc 4560

taagaaagca cggagaggga atgcgtcccc cgcagcgtct ttatagccga gcgcggggcc 4620

gccgcggccg tcctgatccc atccgacggc gcggacgccc agtggaatat gcggggcatc 4680

ggggtcgcca ccgcgctacg agcacacgag cacctcccgg ggacggcttt gttgggaccc 4740

ggagcaggac gggagcgtga ggcggggccc agcgtgactg acctgacctc gttttggccg 4800

ctgcactgcc ggcctgtgca ggccaccgaa aaatatctcg cgtggcgaaa tgccatcctt 4860

gccctcggga gcacacggat cggacggcaa gcggtcgtcg ggtcgggtgg tggatatgga 4920

cacttgctga tgacgtgggt ccacgatggc agtgggcgaa gccaagtcca cgcgtcacag 4980

tgaccgcacc tagtgctcaa aaacgacgga tcggatagga acgaatacat gtcacaattc 5040

actaccttgt aatctaagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa 5100

ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa 5160

tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg 5220

ctagagcagc ttgagcttgg atcagattgt cgtttcccgc cttcagttta gcttcatgga 5280

gtcaaagatt caaatagagg acctaacaga actcgccgta aagactggcg aacagttcat 5340

acagagtctc ttacgactca atgacaagaa gaaaatcttc gtcaacatgg tggagcacga 5400

cacacttgtc tactccaaaa atatcaaaga tacagtctca gaagaccaaa gggcaattga 5460

gacttttcaa caaagggtaa tatccggaaa cctcctcgga ttccattgcc cagctatctg 5520

tcactttatt gtgaagatag tggaaaagga aggtggctcc tacaaatgcc atcattgcga 5580

taaaggaaag gccatcgttg aagatgcctc tgccgacagt ggtcccaaag atggaccccc 5640

acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga 5700

ttgatgtgat atctccactg acgtaaggga tgacgcacaa tcccactatc cttcgcaaga 5760

cccttcctct atataaggaa gttcatttca tttggagaga acacggggga ctcttgacca 5820

tgggatcaag tgcaaaggtc cgccttgttt ctcctctgtc tcttgatctg actaatcttg 5880

gtttatgatt cgttgagtaa ttttggggaa agctagcttc gtccacagtt tttttttcga 5940

tgaacagtgc cgcagtggcg ctgatcttgt atgctatcct gcaatcgtgg tgaacttatt 6000

tcttttatat ccttcactcc catgaaaagg ctagtaatct ttctcgatgt aacatcgtcc 6060

agcactgcta ttaccgtgtg gtccatccga cagtctggct gaacacatca tacgatattg 6120

agcaaagatc tatcttccct gttctttaat gaaagacgtc attttcatca gtatgatcta 6180

agaatgttgc aacttgcaag gaggcgtttc tttctttgaa tttaactaac tcgttgagtg 6240

gccctgtttc tcggacgtaa ggcctttgct gctccacaca tgtccattcg aattttaccg 6300

tgtttagcaa gggcgaaaag tttgcatctt gatgatttag cttgactatg cgattgcttt 6360

cctggacccg tgcagctgcg gtggcaacta gtatgaactc cgtcctcaac agcggccgca 6420

ccaccatctg cgacgcctac aacgtggccg cccacgaccc cttctccttc cagcacaagt 6480

ccctggacac cgttcagaag gagtggaccg agtggaagaa gaacaaccac agcctgtacc 6540

tcgaccccat cgtcggcacc gtggcctcct tcctgctgaa gaaggtcggc tccctcgtcg 6600

gcaagcgtat cctgtccgag ctgcgcaacc tcatcttccc cagcggcagc accaacctga 6660

tgcaggacat cctgcgcgag accgagaagt tcctcaacca gaggctgaac accgacaccc 6720

tggctcgcgt gaacgccgag ctgaccggcc tccaggccaa cgtcgaggag ttcaaccgcc 6780

aggtggacaa cttcctgaac cccaaccgta acgccgtccc cctctccatc acctcctccg 6840

tcaacaccat gcagcagctg ttcctgaacc gcctccccca gttccagatg cagggctacc 6900

agctgctcct gctgcccctc ttcgcccagg ctgccaacct gcacctgtcc ttcatcaggg 6960

acgtcatcct caacgccgac gagtggggca tcagcgccgc caccctgcgc acctaccgcg 7020

actacctgaa gaactacacc cgcgactact ccaactactg catcaacacc taccagagcg 7080

ctttcaaggg cctcaacacc cgtctgcacg acatgctgga gttcaggacc tacatgttcc 7140

tcaacgtgtt cgagtacgtg tccatctggt ccctgttcaa gtaccagagc ctgctcgtct 7200

cctccggcgc caacctgtac gccagcggct ccggccccca gcagacccag agcttcacct 7260

cccaggactg gcccttcctg tactccctct tccaggtcaa ctccaactac gtcctgaacg 7320

gcttcagcgg cgcccgcctg agcaacacct tccccaacat cgtcggcctc cccggctcca 7380

ccaccaccca cgccctgctg gctgcccgcg tgaactactc cggcggcatc tcctccggcg 7440

acatcggcgc cagccccttc aaccagaact tcaactgctc caccttcctc ccccccctgc 7500

tgaccccctt cgtgcgctcc tggctcgact ccggctccga ccgcgagggc gtcgccaccg 7560

tcaccaactg gcagaccgag agcttcgaga ccaccctggg cctgaggtcc ggcgccttca 7620

ccgctcgtgg caacagcaac tacttccccg actacttcat ccgcaacatc tccggcgtcc 7680

ccctcgtcgt gcgcaacgag gacctgcgca ggcccctgca ctacaacgag atccgcaaca 7740

tcgcctcccc cagcggcacc cccggcggcg cccgtgccta catggtgtcc gtccacaacc 7800

gcaagaacaa catccacgcc gtccacgaga acggctccat gatccacctc gctcccaacg 7860

actacaccgg cttcaccatc agccccatcc acgccaccca ggtcaacaac cagacccgca 7920

ccttcatctc cgagaagttc ggcaaccagg gcgacagcct gaggttcgag cagaacaaca 7980

ccaccgcccg ctacaccctg cgcggcaacg gcaactccta caacctctac ctgcgtgtgt 8040

cctccatcgg caacagcacc atccgcgtca ccatcaacgg cagggtgtac accgccacca 8100

acgtcaacac caccaccaac aacgacggcg tcaacgacaa cggcgcccgc ttcagcgaca 8160

tcaacatcgg caacgtggtc gcttcctcca actccgacgt ccccctggac atcaacgtga 8220

ccctcaactc cggcacccag ttcgacctga tgaacatcat gctggtcccc accaacatca 8280

gccccctcta ctaaggtgac cagctcgaat ttccccgatc gttcaaacat ttggcaataa 8340

agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 8400

aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 8460

tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 8520

gcaatccaga tccacgcgca tcacctgcgt caccgtgggc aacgaggtct tctccggcaa 8580

cgacacggcc acgatggcca gcctcctccc cgctatgaag gccgtccacg cggcgctcgc 8640

ggacctgggc ctcggcggcc aggccaccgt ctcgtcggcg cactccgtga acgtgctcgc 8700

cgccagcttc ccgccgtcgt cgggcgcgtt ccgggaggac ctggcggagt acatgaagcc 8760

catcctggac ttccacgccc agacggggtc gccgttcctc atcaacgcct accccttctt 8820

cgcgtacaag gcgagcccgg gcagcgtgtc gctgccgtac gtgctgttcg agcccaaccc 8880

gggggtgcgc gaccccagca ccggcctcag ctacgacaac atgctgtacg cccagatcga 8940

cgccgtgtac gccgccatga aggccatggg gcacacggac gtgggcgtcc ggatctcgga 9000

gaccgggtgg ccgtccaggg gcgacgagga c 9031

<210> 6

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> 5' Forward primer

<400> 6

cgatcgatga acgtgaacaa g 21

<210> 7

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> 5' reverse primer

<400> 7

cagtacatta aaaacgtccg caat 24

<210> 8

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> 3' Forward primer

<400> 8

gtttttatga ttagagtccc gcaat 25

<210> 9

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> 3' reverse primer

<400> 9

caggatgggc ttcatgtact cc 22

<210> 10

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> zSSIIb-F

<400> 10

cggtggatgc taaggctgat g 21

<210> 11

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> zSSIIb-R

<400> 11

aaagggccag gttcattatc ctc 23

<210> 12

<211> 1875

<212> DNA

<213> artificial sequence

<220>

<223> mcry1Ab Gene

<400> 12

tcacacggcc ttctgagccc tctccaggtc gtactcggcc tcgaaggtca cctcggcggg 60

cacgaactcg atgcggtcga tgtacacctc gttgccgctg ttgaacacgt gggcgctcag 120

ggtgaacacg ctgctgccgt tgctgaagtt gaagggggtg gtgaagccca cggtgcggaa 180

gctgccgctc tgcaggttgc tgccgctgct catggtggcg ctgaagttgc cctggttgat 240

ggggcggccg tcgatgctgg tgtggaactg caggttggtg gtgctggcgt agcggatgcg 300

gacgcggtag cgctggctca ggggggcggt gatgttcacg cgcagggtgc tgatctggcc 360

ggggctggtg cggcgcagga tgtcgccgcc ggtgaagccg gggcccttca ccacgctggt 420

gccgctgccc aggttggtgc tcttggtcag ggggatctgg gtgatctggc tgctggggat 480

gatgttgttg aactcggcgg acctgtggat ccaggagaac attggagccc tgatgatgga 540

cacggaggag ttggagaaac cggacctgaa catggacacg tgggaaagcc tgtgggagaa 600

accctgcctt ggtggcacgt tgttgttctg tggtggaatc tcgtcaaggg agtccacggt 660

accggacttc ctgtacacgg cggagggaag gttggaggag gtaccgtagg cgaactcggt 720

accgtcaagc acggaaagct gctggttgtt gataccgatg ttgaagggcc tcctgtaaag 780

ggtggaggaa agggtcctgt acacaccctg accaagctgg gccacgatcc tctgctgtgg 840

agcggcgtta cccatggtac cgtaaagtgg gaaggtgaac tcgggaccgg agaaacccac 900

tgggctggcc atgatctggt ggccggacca gtagtactca ccgcggtggg cgtcggtgta 960

gatggtgatg gagttaagga tgtccataag gtgtgggctc ctgatggacc cctcgatacc 1020

ctgggcggaa ccgcggaagg aaccgtcgaa gttctcaagc actgggttgg tgtagatctc 1080

cctggtaagc tgggacacgg tcctgatagg gtacgtcctg gagtcgtagt tagggaacag 1140

ggacacgatg tcaagcacgg taagggtaag ctccctcctg aactggttgt acctgatcca 1200

gtccctggag tcgggacccc acaccctctc aagaccggtg ttgtaccacc tcacggcgtg 1260

gtcggtgtag ttaccgataa gcctggtaag gtcgttgtac ctggagttga tggtggcggc 1320

gtcgaaaccc cacctctgac cgaacacgga cacgtcccta agcacggaaa ggtgaaggtt 1380

ggcggcctgc acgtacacgg acagcagtgg cacctggtag ttctgcacgg cgaacagtgg 1440

gatggcggtg gtaagggcgg agttcatgtc gttgaactgg atcctcatct cctccctaag 1500

ggcgggattg gtgggatcgg cctcccactc cctgaaggac tcggcgtaga tctggtaaag 1560

gttggaaaga ccctcaagcc tggagatggc ctggttcctg gcgaactcct cgatcctctg 1620

gttgataagc tgctcgatct gcacaaggaa ggcgtcccac tgggagggac cgaagatgcc 1680

ccagatgatg tccacaagac caagcacgaa accggcaccg ggcacgaact cggaaagaag 1740

gaactgggta agggaaaggg agatgtcgat gggagtgtaa ccggtctcga tcctctcacc 1800

accaagcacc tccacctcgg gattggaaag gcagttgtag ggaatgcact cgttgatgtt 1860

gggattgttg tccat 1875

<210> 13

<211> 1902

<212> DNA

<213> artificial sequence

<220>

<223> mcry2Ab Gene

<400> 13

atgaactccg tcctcaacag cggccgcacc accatctgcg acgcctacaa cgtggccgcc 60

cacgacccct tctccttcca gcacaagtcc ctggacaccg ttcagaagga gtggaccgag 120

tggaagaaga acaaccacag cctgtacctc gaccccatcg tcggcaccgt ggcctccttc 180

ctgctgaaga aggtcggctc cctcgtcggc aagcgtatcc tgtccgagct gcgcaacctc 240

atcttcccca gcggcagcac caacctgatg caggacatcc tgcgcgagac cgagaagttc 300

ctcaaccaga ggctgaacac cgacaccctg gctcgcgtga acgccgagct gaccggcctc 360

caggccaacg tcgaggagtt caaccgccag gtggacaact tcctgaaccc caaccgtaac 420

gccgtccccc tctccatcac ctcctccgtc aacaccatgc agcagctgtt cctgaaccgc 480

ctcccccagt tccagatgca gggctaccag ctgctcctgc tgcccctctt cgcccaggct 540

gccaacctgc acctgtcctt catcagggac gtcatcctca acgccgacga gtggggcatc 600

agcgccgcca ccctgcgcac ctaccgcgac tacctgaaga actacacccg cgactactcc 660

aactactgca tcaacaccta ccagagcgct ttcaagggcc tcaacacccg tctgcacgac 720

atgctggagt tcaggaccta catgttcctc aacgtgttcg agtacgtgtc catctggtcc 780

ctgttcaagt accagagcct gctcgtctcc tccggcgcca acctgtacgc cagcggctcc 840

ggcccccagc agacccagag cttcacctcc caggactggc ccttcctgta ctccctcttc 900

caggtcaact ccaactacgt cctgaacggc ttcagcggcg cccgcctgag caacaccttc 960

cccaacatcg tcggcctccc cggctccacc accacccacg ccctgctggc tgcccgcgtg 1020

aactactccg gcggcatctc ctccggcgac atcggcgcca gccccttcaa ccagaacttc 1080

aactgctcca ccttcctccc ccccctgctg acccccttcg tgcgctcctg gctcgactcc 1140

ggctccgacc gcgagggcgt cgccaccgtc accaactggc agaccgagag cttcgagacc 1200

accctgggcc tgaggtccgg cgccttcacc gctcgtggca acagcaacta cttccccgac 1260

tacttcatcc gcaacatctc cggcgtcccc ctcgtcgtgc gcaacgagga cctgcgcagg 1320

cccctgcact acaacgagat ccgcaacatc gcctccccca gcggcacccc cggcggcgcc 1380

cgtgcctaca tggtgtccgt ccacaaccgc aagaacaaca tccacgccgt ccacgagaac 1440

ggctccatga tccacctcgc tcccaacgac tacaccggct tcaccatcag ccccatccac 1500

gccacccagg tcaacaacca gacccgcacc ttcatctccg agaagttcgg caaccagggc 1560

gacagcctga ggttcgagca gaacaacacc accgcccgct acaccctgcg cggcaacggc 1620

aactcctaca acctctacct gcgtgtgtcc tccatcggca acagcaccat ccgcgtcacc 1680

atcaacggca gggtgtacac cgccaccaac gtcaacacca ccaccaacaa cgacggcgtc 1740

aacgacaacg gcgcccgctt cagcgacatc aacatcggca acgtggtcgc ttcctccaac 1800

tccgacgtcc ccctggacat caacgtgacc ctcaactccg gcacccagtt cgacctgatg 1860

aacatcatgc tggtccccac caacatcagc cccctctact aa 1902

<210> 14

<211> 552

<212> DNA

<213> artificial sequence

<220>

<223> Bar Gene

<400> 14

tcaaatctcg gtgacgggca ggaccggacg gggcggtacc ggcaggctga agtccagctg 60

ccagaaaccc acgtcatgcc agttcccgtg cttgaagccg gccgcccgca gcatgccgcg 120

gggggcatat ccgagcgcct cgtgcatgcg cacgctcggg tcgttgggca gcccgatgac 180

agcgaccacg ctcttgaagc cctgtgcctc cagggacttc agcaggtggg tgtagagcgt 240

ggagcccagt cccgtccgct ggtggcgggg ggagacgtac acggtcgact cggccgtcca 300

gtcgtaggcg ttgcgtgcct tccaggggcc cgcgtaggcg atgccggcga cctcgccgtc 360

cacctcggcg acgagccagg gatagcgctc ccgcagacgg acgaggtcgt ccgtccactc 420

ctgcggttcc tgcggctcgg tacggaagtt gaccgtgctt gtctcgatgt agtggttgac 480

gatggtgcag accgccggca tgtccgcctc ggtggcacgg cggatgtcgg ccgggcgtcg 540

ttctgggctc at 552

<210> 15

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> TAIL-PCR primer

<400> 15

acgatggact ccagagcggc cgcvnvnnng gaa 33

<210> 16

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> TAIL-PCR primer

<400> 16

acgatggact ccagagcggc cgcbnbnnng gtt 33

<210> 17

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> TAIL-PCR primer

<400> 17

acgatggact ccagagcggc cgcvvnvnnn ccaa 34

<210> 18

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> TAIL-PCR primer

<400> 18

acgatggact ccagagcggc cgcbdnbnnn cggt 34

<210> 19

<211> 16

<212> DNA

<213> artificial sequence

<220>

<223> TAIL-PCR primer

<400> 19

acgatggact ccagag 16

<210> 20

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> TAIL-PCR primer

<400> 20

ctgttgccgg tcttgcgatg attat 25

<210> 21

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> TAIL-PCR primer

<400> 21

ttctgttgaa ttacgttaag catgt 25

<210> 22

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> TAIL-PCR primer

<400> 22

ggtttttatg attagagtcc cgcaa 25

<210> 23

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> TAIL-PCR primer

<400> 23

ctgcccgtca ccgagatttg 20

<210> 24

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> TAIL-PCR primer

<400> 24

tcctataggg tttcgctcat gtgtt 25

<210> 25

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> TAIL-PCR primer

<400> 25

gtactaaaat ccagatcccc cgaat 25

Claims

1. An article of manufacture comprising a maize plant or part, seed, cell, or progeny thereof having incorporated into its genome an exogenous nucleic acid molecule comprising Cry1Ab and Cry2Ab genes and a Bar gene, wherein:

(1) The exogenous nucleic acid molecule is flanked by 5 'flanking regions consisting of the nucleotide sequence from positions 1 to 432 or from positions 300 to 432 of SEQ ID NO. 5, and the exogenous nucleic acid molecule is flanked by 3' flanking regions consisting of the nucleotide sequence from positions 8532 to 9031 or from positions 8532 to 8800 of SEQ ID NO. 5; or,

(2) The exogenous nucleic acid molecule is flanked by a 5 'flanking region and a 3' flanking region, the sequence of the part comprising the 5 'flanking region and the exogenous nucleic acid molecule adjacent thereto is shown in SEQ ID NO. 1, and the sequence of the part comprising the 3' flanking region and the exogenous nucleic acid molecule adjacent thereto is shown in SEQ ID NO. 2;

and, the product is a food, feed, cosmetic, pharmaceutical or industrial product, which product is not reproducible.

2. The article of manufacture of claim 1, wherein the exogenous nucleic acid molecule is located in the genome at a position corresponding to between chr3:179141694bp-179141724bp of a B73V4 version reference genomic sequence.

3. The article of manufacture of claim 1, wherein the nucleotide sequences of the Cry1Ab and Cry2Ab genes are codon optimized for expression in monocot plant cells.

4. The article of manufacture of claim 3, wherein said Cry1Ab gene is set forth in SEQ ID No. 12; and/or, the Cry2Ab gene is shown as SEQ ID NO. 13.

5. The article of manufacture of claim 1, wherein the exogenous nucleic acid molecule comprises a 35S polyA terminator, a Bar gene, a CAMV 35S promoter, a nos polyA terminator, a Cry1Ab gene, a Gly promoter, a CAMV 35S promoter, an adh1 enhancer, a Cry2Ab gene, and a nos polyA terminator.

6. The article of claim 1, wherein the exogenous nucleic acid molecule consists of the sequence of nucleotides 433-8531 of SEQ ID No. 5 or the complement thereof.

7. The article of manufacture of claim 1, wherein the genome of the maize plant or part, seed, cell or progeny thereof comprises the sequence set forth in any one of SEQ ID NOs 1-5 or the complement thereof.

8. The article of manufacture of claim 1, wherein the corn seed from which the corn plant or portion, seed, cell or progeny thereof is produced is deposited at the China general microbiological culture collection center (CGMCC) and has a deposit number of CGMCC No.17848.

9. The article of manufacture of claim 1, wherein the maize plant or part, seed, cell, or progeny thereof is resistant to insect infestation.

10. The article of claim 9, wherein the insect is selected from lepidopteran insects.

11. The article of claim 9, wherein the insect is selected from one or more of the group consisting of armyworms, corn borers, cotton bollworms, carpopodoptera frugiperda, and spodoptera frugiperda.

12. An article of manufacture comprising corn seeds or corn plants produced from said corn seeds or parts, seeds, cells or progeny thereof, wherein said corn seeds are deposited at the China general microbiological culture collection center (CGMCC) and have a deposit number of CGMCC No.17848;

13. The article of manufacture of claim 1, wherein the article of manufacture comprises genomic DNA of the maize plant or part, seed, cell, or progeny thereof.

14. The article of manufacture of claim 12, wherein the article of manufacture comprises genomic DNA of the corn seed or a corn plant produced from the corn seed or a part, seed, cell, or progeny thereof.

15. The article of manufacture of any one of claims 1-14, wherein the article of manufacture comprises a sequence selected from the group consisting of the sequences set forth in any one of SEQ ID NOs 1-5 or the complement thereof.

16. The preparation of any one of claims 1 to 14, wherein the nucleic acid contained in the preparation is amplified with SEQ ID No. 6 and SEQ ID No. 7 as forward and reverse primers, respectively, to produce an amplicon having a length of 200-300 bp; and/or, using SEQ ID NO. 8 and SEQ ID NO. 9 as forward primer and reverse primer, respectively, to amplify the nucleic acid contained in the preparation to generate an amplicon with the length of 250-350 bp.

17. The article of any one of claims 1-14, wherein the article is selected from the group consisting of corn ears, bract-removed corn, corn cobs, corn pollen, corn grits, corn flour, crushed corn, corn flour, corn oil, corn starch, corn slurry, corn malt, corn sugar, corn syrup, margarine produced from corn oil, corn flakes, popcorn, ethanol and/or juice produced from corn (liquor), distillers dried grains produced from corn fermentation (DDGS), animal feed from corn, cosmetics, and bulking agents.

18. The article of claim 17, wherein the corn oil is an unsaturated corn oil or a saturated corn oil.

19. The article of any one of claims 1-14, wherein the portion of the corn plant is selected from the group consisting of kernels, pollen, ovules, flowers, shoots, roots, stems, silks, inflorescences, ears and leaves.

20. A primer pair comprising a first primer and a second primer, wherein the first primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 1 to 432 of SEQ ID NO. 5 or a complement thereof, said second primer consisting of: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 483 to 8524 of SEQ ID NO. 5 or the complement thereof.

21. The primer pair of claim 20, wherein the first primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 200 to 432 of SEQ ID NO. 5 or the complement thereof.

22. The primer pair of claim 20, wherein the second primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 483 to 1000 of SEQ ID NO. 5 or the complement thereof.

23. The primer pair of claim 20, wherein amplification of the sequence set forth in SEQ id No. 5 using the first and second primers yields an amplicon of 200-300bp in length.

24. The primer pair of claim 23, wherein the amplicon is about 258bp in length.

25. The primer pair of claim 20, wherein the first primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 300 to 400 of SEQ ID NO. 5 or a complement thereof, said second primer consisting of: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 500 to 600 of SEQ ID NO. 5 or the complement thereof.

26. The primer pair of claim 25, wherein the first primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 300 to 350 of SEQ ID NO. 5 or the complement thereof.

27. The primer pair of claim 25, wherein the second primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 520 to 570 of SEQ ID NO. 5 or the complement thereof.

28. The primer pair of claim 25, wherein the first primer consists of the sequence set forth in SEQ ID No. 6 and the second primer consists of the sequence set forth in SEQ ID No. 7.

29. A primer pair comprising a first primer and a second primer, wherein the first primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 483 to 8524 of SEQ ID NO. 5 or a complement thereof, said second primer consisting of: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 8532 to 9031 of SEQ ID NO. 5 or the complement thereof.

30. The primer pair of claim 29, wherein the first primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 8300-8524 of SEQ ID NO. 5 or the complement thereof.

31. The primer pair of claim 29, wherein the second primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 8532 to 8800 of SEQ ID NO. 5 or the complement thereof.

32. The primer pair of claim 29, wherein amplification of the sequence set forth in SEQ id No. 5 using the first and second primers produces an amplicon having a length of 250-350 bp.

33. The primer pair of claim 32, wherein the amplicon is about 310bp in length.

34. The primer pair of claim 29, wherein the first primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 8400-8500 of SEQ ID NO. 5 or a complement thereof, said second primer consisting of: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 8700-8800 of SEQ ID NO. 5 or the complement thereof.

35. The primer pair of claim 34, wherein the first primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 8450-8500 of SEQ ID NO. 5 or the complement thereof.

36. The primer pair of claim 34, wherein the second primer consists of the sequence: a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 8750 to 8800 of SEQ ID NO. 5 or the complement thereof.

37. The primer pair of claim 34, wherein the first primer consists of the sequence set forth in SEQ ID No. 8 and the second primer consists of the sequence set forth in SEQ ID No. 9.

38. A method of detecting a nucleic acid molecule unique to maize event 2A-7 in a sample comprising maize nucleic acid, comprising:

(1) Contacting the primer pair of any one of claims 20-37 with the sample;

(2) Performing a nucleic acid amplification reaction; and

(3) Detecting the product of step (2) by gel electrophoresis;

wherein the maize event 2A-7 is a maize plant or part, seed, cell or progeny thereof as defined in any one of claims 1-12.

39. The method of claim 38, comprising:

(1) Contacting the primer pair of any one of claims 20-28 with the sample;

(2) Performing a nucleic acid amplification reaction; and

(3) Detecting the product of step (2) by gel electrophoresis;

wherein detection of an amplicon having a length of 250-260bp indicates the presence of a nucleic acid molecule in the sample that is unique to maize event 2A-7.

40. The method of claim 39, wherein detection of an amplicon having a length of about 258bp indicates the presence of a nucleic acid molecule unique to maize event 2A-7 in the sample.

41. The method of claim 39, wherein the primer pair comprises: a first primer consisting of the sequence shown in SEQ ID NO. 6 and a second primer consisting of the sequence shown in SEQ ID NO. 7.

42. The method of claim 38, comprising:

(1) Contacting the primer pair of any one of claims 29-37 with the sample;

(2) Performing a nucleic acid amplification reaction; and

(3) Detecting the product of step (2) by gel electrophoresis;

wherein detection of an amplicon having a length of 305-315bp indicates the presence of a nucleic acid molecule in the sample that is unique to maize event 2A-7.

43. The method of claim 42, wherein detection of an amplicon having a length of about 310bp indicates the presence of a nucleic acid molecule unique to maize event 2A-7 in the sample.

44. The method of claim 42, wherein the primer pair comprises: a first primer consisting of the sequence shown in SEQ ID NO. 8 and a second primer consisting of the sequence shown in SEQ ID NO. 9.

45. A method of detecting a nucleic acid molecule unique to maize event 2A-7 in a sample comprising maize nucleic acid, comprising:

(1) Contacting a primer pair with the sample; wherein the primer pair is used to amplify nucleic acid of genomic DNA of maize event 2A-7 to generate an amplicon comprising a nucleotide sequence selected from the group consisting of: the sequence shown in any one of SEQ ID NOs 1-5 or a complementary sequence thereof;

(3) Detecting the amplicon;

46. A method of detecting the presence of a nucleic acid molecule unique to corn event 2A-7 in a sample comprising corn nucleic acid, comprising:

(3) Detecting hybridization of the nucleic acid probe to a sample;

47. The method of claim 46, wherein the nucleic acid probe comprises: sequences comprising the 5' flanking region and portions of the exogenous nucleic acid molecule adjacent thereto.

48. The method of claim 46, wherein the nucleic acid probe comprises the sequence set forth in SEQ ID NO. 1 or 2 or a sequence complementary thereto.

49. The method of claim 46, wherein the nucleic acid probe is fluorescently labeled.

50. The method of any one of claims 38-49, wherein the sample is selected from the group consisting of corn ear, bract-removed corn, corn silk, corn pollen, corn grits, corn flour, crushed corn, corn flour, corn oil, corn starch, corn slurry, corn malt, corn sugar, corn syrup, margarine produced from corn oil, corn flakes, popcorn, ethanol and/or juice produced from corn (liquor), distillers dried grains produced from corn fermentation (DDGS), animal feed from corn, cosmetics, and bulking agent.

51. The method of claim 50, wherein the corn oil is an unsaturated corn oil or a saturated corn oil.

52. A kit, comprising: (i) The primer pair of any one of claims 20 to 37, and/or (ii) a nucleic acid probe specific for a sequence set forth in any one of SEQ ID NOs 1 to 5 or a complement thereof.

53. The kit of claim 52, wherein the nucleic acid probe is as defined in any one of claims 47 to 49.

54. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: SEQ ID NOs: any of the sequences shown in SEQ ID NOs 1 to 5 or a complement thereof.

55. A method of producing an insect-resistant corn plant comprising:

(1) Crossing a first parent corn plant with a second parent corn plant; wherein the first or second parent corn plant is a corn plant as defined in any one of claims 1-12;

(2) Obtaining a first generation progeny plant from the crossing of (1); and

(3) Selecting from among these first generation progeny plants a progeny plant that is resistant to the insect, wherein satisfaction of at least one of the following (3 a) - (3 d) indicates that it is an insect-resistant maize plant:

(3b) The genome DNA of the sub-generation plant is amplified by using SEQ ID NO. 6 and SEQ ID NO. 7 as a forward primer and a reverse primer respectively to generate an amplicon with the length of 200-300 bp; and/or the number of the groups of groups,

(3c) Amplifying genome DNA of the sub-generation plant by using SEQ ID NO. 8 and SEQ ID NO. 9 as forward primers and reverse primers respectively to generate an amplicon with the length of 250-350 bp; or alternatively, the first and second heat exchangers may be,

(3d) Genomic DNA of the progeny plant is detected and hybridization detected using nucleic acid probes specific for the sequence set forth in any one of SEQ ID NOs 1-5 or the complement thereof.

56. The method of claim 55, wherein the genomic DNA of the progeny plant is amplified using SEQ ID NO. 6 and SEQ ID NO. 7 as forward and reverse primers, respectively, to generate an amplicon having a length of about 258 bp; and/or, the genome DNA of the progeny plant is amplified by using SEQ ID NO. 8 and SEQ ID NO. 9 as a forward primer and a reverse primer respectively to generate an amplicon with the length of about 310 bp.

57. A method according to claim 55, wherein the insect is selected from lepidopteran insects.

58. The method of claim 57, wherein the insect is selected from one or more of the group consisting of armyworms, corn borers, cotton bollworms, carpopodoptera frugiperda, and spodoptera frugiperda.

59. The method of claim 55, wherein the method further comprises the steps of:

(5) Selecting a plant from these second generation progeny plants that is resistant to insects, wherein satisfaction of said plant of at least one of (3 a) - (3 d) indicates that it is an insect-resistant maize plant.

60. A method of producing hybrid corn seed grown as an insect-resistant corn plant comprising: crossing a first parent corn plant with a second parent corn plant and harvesting the resulting hybrid seed, wherein the first parent corn plant and/or the second parent corn plant is a corn plant as defined in any one of claims 1-12.

61. The method of claim 60, wherein the insect is selected from the group consisting of lepidopteran insects.

62. The method of claim 60, wherein the insect is selected from one or more of the group consisting of armyworms, corn borers, cotton bollworms, carpopodoptera frugiperda, and spodoptera frugiperda.

63. The method of claim 60, comprising:

(1) Planting seeds of a first inbred maize line, the inbred maize line being a maize plant as defined in any of claims 1-12; and planting seeds of a second inbred line having a different genotype;

(3) Emasculating flowers of plants of one of the maize inbred lines;

(4) Sexual crossing the two different inbred lines with each other; and

(5) Harvesting the hybrid seed thus produced.

64. Use of a maize plant or part, seed, cell or progeny thereof for the production of a food, feed, cosmetic, pharmaceutical or industrial product, wherein the maize plant or part, seed, cell or progeny thereof is as defined in any one of claims 1 to 11.

65. Use of a corn seed or a corn plant produced from said corn seed or a part, seed, cell or progeny thereof for the production of a food, feed, cosmetic, pharmaceutical or industrial product, wherein said corn seed is deposited with the China general microbiological culture collection center (CGMCC) and has a accession number of CGMCC No.17848.