CN112280743A

CN112280743A - Corn event 2A-7 and methods for identifying same

Info

Publication number: CN112280743A
Application number: CN202011220206.7A
Authority: CN
Inventors: 赖锦盛; 赵海铭; 宋伟彬
Original assignee: China Agricultural University
Current assignee: Beijing Liangyuan Biotechnology Co ltd
Priority date: 2020-11-04
Filing date: 2020-11-04
Publication date: 2021-01-29
Anticipated expiration: 2040-11-04
Also published as: CN112280743B

Abstract

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

Description

Corn event 2A-7 and methods for identifying same

Technical Field

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

Background

Corn is the cereal crop with the widest planting range and the largest yield all over the world and is the first of the three grains (corn, wheat and rice). However, the current corn insect pests are serious, about 350 corn pests are worldwide, wherein the lepidoptera pests with stem eating and leaf eating, namely the corn borer, has the widest distribution and the most serious harm and is a worldwide important corn pest, the occurrence and the harm of the lepidoptera pest seriously affect the yield and the quality of the corn, and huge economic losses are caused to the corn production. Therefore, the problem of increasing the corn yield and increasing the income of farmers needs to be solved urgently by adopting effective measures to control the harm of the corn.

The main method of pest control has long been the spraying of chemical pesticides during growth, due to the lack of suitable pest-resistant varieties. However, chemical insecticides kill not only pests but also natural enemies of the pests, causing ecological balance damage 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 a corn variety through a transgenic technology, so that the insect resistance of the transgenic corn is improved, and meanwhile, each plant of the transgenic corn has a certain degree of resistance, so that the insect-resistant effect of the transgenic corn is remarkably and stably better than the control effect of pesticide spraying, the investment of manpower and material resources can be saved, and the social resources are effectively saved.

However, the integration of the foreign gene in the host plant genome is random. If the foreign gene happens to be inserted into a very important host gene, and is therefore mutated, the random insertion event can be lethal to the recipient plant. In addition, even if random insertion events do not impair the function of the host cell gene, the expression of the inserted foreign gene can be influenced by "position effects" caused by the surrounding genomic DNA. In some cases, the gene is inserted into sites where the 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 characterized by optimal expression of the introduced gene of interest. It is necessary to identify one or more unique sequences that are only associated 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 stably integrated in its genome a nucleic acid molecule encoding a Cry1Ab and Cry2Ab proteins that confer 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 the 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 the pressure of insects, the transgenic corn plant has obviously improved performance of resisting the invasion of insects such as corn borer, armyworm, cotton bollworm, dichocrocis punctiferalis and the like compared with non-transgenic corn plants.

Maize transformant 2A-7 and products thereof

Thus, 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 the Cry1Ab and Cry2Ab genes, wherein said exogenous nucleic acid molecule is flanked by a5 'flanking region, said 5' flanking region having the nucleotide sequence at positions 1-432 or position 300-432 of SEQ ID NO:5 and said exogenous nucleic acid molecule is flanked by a3 'flanking region, said 3' flanking region having the nucleotide sequence at positions 8532-9031 or 8532-8800 of SEQ ID NO: 8805.

The invention also provides a maize plant or a part, seed, cell or progeny thereof having incorporated into its genome an exogenous nucleic acid molecule comprising Cry1Ab and Cry2Ab genes, wherein the exogenous nucleic acid molecule is flanked by a5 'flanking region and a 3' flanking region, the sequence comprising the 5 'flanking region and portions of the exogenous nucleic acid molecule adjacent thereto is set forth in SEQ ID NO:1, and the sequence comprising the 3' flanking region and portions of the exogenous nucleic acid molecule adjacent thereto is set forth in SEQ ID NO: 2.

The maize plants of the invention, or parts, seeds, cells or progeny thereof, are resistant to insect infestation. 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 myxoplasma spp, ostrinia nubilalis (e.g., ostrinia furnacalis), cotton bollworm, dichocrocis punctiferalis, spodoptera frugiperda, and the like. In some embodiments, the corn plants of the invention, or parts, seeds, cells, or progeny thereof, also have resistance to herbicides (e.g., glyphosate, glufosinate, and the like).

In certain embodiments, the exogenous nucleic acid molecule is located in the genome at a position corresponding to between Chr3:179141694bp-179141724bp of the B73 reference genomic sequence (V4 version). In certain embodiments, integration of the exogenous nucleic acid molecule results in deletion of a portion of the original genome from which Chr3:179141695bp-179141723bp (B73 reference genome V4 version), for a total of 29bp, the deleted region being the maize endogenous gene Zm00001d042767, whose function is predicted to be Glucan endo-13-beta-glucopyranosase 14, and the insertion of the target fragment and deletion of Chr3:179141695bp-179141723bp sequences will inactivate the function of this gene. The gene is double-copy in corn, and the gene number of the other homologous gene is Zm00001d 012292.

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 set forth in SEQ ID NO. 12. In certain embodiments, the Cry2Ab gene is 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 in the course of integration of a foreign nucleic acid into the genome, unintended integrated sequences are produced, i.e.non-genomic sequences as well as the desired insert (e.g.T-border sequences), for example sequences which result from genome repair on integration of the insert. Thus, an exogenous nucleic acid molecule incorporated into the genome of a maize plant of the invention, or a part, seed, cell, or progeny thereof, can comprise the 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 the 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 the complement thereof.

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

In certain embodiments, 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 accession number of CGMCC No. 17848. In certain embodiments, the corn plant, or a portion, seed, cell, or progeny thereof is propagated and/or bred from a corn plant grown from the corn seed.

In a second aspect, the present 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 a maize seed according to 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 the maize seed of the second aspect.

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

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

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

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

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

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

In certain embodiments, the article is selected from the group consisting of corn ear, corn with bract removed, corn ear silk, corn pollen, corn grits, corn flour, crushed corn, corn flour, corn oil, corn starch, corn steep liquor, 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 corn fermentation (DDGS), animal feed from corn, cosmetics, and bulking agents.

In certain embodiments, the part of a maize plant according to any of the preceding aspects is selected from the group consisting of grain, pollen, ovule, flowers, shoots (shoots), roots, stems, silks, inflorescences (tassels), ears and leaves.

The invention also relates to the use of a maize plant of any of the preceding aspects or parts, seeds, cells or progeny thereof for the production of food, agricultural products (e.g. feed), cosmetics, pharmaceuticals or industrial products.

In certain embodiments, the maize plant or part, seed, cell or progeny thereof is used for the production of food, feed, starch or for brewing.

In certain embodiments, the part of the maize plant is selected from the group consisting of grain, pollen, ovule, flower, shoot, root, stem, silk, inflorescence, ear and leaf.

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, corn with bract removed, corn ear silk, corn pollen, corn grits, corn flour, crushed corn, corn flour, corn oil, corn starch, corn steep liquor, 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 corn fermentation (DDGS), animal feed from corn, cosmetics, and bulking agents.

Methods for identifying event 2A-7

Due to the random integration of the exogenous fragments in the plant genome in the transgenic events, the exogenous fragments of different transgenic events have different insertion sites in the genome. The flanking sequences are specific for a particular transgenic event. Thus, the use of flanking sequences of the insert allows for the specific detection of the transgenic event. For example, hybridization is performed using a probe containing a part of the flanking sequence and a part of the foreign insert sequence, or PCR amplification is performed by designing a specific primer containing a part of the flanking sequence and a part of the foreign insert sequence.

Thus, in a fourth aspect, the present invention provides a primer pair comprising a first primer and a second primer, wherein 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 from nucleotide 1-432 (e.g., nucleotide 200-432) of SEQ ID NO. 5 or a complementary sequence 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 compared to the nucleotide sequence, and the second primer comprises a sequence of at least 15 consecutive nucleotides (e.g., at least 18; e.g., 15-30 consecutive nucleotides) of the sequence from nucleotide 483-8524 (e.g., nucleotide 483-1000) of SEQ ID NO. 5 or a complementary sequence thereof Nucleotides, or 18-30 contiguous nucleotides) or has 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.

In certain embodiments, an amplicon having a length of 100-500bp is generated when the sequence depicted in SEQ ID NO. 5 is amplified using the first and second primers. In certain embodiments, the amplicon has a length of 200-400bp, such as 200-300bp, such as about 258 bp.

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 at nucleotide position 300-400 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, 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 at nucleotide position 500-600 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.

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 at nucleotide position 300-350 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 compared 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 at nucleotide position 520-600 (e.g., nucleotide position 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 compared 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 present invention provides a primer pair comprising a first primer and a second primer, wherein the first primer comprises a nucleotide sequence consisting of the 483-8524-position nucleotide (e.g., the 8000-8524-position nucleotide) of SEQ ID NO:5 or at least 15 consecutive nucleotides (e.g., at least 18; e.g., 15-30 consecutive nucleotides, or 18-30 consecutive nucleotides) of its complementary sequence 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 compared to the nucleotide sequence, the second primer comprises a sequence consisting of the 8532-9031-position nucleotide (e.g., the 8532-8800-position nucleotide) of SEQ ID NO:5 or at least 15 consecutive nucleotides (e.g., at least 18; e.g., 15-30 consecutive nucleotides of its complementary sequence, or 18-30 contiguous nucleotides) or has 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.

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

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-, 80% -100%, or 90% -100%; or at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity.

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, 8450-8550, or 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 compared 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 at nucleotide 8500-9000 (e.g., nucleotide 8500-8800, nucleotide 8700-8800, or nucleotide 8750-8800) or a complement thereof of SEQ ID NO. 5 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 compared 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 corn event 2A-7 in a sample comprising corn nucleic acids, 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, the exogenous nucleic acid molecule flanked by a5 'flanking region and a 3' flanking region, the sequence comprising the 5 'flanking region and portions of the exogenous nucleic acid molecule adjacent thereto being set forth in SEQ ID No.1, and the sequence comprising the 3' flanking region and portions of the exogenous nucleic acid molecule adjacent thereto being set forth 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 the complement thereof.

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

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

In certain embodiments, the primer pair comprises: a first primer comprising 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 a second primer comprising 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 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 the presence of a nucleic acid molecule unique to maize event 2A-7 in the sample is indicated when an amplicon having a length of about 305-315bp (e.g., about 310bp) is detected.

In certain embodiments, the primer pair comprises: a first primer comprising 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 a second primer comprising 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 seventh aspect, the invention provides a method of detecting a nucleic acid molecule unique to corn event 2A-7 in a sample comprising corn nucleic acids, comprising:

(1) contacting a primer pair with the sample; wherein, when the primer pair is used to amplify a nucleic acid of the genomic DNA of maize event 2A-7, an amplicon comprising a nucleotide sequence selected from the group consisting of: 1-5 or a complement 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 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 said sample is indicated.

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

(1) contacting said sample with a nucleic acid probe specific for a target sequence comprising a nucleotide sequence selected from the group consisting of: 1-5 or a complement thereof;

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

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

In certain embodiments, when hybridization is detected, the presence of a nucleic acid molecule unique to maize event 2A-7 in the sample 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%, such as at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity compared to said 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 any one thereof, 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 shown in

SEQ ID NO

1 or 2 or a complement thereof.

In certain embodiments, the nucleic acid probe carries a fluorescent label, such as a Taqman probe.

In this context, the term "primer" is an isolated nucleic acid that is annealed to a complementary (complementary) target DNA strand by nucleic acid hybridization to form a 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). 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 conventional 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 event 2A-7. Probes of the invention include not only deoxyribonucleic or ribonucleic acids, 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, 2 nd edition, Vol.1-3, edited by Sambrook et al, Cold Spring Harbor Laboratory Press, Cold Spring Ha r bor, NY, 1989 (hereinafter, "Sam brook et al, 1989"); current Protocols in Molecular Biology, edited by 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. PCR Primer pairs can be derived from known sequences, for example, by using a computer program intended for this purpose, such as Primer (version 0.5, 1991, Whitehead Institute for biological Research, Cambridge, MA).

The nucleic acid probes and primers of the invention hybridize to a target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA for a transgenic event in a sample. Herein, two nucleic acid molecules are said to be capable of specifically hybridizing to each other if they are capable of forming an antiparallel double-stranded nucleic acid structure. If the nucleic acid molecules show complete complementarity, one nucleic acid molecule is referred to as the "complement (complementary sequence)" of the other nucleic acid molecule. Herein, molecules are said to exhibit "perfect 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 can hybridize to each other with sufficient stability to allow them to remain annealed to each other under at least conventional "low stringency" conditions. Similarly, molecules are said to be "complementary" if they can hybridize to each other with sufficient stability to allow them to remain annealed to each other under conventional "high stringency" conditions. Conventional stringent conditions are described by Sambrook et al, 1989 and Haymes et al, in: nucleic acid hybridization, 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 molecules to form double-stranded structures. In order for a nucleic acid molecule to be useful 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 concentrations used.

Suitable stringency conditions for facilitating DNA hybridization, such as 6.0 XSSC at about 45 ℃ followed by a wash with 2.0 XSSC 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 can be selected from the low stringency 50 ℃ about 2.0 XSSC to the high stringency 50 ℃ about 0.2 XSSC. In addition, the temperature of the washing step can also be raised from low stringency conditions at room temperature of about 22 ℃ to high stringency conditions of about 65 ℃. Both temperature and salt concentration are variable, or either temperature or salt concentration may be held constant while the other variable is varied. In certain embodiments, the probes or primers of the invention specifically hybridize to the 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 are identical to SEQ ID NO:1 or SEQ ID NO:2 or a complement thereof or a fragment of any one thereof, 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. 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, which may include, but are not limited to, fluorescent labeling, radioactive labeling, antibody-based labeling, and chemiluminescent labeling.

With respect to amplification of a target nucleotide sequence (e.g., by PCR) using a particular pair of amplification primers, "stringent conditions" refer to conditions such that: in a DNA thermal amplification reaction, the primer pair is allowed to hybridize only to the target nucleic acid sequence to which the primer having the corresponding wild-type sequence (or its complement) should bind and preferably produces 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 the nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a corn plant results from a sexual cross containing transgenic event genomic DNA of a corn plant of the invention, a nucleic acid amplification method can be performed on DNA extracted from a corn plant tissue sample using a primer pair comprising a flanking sequence in the plant genome immediately adjacent to the insertion site into which the heterologous DNA was inserted 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 can be within the combined length of the primer pair plus one nucleotide base pair, preferably plus about 50 nucleotide base pairs, more preferably plus about 250 nucleotide base pairs, and even more preferably plus about 450 nucleotide base pairs. Alternatively, the primer pair may be derived from flanking sequences on both sides of the inserted DNA so as to produce an amplicon comprising the entire inserted nucleotide sequence. Members of a primer pair derived from a plant genomic sequence can be located a distance from the inserted DNA molecule that can 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 amplification methods are known in the art, and are described, inter alia, in U.S. Pat. nos. 4,683,195 and 4,683,202 and PCR Protocols: a Guide to Methods and Applications, edited by Innis et al, Academic Press, San Diego, 1990. PCR amplification methods have been developed to amplify genomic DNA of up to 22kb and phage DNA of up to 42kb (Cheng et al, Proc. Natl. Acad. Sci. USA 91: 5695-. These methods, as well as other methods known in the art of DNA amplification, can be used to practice the present invention. The heterologous DNA insert or flanking sequence from maize events 2A-7 can be tested as follows: these sequences from the events are amplified using primers derived from the sequences provided herein, 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 (Nikiforov et al, Nucleic Acid Res.22: 4167-4175, 1994) in which DNA oligonucleotides are designed which overlap both the adjacent flanking genomic DNA sequence and the inserted DNA sequence. Oligonucleotides are immobilized in wells of a microtiter plate. After PCR of the region of interest (using one primer at the insert and one at the adjacent flanking genomic sequence), the single stranded PCR product can be hybridized to an 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 sequence due to successful amplification, hybridization and single base extension.

Another method is pyrosequencing technology (pyrosequencing technology) described by Winge (Innov. Pharma. Tech.00: 18-24, 2000). In this method, oligonucleotides are designed that overlap the junction of adjacent genomic DNA and insert DNA. The oligonucleotides are hybridized to single-stranded PCR products from the region of interest (one primer located at the insert sequence and the other primer located at 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 separately and the optical signal generated by incorporation was measured. The light signal indicates the presence of the transgene insert/flanking sequence due to successful amplification, hybridization and single or multiple base extension.

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

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

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

In the methods of any of the above aspects, the sample can be any sample comprising corn event 2A-7DNA, provided that the sample comprises at least a detectable amount of a polynucleotide that is diagnostic for 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 corn ear, corn with bract removed, corn ear silk, corn pollen, corn grits, corn flour, crushed corn, corn flour, corn oil, corn starch, corn steep liquor, 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 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 that specifically hybridize under high stringency conditions to a target sequence set forth in any one of SEQ ID NOs: 1-5. In certain embodiments, the kit further comprises and additional materials necessary to enable nucleic acid hybridization or amplification methods to be performed.

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 corn event 2A-7, or for detecting the presence of corn event 2A-7, in a sample comprising corn nucleic acid.

The invention also relates to an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: 1-5 or the 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 maize plant is as defined in the first or second aspect;

(2) obtaining a first generation progeny plant from the cross of (1); and

(3) selecting from these first generation progeny plants a progeny plant that is resistant to insects, wherein the progeny plant is indicated to be an insect-resistant corn plant when it meets at least one of the following (3a) - (3 c):

(3a) the progeny plant comprises in its genome the sequence shown in any one of SEQ ID NOs:1-5 or a complement thereof;

(3b) when the genomic DNA of progeny plants is amplified with SEQ ID NO 6 and SEQ ID NO 7 as forward and reverse primers, respectively, an amplicon having a length of about 200-300 (e.g., about 258bp) bp is generated; and/or the presence of a gas in the gas,

(3c) when genomic DNA of progeny plants 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 310bp) is produced; or the like, or, alternatively,

(3d) hybridization can be detected when genomic DNA of progeny plants is detected using a nucleic acid probe specific for the sequence shown in any one of SEQ ID NOs:1-5 or a sequence complementary thereto (e.g., a nucleic acid probe comprising the sequence shown in SEQ ID NO:1 or 2 or a sequence complementary thereto).

In certain embodiments, the first or second parent maize plant comprises incorporated into its genome an exogenous nucleic acid molecule encoding mcry1Ab and mcry2Ab flanked by a5 'flanking region and a 3' flanking region, the sequence comprising the 5 'flanking region and portions of the exogenous nucleic acid molecule adjacent thereto is set forth in SEQ ID No.1, and the sequence comprising the 3' flanking region and portions of the exogenous nucleic acid molecule adjacent thereto is set forth 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 plants 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 when said plant satisfies at least one of said (3a) - (3c), it is indicated as an insect-resistant corn plant.

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

In certain embodiments, the first and/or second parent maize plant comprises incorporated into its genome an exogenous nucleic acid molecule encoding mcry1Ab and mcry2Ab, flanked by a5 'flanking region and a 3' flanking region, the sequence comprising the 5 'flanking region and portions of the exogenous nucleic acid molecule adjacent thereto is set forth in SEQ ID No.1, and the sequence comprising the 3' flanking region and portions of the exogenous nucleic acid molecule adjacent thereto is set forth in SEQ ID No. 2.

In certain embodiments, the first parent maize plant and/or the 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 comprises:

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

(2) growing corn plants produced from said planting until a period of flowering;

(3) emasculating a flower of a plant of one of the maize inbred lines;

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

(5) the hybrid seed thus produced is harvested.

In certain embodiments, the first inbred maize line comprises incorporated into its genome an exogenous nucleic acid molecule encoding mcry1Ab and mcry2Ab flanked by a5 'flanking region and a 3' flanking region, the sequence comprising the 5 'flanking region and portions of the exogenous nucleic acid molecule adjacent thereto is set forth in SEQ ID No.1, and the sequence comprising the 3' flanking region and portions of the exogenous nucleic acid molecule adjacent thereto is set forth 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 corn line provides the female parent. In certain embodiments, the first inbred corn line provides the male parent.

In the method of the ninth or tenth aspect, the insect may be selected from lepidopteran insects such as armyworm, ostrinia nubilalis (e.g., ostrinia nubilalis), cotton bollworm, dichocrocis punctiferalis, spodoptera frugiperda, and the like.

One of ordinary skill in the art will recognize that the transgenic genotype of 2A-7 can be introgressed by breeding into other maize lines containing different transgenic genotypes to achieve a characteristic packing in the resulting seed and progeny plants and give these plants an increased activity profile.

The transgenic genotypes of the invention can be introgressed into any maize inbred or hybrid using art-recognized breeding techniques. The goal of plant breeding is to combine different desired characteristics in 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 maturity, greater yield, and better agronomic quality. With the mechanical harvesting of many crops, uniformity of plant characteristics (e.g., germination and stand establishment, growth rate, maturity, and height of plants and ears) 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 flower 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-pollination and cross-pollination techniques. Maize has separate male and female flowers on the same plant, located on the tassel and ear, respectively. Natural pollination of maize occurs when wind blows pollen from the tassel to the filaments that protrude from the ear tip.

Reliable methods of controlling male fertility in plants provide opportunities for improved plant breeding. This is particularly 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 detasseling (or detasseling of the tassel), cytoplasmic male sterility, genetic male sterility, gametocides, and the like.

Hybrid corn seed is typically produced by a male sterility system that incorporates manual or mechanical detasseling.

Two maize inbreds were planted on land in staggered strips and pollen-bearing tassels were removed from one inbred (female). If sufficient isolation from the foreign corn pollen source is achieved, the ear of the detasseled inbred will only be fertilized from the other inbred (male) and the resulting seed is a hybrid and a hybrid plant will be formed.

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 enhanced restriction length polymorphisms, selection for enhanced genetic markers, and transformation.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the 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 bred with corn, including wild maize species.

As used herein, the term "Cry 2Ab gene" has a meaning well known to those skilled in the art, which is an endotoxin gene found in the entomopathogenic bacterium Bacillus thuringiensis (Bt for short), and can be found in various public databases (e.g., GenBank: M23724). The inventor optimizes and modifies the codon of Cry2Ab aiming at the sequence characteristics of monocotyledons to obtain a sequence (mCry2Ab) particularly suitable for expression in corn cells, wherein the sequence of mCry2Ab is shown as SEQ ID NO. 13 and can be referred to Chinese patent application CN 201410483143.2.

As used herein, the term "Cry 1Ab gene" has a meaning well known to those skilled in the art, which is an endotoxin gene found in the entomopathogenic bacterium Bacillus thuringiensis (Bt for short), and can be found in various public databases (e.g., GenBank: M13898). The inventor optimizes and modifies the codon of Cry1Ab aiming at the sequence characteristics of monocotyledons to obtain a sequence (mCry1Ab) particularly suitable for expression in corn cells, wherein the sequence of mCry1Ab is shown as SEQ ID NO:12 and can be referred to Chinese patent application CN 201710701976.5.

As used herein, the term "Bar gene" has a meaning well known to those skilled in the art and is an herbicide-resistant gene derived from S.hygroscopicus. Bar gene is often used as a selective marker gene, which can generate phosphorylation acetyltransferase, and can acetylate free amino of glufosinate herbicide to achieve the aim of detoxification, thereby facilitating the screening of positive callus in genetic transformation. The Bar gene sequence is known in the art, see for example Thompson CJ et al, EMBO j.1987 sep; 6(9):2519-23.. In certain exemplary embodiments, the Bar gene has the sequence shown in SEQ ID NO 14.

As used herein, the terms "corn event", "transgenic event" are used interchangeably to 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" comprises the original transformant of the heterologous DNA and/or progeny of the transformant. The term "event" also encompasses progeny produced by 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 cross. 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 to progeny that have received 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 results in multiple events, each event representing the insertion of a DNA construct into a different location in the genome of a plant cell. Based on the expression of the transgene or other desired characteristic, a particular event is selected. Thus, "events 2A-7", "2A-7", or "2A-7 events" 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. Specifically, "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 be present as an extrachromosomal molecule. Such extrachromosomal molecules are capable of autonomous replication. Transformed cells, tissues or plants are understood to comprise not only the end product of the transformation process, but also the progeny of the transgenes thereof. A "non-transformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism, i.e., a bacterium or a plant, which does not contain a heterologous nucleic acid molecule. As used herein, "transgenic" refers to a plant, plant cell, or a plurality of plant cells, structured or unstructured, that have integrated into the plant genome, and typically into the chromosome, mitochondria or other chromosome-containing organelle of the nucleus, at a site that is different from, or more than, the number of copies that normally occur in the native plant or plant cell, a nucleic acid sequence representing the gene of interest, by well-known gene manipulation and gene insertion techniques. Transgenic plants result from the manipulation and insertion of such nucleic acid sequences (as opposed to naturally occurring mutations) 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.

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

As used herein, the term "genotype" is genetic material inherited by a parent corn plant, and not all of the 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 the plant in maize event 2A-7 as well as the 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 of the 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 matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 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 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, Needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of E.Meyers and W.Miller (Compout.appl biosci., 4:11-17(1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a 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-.

Advantageous effects of the invention

The events 2A-7 provided by the invention can significantly resist the invasion of lepidoptera insects and have 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 no insect pressure. The events 2A-7 of the invention have wide application prospects, and realize positive social and ecological benefits.

Drawings

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

FIG. 2 shows the results of electrophoresis of the PCR products 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 (Zm00001d052263) plasmid (10 pg); lane M: molecular weight marker DL2000 plus.

FIG. 3 shows the results of electrophoresis of PCR products 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: a T + 2A-75 'plasmid (10pg) containing the 5' flanking sequence and the insert cloned in the previous stage; lane M: molecular weight marker DL2000 plus.

FIG. 4 shows the results of electrophoresis of PCR products 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: a T + 2A-73 'plasmid (10pg) containing a 3' flanking sequence and an insertion sequence; lane M: molecular weight marker DL2000 plus.

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

FIG. 6A shows the results of the identification of resistance of ears 2A-7 to corn borer in example 3. Wherein, 2A-7, Zheng 58 and Zheng 958 are arranged from top to bottom in sequence.

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

FIG. 7 shows the results of the identification of the resistance of 2A-7 ears to Helicoverpa armigera in example 3. Wherein, 2A-7, Zheng 58 and Zheng 958 are arranged from top to bottom in sequence.

FIG. 8 shows the results of indoor bioassay of Spodoptera frugiperda for leaves 2A-7 in example 3. Wherein the upper part is the control Zheng 58 leaves, and the lower part is the 2A-7 leaves.

FIG. 9 shows the results of indoor bioassay of Spodoptera frugiperda for 2A-7 filaments in example 3. Wherein the upper part is contrast Zheng 58 filaments, and the lower part is 2A-7 filaments.

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

Sequence information

Information on the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequences

Description of biological Material preservation

The invention relates to the following biological materials which are preserved in China general microbiological culture Collection center (CGMCC) (No. 3 Xilu-Beijing university facing Yang district Beichen No. 1):

the seed of corn (Zea mays)2A-7, which has the preservation number of CGMCC NO.17848 and the preservation 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.

Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. In addition, those whose specific conditions are not specified in the examples are conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration 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 events 2A-7

A selection marker gene bar (SEQ ID NO:14), a target gene mCry1Ab (SEQ ID NO:12) and mCry2Ab (SEQ ID NO:13) and matched regulatory elements thereof are inserted into T-DNA of pCAMBIA3301 (purchased from lake Nanfeng Hui biological science and technology Co., Ltd.), a plant expression vector is constructed according to a conventional molecular biology method and is named as pCAMBIA3301+ mCry1Ab + mCry2Ab, the matched regulatory elements of each inserted gene are shown in the following table, and a map is shown in FIG. 1, wherein a promoter Gly can be referred to Chinese patent application CN 201710702435.4. And (3) identifying positive clones by enzyme digestion and bidirectional sequencing for corn transformation.

Table 2: regulatory elements in transformation vectors

Regulatory element	Function(s)	Source
			Gly	Promoter of target gene mcry1Ab	Corn (corn)
nos	Terminator of target gene mcry1Ab	Agrobacterium tumefaciens
			CaMV35s	Promoter of target gene mcry2Ab	CauliflowerMosaic virus
adh1	Enhancing expression of mCry2Ab	Corn (corn)
			nos	Terminator of target gene mcry2Ab	Agrobacterium tumefaciens
CaMV35s	Promoter of the selectable marker Gene Bar	Cauliflower mosaic virus
			35S ployA	Terminator of selectable marker gene bar	Cauliflower mosaic virus

The vector is transformed into agrobacterium EHA105 (purchased from Beijing Huayue Biotechnology Co., Ltd.), and the agrobacterium containing target genes is used for infecting maize immature embryos, and the specific transgenic method is as follows:

the recipient used in the transgenic process was hybrid F1 generation of inbred lines HiIIA and HiIIB (publicly available from Maize genetic resource elite center (Maize GDB, 2010)). Firstly, planting inbred lines HiIIA and HiIIB in a field, and respectively bagging when the inbred lines loose powder; then, pollination is prepared, and two pollination modes are adopted: HiIIA is used as a female parent, and HiIIB is used as a male parent; HiIIA is used as a male parent, HiIIB is used as a female parent, immature embryos on pollinated cluster grains are taken 9-11 days after pollination, then agrobacterium infection is carried out indoors, the immature embryos invaded by agrobacterium are placed on a selective culture medium for multiple times of screening to obtain resistant calli, and the resistant calli are regenerated into seedlings to obtain transgenic T0 generation plants. After transgenic T0 generation is obtained, some female parents of seed production, such as Zheng 58, are hybridized with pollen of transgenic plants of T0 generation. The insertion sequence is introduced into the immature embryo of the receptor plant by adopting an agrobacterium infection method, and the transgenic plant is obtained after screening of herbicide dipropylamine phosphate. More than 2000T 0 generation transformants are obtained through multiple transformations, and the maize transformants 2A-7 with high expression, single copy and genetic stability of mcry1Ab and mcry2Ab are obtained through detecting the protein content of mcry1Ab and mcry2Ab, screening the copy number of the inserted fragment, detecting the stability of continuous multiple generations and identifying the agronomic characters at different places.

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

Table 3: primers used in TAIL-PCR

Note: n ═ a/T/C/G, B ═ G/T/C, V ═ a/G/C, D ═ a/G/C

The PCR product was ligated with B vector (Beijing Panzhijin Biotechnology Co., Ltd., CB101-01), and the ligation product was transformed into E.coli competent cells. Selecting single clone for PCR identification, and sending the amplified product to Beijing Okkanseng Biotechnology Limited for sequencing. And (3) comparing the sequencing result with the T-border sequence and the corn genome, and analyzing the integration condition of the exogenous fragments.

It was determined that integration of the exogenous DNA resulted in deletion of a portion of the sequence in the receptor genome, which was Chr3:179141695bp-179141723bp (B73 reference genome V4 version), for a total of 29bp, with the deletion region being the maize endogenous gene Zm00001d042767, whose function was predicted to be Glucan endo-13-beta-glucopyranosase 14, and that insertion of the target fragment and deletion of the sequence of Chr3:179141695bp-179141723bp would functionally inactivate this gene. The gene is double-copy in corn, and the gene number of the other homologous gene is Zm00001d 012292. The 2A-7 transformant was inserted between Chr3:179141694bp-179141724bp (B73 reference genome V4 version), the sequence of T-border integration into the genome is shown as 483-8524 of SEQ ID NO:5, and the composition of SEQ ID NO:5 is shown in the following table. In the 2A-7 genome, the exogenous sequence is flanked by a5 'flanking region having nucleotides 1-432 of SEQ ID NO. 5 and a 3' flanking region having 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 in 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 in 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: (1) non-expected integration sequences, i.e., non-genomic sequences and non-T-border sequences, which may be sequences resulting from genomic repair upon integration of the insert; (2) the positions not labeled in the table are the T-border region intergenic sequences.

2A-7 seed preparation

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 are preserved in China general microbiological culture Collection center (CGMCC), wherein the preservation number is CGMCC NO.17848, and the preservation date is 2019, 10 months and 28 days.

Example 2 identification of maize events 2A-7

1. Materials and methods

1.1 extraction method of corn genome DNA

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

(2) taking about 0.1g of fresh corn leaves, cutting into pieces, placing in a precooled mortar, quickly grinding into powder in liquid nitrogen and immediately transferring into a precooled 2mL EP tube (generally not exceeding 1/2 tube volume);

(3) quickly adding 0.8mL of CTAB buffer solution which is subjected to warm bath at 65 ℃ into an EP tube, gently shaking uniformly, and carrying out water bath at 65 ℃ for 30min, and gently shaking when not needed;

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

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

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

(7) the supernatant was aspirated into a new 1.5mL EP tube;

(8) adding equal volume of pre-cooled isopropanol (pre-cooled at 4 ℃);

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

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

(11) placing the mixture 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 for further use.

1.2 transformation event-specific PCR methods

The 5 'and 3' flanking sequences of events 2A-7 are shown at nucleotides 1-432 and 8532-9031, respectively, of SEQ ID NO 5. Forward and reverse primers (tables 5-6) were respectively involved in the 5 '-end and 3' -end insertion site sequences of maize transformants 2A-7, and PCR was carried out under the conditions and reaction systems shown in tables 7-8 below.

Table 5: 5' end primer information

Table 6: 3' end 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 samples (50ng, 25 ng/. mu.L)	2.5ng/μL	2
Total volume		20

Meanwhile, a maize endogenous gene zSSIIb (Zm00001d052263) is used as an internal reference gene, a forward primer zSSIIb-F is shown as SEQ ID NO:10, and a reverse primer zSSIIb-R is shown as SEQ ID NO: 11.

The PCR amplification products were detected by electrophoresis on ethidium bromide-stained agarose gel (3%). And adding a proper molecular mass standard during electrophoresis to determine the size of the amplified product, and using a gel imaging system to visualize the PCR amplified product.

2. Method verification data

About 1000ng of genomic DNA extracted from the following samples is used as a template for PCR amplification of a corn transformant 2A-7 specific system and an internal standard system at a5 'end and a 3' end so as 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 and MON87460, mixed to make 1 sample, the content is 1% each)

2) Industrialized transgenic soybean (MON87769, 356043, 305423, CV127, MON89788, A5547-127 and A2704-12, mixed to make 1 sample, content 1% each)

3) Industrialized transgenic rape (MS1, MS8, RF1, RF2, RF3, T45, Oxy235, Topas19/2 are mixed to prepare 1 sample, the content is 1 percent respectively)

4) Industrialized transgenic cotton (MON1445, MON531, MON15985, LLCOTTON25, MON88913, mixing to make 1 sample, each content is 1%)

5) Industrialized transgenic rice (KF-6, KMD-1, M12, KF-2, KF-8 were mixed to make 1 sample, content 1% each).

The results of the detection of the reference gene are shown in fig. 2, and show that all corn samples showed the expected amplification product and all non-corn samples showed no amplification product.

The results of the detection of amplification using the 5' end primer pair shown in Table 5 are shown in FIG. 3, and show that clear single bands consistent with the expected size of the fragment were observed for all samples containing transformants 2A-7, and no non-specific amplification was observed; for all other maize and non-maize samples, no expected amplification product was observed.

The results of the detection of amplification using the pair of 3' end primers shown in Table 6 are shown in FIG. 4, and show that clear single bands consistent with the expected size of the fragment were observed for all samples containing transformants 2A-7, and no non-specific amplification was observed; for all other maize and non-maize samples, no expected amplification product was observed.

Since the two primers of the specific PCR bind to specific regions of the T-DNA and receptor genomes, respectively, the PCR amplification process can only be completed when the two binding regions are adjacent. While integration of T-DNA during the transgene was random, the binding domains of the other transformants were nearly impossible to border except for the 2A-7 transformant, and even if they were, the products would not match the expected size. Therefore, the 5 'end primer pair and the 3' end primer can be used for detecting the corn transformant 2A-7.

In addition, 2A-7 maize events can also be identified by detecting the presence of the 5 'junction sequence or the 3' junction sequence using a DNA probe that specifically hybridizes thereto given the uniqueness of the 5 'junction sequence and the 3' junction sequence.

Example 3 identification of field insect resistance traits for corn events 2A-7

1. Basis of experiment

The basis of this test reference is: notice No. 953 of Ministry of agriculture-10.1-2007.

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 young embryo hybridized by HiIIA and HiIIB by using agrobacterium containing a target vector to obtain T0 generation transgenic plant, hybridizing and backcrossing by using Zheng as recurrent parent to obtain T3 generation, and then continuously selfing for 2 generation to obtain homozygous 2A-7 of Zheng 58 background;

(2) receptor control zheng 58; and

(3) zhengdan 958, a conventional corn variety used in local production.

The quality of the material reaches the requirement of GB4404.1 that the quality is not lower than that of second-grade corn seeds.

2.2 test insects

Mythimna separata: myxozoon population bred by indoor artificial feed or corn seedlings for initial larvae incubation of myxozoon (incubation time 12-24 h);

asiatic corn borer Ostrinia furnacalis: the corn borer primary larva (hatching time is 2-12h) is an indoor artificially-fed Asian corn borer population;

helicoverpa armigera: the cotton bollworm initial larva (incubation time is 12-24h) is a cotton bollworm population artificially fed indoors.

Spodoptera frugiperda: the Spodoptera frugiperda is initially hatched larvae (hatching time is 12-24h) and is an indoor artificially-bred Spodoptera frugiperda population.

3. Isolation measures

The isolation of the test field is carried out in a way of isolating the flowering phase within 300 meters for more than 25 days.

4. Experimental methods

And (3) insect-resistant artificial inoculation identification: according to the standard of No. 953 bulletin-10.1-2007 of the Ministry of agriculture.

The field experiment design adopts a random block design, the three times of the design are repeated, and the area of a cell is 30m²(5m multiplied by 6m), row spacing of 60cm, plant spacing of 25cm, soil fertility level and cultivation management are the same as field production, and no pesticide is sprayed in the whole growth period. The interval of 2m is arranged between different insect pest inoculation test districts, so that the insect pests are prevented from spreading among different districts. Before and after inoculation, the field should be kept at a certain humidity, and if drought occurs, watering should be carried out in time.

4.1 myxozoa

The method for inoculating the insects comprises the following steps: the resistance identification of armyworm is carried out in the heart leaf stage, the corn plant is developed to the 4-6 leaf stage, and at least 40 plants are artificially inoculated in each cell. And inoculating 30-40 heads of artificially-fed first-hatched larvae to the heart leaves of the corn, inoculating the larvae for 3 days, then inoculating the larvae for the second time, wherein the quantity and the method of the inoculated larvae are the same as those of the first time, and the inoculation is carried out in the evening.

Survey recording: after 14 days of inoculation, corn leaf pest damage and larva survival number of armyworm are investigated.

The results show that: according to the damage degree of the corn leaves to the armyworms, the average value of the damage level (leaf eating level) of the armyworms to the corn leaves in each cell is calculated, the judgment standard is shown in table 9, and then the resistance level of the transgenic insect-resistant corn to the armyworms is judged according to the specification of table 10.

Table 9: grading standard of degree of damage of corn leaves by armyworms

Table 10: evaluation standard of resistance of corn to armyworm

Average value of leaf eating grade in heart leaf stage	Type of resistance
		1.0～2.0	High resistance HR
2.1～4.0	anti-R
		4.1～6.0	anti-MR
6.1～8.0	Feeling S
		8.1～9.0	High-sensitivity HS

4.2 Asian corn borer

The method for inoculating the insects comprises the following steps: the resistance identification of the target pest Asiatic corn borer is carried out on a leaf stage generation and a ear stage generation, artificial inoculation is carried out respectively in a leaf stage (a small trumpet stage, a corn plant grows to 8-10 leaf stage) and a spinning stage, each artificial inoculation is carried out for 2 times in each stage, no less than 40 artificial inoculations are carried out in each cell in each stage, and each artificial inoculation is respectively inoculated with 60-80 larvae of the Asiatic corn borer which are hatched initially. The inoculation is carried out in the evening, and if the insects meet the weather above the middle rain after inoculation, the insects need to be inoculated for 1 time.

Heart and leaf stage survey record: and (4) investigating the condition that the middle and upper leaves are eaten by the corn borers one by one after inoculating the corn borers for 2-3 weeks at the heart leaf stage. Each identification material is selected from 15-20 plants/row at random, and leaf eating grades of the corn borers are recorded one by one according to descriptions in table 11 (the leaf eating grades are divided according to the diameter and the number of leaf worm holes formed after the corn borers take core leaves).

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

Calculating the average value of the damage degree (leaf eating grade) of the corn borers to the leaves of the identification material population. The calculation method is as follows:

average leaf eating grade ═ sigma (leaf eating grade x number of plants at that grade)/total number of plants investigated

Heart lobe stage results show: insect pest grade of each identified material was divided according to the average leaf eating grade, see table 12.

Table 12: evaluation standard of resistance of corn to corn borer

Insect pest grade	Average value of leaf eating grade in heart leaf stage	Resistance to
			1	1.0～2.9	High resistance HR
3	3.0～4.9	anti-R
			5	5.0～6.9	anti-MR
7	7.0～8.9	Feeling S
			9	9.0	High-sensitivity HS

Recording the spinning period survey: the damage condition of the corn ear, the number of wormholes, the tunnel length (cm) of the wormhole and the age and the survival number of the survival larva are investigated, and the damage degree of the ear and the damage condition of the plant are evaluated.

Laying period results show that: evaluation of insect resistance in ear stage of corn the average value of the level of damage of resistance of corn borers to ears in ear stage of each cell was calculated from the damage of ears, the number of wormholes, the tunnel length (cm) of wormholes, the age stage of living larvae and the number of living larvae, the judgment criteria are shown in table 13, and the level of resistance of corn borers in ear stage was judged according to the specifications of table 14.

Table 13: grading standard for degree of damage of Asiatic corn borers in corn ear period

Grade of damage to ears	Description of the 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 damaged50 percent; the larva survives 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 larvae survive, the age is less than or equal to 3 years
		5	The damage of spike tip is less than or equal to 2 cm; or the larvae survive and the age is less than or equal to 4 years old; the length of the tunnel is less than or equal to 2cm
6	The damage to the spike tip is less than or equal to 3 cm; or the larvae survive and the age is more than or equal to 4 years old; the length of the tunnel is less than or equal to 4cm
		7	The damage of spike tip is less than or equal to 4cm, the length of tunnel is less than or equal to 6cm
8	The damage of spike tip is less than or equal to 5cm, the length of tunnel is less than or equal to 8cm
		9	The damage of the spike tip is more than 5cm, the tunnel length is more than 8cm

Table 14: resistance evaluation standard of corn ears to Asiatic corn borers

Average grade of damaged leaves of female ear	Type of resistance
		1.0～2.0	High resistance HR
2.1～3.0	anti-R
		3.1～5.0	anti-MR
5.1～7.0	Feeling S
		≥7.1	High-sensitivity HS

4.3 Cotton bollworm

The method for inoculating the insects comprises the following steps: the resistance identification of the cotton bollworms is carried out in the stage of silking and powder scattering, and each plant is inoculated with 20-30 newly hatched larvae. At least 40 artificial insects are inoculated in each cell, the artificial insects are inoculated on the corn silks, after 3 days, the second inoculation is carried out, and the number of the inoculated insects is the same as that of the first inoculation. The inoculation is carried out in the evening, and if the insects meet the weather above the middle rain after inoculation, the insects need to be inoculated for 1 time.

Hazard level investigation: and (4) carrying out artificial inoculation on 14-21 days, and investigating the damage rate of the female ears one by one, wherein the number of living larvae of each female ear and the damage length of the female ears are determined.

The results show that: the insect resistance evaluation of the corn ear period is carried out by calculating the average damage level of cotton bollworms to the ears in the corn ear period of each cell according to the ear damage rate, the number of surviving larvae and the ear damage length (cm), the judgment standard is shown in table 15, and the resistance level of the corn ear period to the cotton bollworms is judged according to the specification of the table 16.

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

Female earLevel of damage	Description of the symptoms
		0	The ear of the female ear is not damaged
1	Only the filament is damaged
		2	Damage to ear tip of 1cm
3+	The damage level under the top of the spike is increased by 1cm every time the damage level under the top of the spike is increased by 1 level
		…N

Table 16: evaluation standard of resistance of corn ears to cotton bollworms

Average grade of damaged leaves of female ear	Type of resistance
		0～1.0	High resistance HR
1.1～3.0	anti-R
		3.1～5.0	anti-MR
5.1～7.0	Feeling S
		≥7.1	High-sensitivity HS

4.4 Spodoptera frugiperda

The method for inoculating the insects comprises the following steps: the resistance identification of the target pest Spodoptera frugiperda is divided into a heart leaf period generation and a ear period generation, artificial inoculation is carried out respectively in the heart leaf period (small trumpet mouth period, corn plant development to 8-10 leaf period) and the spinning period, each artificial inoculation is carried out for 2 times, no less than 40 artificial inoculation strains are inoculated in each cell in each period, and each artificial inoculation strain is inoculated with 20-30 initial larvae of Spodoptera frugiperda. The inoculation is carried out in the evening, and if the insects meet the weather above the middle rain after inoculation, the insects need to be inoculated for 1 time.

Survey recording: after 14 days of inoculation, the damage degree of the corn leaves by spodoptera frugiperda is investigated.

The results show that: according to the damage degree of the Spodoptera frugiperda to the corn leaves, calculating the average value of the damage degree (leaf eating degree) of the Spodoptera frugiperda to the corn leaves in each cell, wherein the judgment standard is shown in the following table, and then judging the resistance level of the transgenic insect-resistant corn to the Spodoptera frugiperda according to the regulations in the tables 17 and 18.

Table 17: grading standard for degree of damage of corn leaves by spodoptera frugiperda

Grade of eating leaves	Description of the symptoms
		1	The leaves are not damaged, so that the utility model has no harm to the leaves,or only the leaves are provided with needle-punched (less than or equal to 1mm) wormholes
2	Only a few bug holes with the spring hole size (less than or equal to 5mm) are arranged on the individual leaves
		3	A small number of leaf blades are provided with wormholes with spring hole sizes (less than or equal to 5mm)
4	The upper part of the individual leaf is carved (less than or equal to 10mm)
		5	A small number of blades are provided with notches (less than or equal to 10mm)
6	The partial blade is provided with a notch (less than or equal to 10mm)
		7	The part of each leaf is eaten, and a small number of leaves are provided with large scale (less than or equal to 10mm)
8	A small number of leaves are eaten, and a large number of notches (less than or equal to 10mm) are arranged on part of the leaves
		9	Most of the leaves are eaten

Table 18: evaluation standard of resistance of corn to spodoptera frugiperda

Laying period results show that: the evaluation of the insect resistance of the maize ear stage includes calculating the average value of the level of the insect resistance of spodoptera frugiperda to the maize ear in each cell ear stage according to the damage condition of the maize ear, the number of wormholes, the tunnel length (cm) of the wormholes, the age stage of the living larvae and the number of living larvae, the judgment standard is shown in table 19, and the resistance level of the maize ear stage to the spodoptera frugiperda is distinguished according to the specification of the table 20.

Table 19: grading standard of degree of damage caused by Spodoptera frugiperda in ear stage of corn

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

Table 20: evaluation standard of resistance of corn ears to spodoptera frugiperda

5. Analysis of results

5.1 analysis of armyworm resistance identification result

And investigating the result of the inoculation identification 2-3 weeks after the inoculation. The results are shown in fig. 5 and table 21, and analysis of parameters such as the leaf hazard rate, the wormhole or the size of the notch shows that the leaf hazard rate, the wormhole or the size of the notch of the transgenic corn 2A-7 is significantly lower than that of the corresponding non-transgenic corn variety and the local common cultivated corn variety within the 5% significant level range, and the significant difference level is achieved. Experimental results show that the transgenic corn 2A-7 has a good control effect on target pests, namely armyworms.

Table 21: investigation result of damage level of armyworm to leaves

5.2 analysis of the results of resistance identification of corn borer

5.2.1 leaf hazard level survey

Inoculating insects at heart and leaf stage (V6-V8), and investigating the grade of eating leaves 14 days after inoculating insects. Leaf hazard level survey results show that the transgenic 2A-7 corn has a good control effect on the target pest corn borer, and within a 5% obvious level range, the 2A-7 leaf hazard level is obviously lower than that of a corresponding non-transgenic corn variety and a local common cultivated corn variety and reaches a significant difference level (Table 22). The results show that the resistance of the transgenic corn 2A-7 Asiatic corn borers is superior to that of the corresponding non-transgenic corn variety and local common cultivated corn variety.

Table 22: investigation result of damage level of corn borers to leaves in heart leaf stage

5.2.2 ear hazard level investigation

Inoculating insects in the spinning stage, and investigating the result of insect inoculation identification before harvesting. As shown in fig. 6A-6B and tables 23-24, the analysis of five parameters, such as the damage rate of the ears, the number of holes eaten by the individual plants, the length of the tunnels, the number of live insects, the age of the living larvae, etc., shows that the damage rate of the ears, the number of holes eaten by the individual plants, the length of the tunnels, the number of live insects, and the age of the living larvae of the transgenic corn 2A-7 are all significantly lower than those of the corresponding non-transgenic corn varieties and local common cultivated corn varieties within the 5% significant level range, and achieve significant difference levels. Experimental results show that the transgenic corn 2A-7 has a good control effect on the target pest Asiatic corn borer.

Table 23: effect of resisting corn borer in spinning stage

Table 24: survey result of damage level of female ear by corn borer

From the above results, the average damage levels of the 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 corns on the corn borers are obviously different, and the resistance effects are expressed as high resistance levels.

5.2.3 ear Stem hazard level survey

The ear stalks are used as the only channel for nutrient transportation in corn filling, play an important role in grain development, and simultaneously play a supporting role in the ears in the harvesting period, thereby providing an important guarantee for mechanized harvesting. Therefore, parameters such as the stalk damage rate, the number of stalk survival larvae, the stalk damage length (cm) and the like are investigated, and analysis shows that the stalk damage rate, the number of stalk survival larvae and the stalk damage length (cm) of the transgenic corn 2A-7 are all significantly lower than those of corresponding non-transgenic corn varieties and local common cultivated corn varieties within the 5% significant level range, and achieve significant difference levels (Table 25). The experimental result shows that the transgenic corn 2A-7 has better control effect on the target pest corn borer.

Table 25: insect-resistant effect of stipe on corn borer

5.3 analysis of Cotton bollworm resistance identification result

Inoculating insects in the spinning stage, and investigating and identifying results 2-3 weeks after inoculating insects. As shown in fig. 7 and table 26, the analysis of parameters of the ear damage rate, the number of surviving larvae, and the ear damage length (cm) shows that the ear damage rate, the number of surviving larvae, and the ear damage length (cm) of the transgenic corn 2A-7 are significantly lower than those of the corresponding non-transgenic corn variety and the corresponding local general cultivated corn variety within the 5% significant level range, and achieve significant difference levels. Experimental results show that the transgenic corn 2A-7 has a good control effect on the target pest cotton bollworm.

Table 26: bollworm resisting effect in silk spitting period

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

5.4 Spodoptera frugiperda resistance identification results

5.4.1 leaf hazard class survey

Inoculating the insects at the heart-leaf stage (the small trumpet stage, the corn plant develops to the 8-leaf to 10-leaf stage), and investigating the result of the identification of the inoculated insects 2 to 3 weeks after inoculation. Results are shown in table 27, and analysis of parameters such as leaf hazard rate, wormhole or nick size indicates that, within the 5% significant level range, 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, and reaches significant difference level. Experimental results show that the transformant leaves have a good control effect on the target pest Spodoptera frugiperda, and a high resistance level is achieved.

Table 27: investigation result of damage level of Spodoptera frugiperda to leaves in cardiac leaf period

5.4.2 ear hazard level investigation

Inoculating insects in the spinning stage, and investigating the result of insect inoculation identification before harvesting. Results shown in tables 28-29 show that the damage rate of the ears, the number of holes eaten by the single plant, the length of the tunnel of the single plant, the number of live insects and the age of the living larvae are all significantly lower than that of the corresponding non-transgenic corn varieties and local common cultivated corn varieties within the 5% significant level range, and achieve significant difference levels through analysis of five parameters, namely the damage rate of the ears, the number of holes eaten by the single plant, the length of the tunnel of the single plant, the number of live insects and the age of the living larvae. Experimental results show that the transgenic corn 2A-7 has a good control effect on the target pest Spodoptera frugiperda.

Table 28: spodoptera frugiperda resistance effect in spinning period

Table 29: survey result of level of damage of female ears by spodoptera frugiperda

In addition, FIGS. 8-9 show the results of indoor bioassay of resistance of 2A-7 leaves or filaments to Spodoptera frugiperda, respectively. FIG. 10 shows the results of field bioassay of 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 Glucone event 2A-7 Glufosinate resistance

1. Test protocol

1.1 test materials

The test was performed up to Basta (18% glufosinate-ammonium solubles), produced by Bayer corporation.

2A-7: the transformant T5 produced the same material as in example 3.

1.2 design of the experiment

(1) Design of experiments

Randomized block design, 3-4 replicates. 1.0m wide isolation zone is arranged among cells, and the area of each cell is not less than 24m²The processing comprises the following steps: the transgenic corn is not sprayed with herbicide; spraying a target herbicide on the transgenic corn; the corresponding non-transgenic corn is not sprayed with herbicide; spraying target herbicide on corresponding non-transgenic corn.

(2) Glufosinate application dose

The application doses of the herbicides used were divided into: the pesticide registration label has medium dose (600g of effective component/hectare), medium dose 2 times (1200g of effective component/hectare), and medium dose 4 times (2400g of effective component/hectare). For water amount 450L/hectare.

1.3 administration period

Application was at the recommended time for glufosinate-resistant corn.

The glufosinate-ammonium is treated on stem leaves after seedlings, and general resistance identification is used in 3-5 leaf stages of corns.

1.4 spray Instrument requirements

(1) Selection of sprayers

Manual knapsack sprayer with constant pressure, wide spray width and stable flow rate or CO₂A compressed atomizer. The spraying is uniform.

(2) Spray head

A fan-shaped spray head is selected.

(3) Spraying method

Each treatment needs to be completed by one-time spraying. The dosage is calculated according to the actual area of the spraying cell. The test should be repeated within 12 hours of rainfall after dosing.

1.5 resistance identification survey

Corn seedling rate, plant height, phytotoxicity symptoms were investigated and recorded at 1 week, 2 weeks and 4 weeks after application, respectively. 15 maize plants were taken per cell.

After the corns are harvested, the middle 2 rows of corns in each cell are taken for measuring the yield.

1.6 analysis and presentation of results

The herbicide injury rate was calculated as follows.

In the formula:

x-damage in percent (%);

n-number of victim strains at the same level;

s-number of levels;

t is the total number of plants;

m-highest level.

The phytotoxicity symptoms are classified according to GB/T17980.42-2000.

Level 1: the corn grows normally and has no any harmful symptoms;

and 2, stage: slight phytotoxicity of corn is less than 10%;

and 3, level: the corn can be recovered later due to medium phytotoxicity, and the yield is not influenced;

4, level: the corn has serious phytotoxicity and is difficult to recover, thereby causing the reduction of yield;

and 5, stage: the corn has serious phytotoxicity and can not be recovered, thereby causing obvious yield reduction or no yield.

2. Results of the experiment

2.1 Damage Rate

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

Table 30: glufosinate treatment damage rate

From the above table, it can be seen that after spraying 2 times of the medium dosage glufosinate ammonium, the phytotoxicity is slight and less than 10%, and after spraying 4 times of the medium dosage glufosinate ammonium, the phytotoxicity in 2A-7 of the transgenic corn is moderate and more than 10%.

2.22A-7 plant height survey

The plant height of the transformants was investigated at 1 week, 2 weeks and four weeks of spraying, respectively, and the results are shown in the following table.

Table 31: effect of Glufosinate treatment on plant height

As can be seen from the table above, the transgenic corn 2A-7 has a small reduction in height after spraying glufosinate at different concentrations compared with the control plant which is not sprayed, but has no significant difference.

2.32A-7 yield survey

The yield and moisture content of the single ear grains at harvest were investigated and the results calculated per mu (14% moisture) are shown in the table below.

Table 32: effect of Glufosinate treatment on yield

As can be seen from the above table, the yield of the transgenic corn 2A-7 is reduced after spraying glufosinate with different concentrations compared with the control which is not sprayed, but has no significant difference.

Based on the above results, the phytotoxicity symptom classification results according to GB/T17980.42-2000 are shown in the following table. After the glufosinate ammonium of the medium dosage is sprayed on the transgenic corns 2A-7, the corns grow normally and have no harm symptom, the resistance grade is 1 grade, the slight phytotoxicity of the corns is less than 10 percent after the glufosinate ammonium of the medium dosage is sprayed by 2 times, the resistance grade is 2 grade, the moderate phytotoxicity of the corns is reduced after the glufosinate ammonium of the medium dosage is sprayed by 4 times, the plant height can be recovered, the yield is reduced compared with that of the corns subjected to micro-spraying, and the resistance grade is 4 grade. The results show that 2A-7 has excellent resistance to herbicides such as glufosinate-ammonium.

Table 33: glufosinate resistance grading

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be 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> university of agriculture in China

<120> maize event 2A-7 and methods for identifying same

<130> IDC190193

<160> 25

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> 5' joining sequence

<400> 1

ggcagtgggt tgcccacggt 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> 3' joining sequence

<400> 2

caatccagat ccacgcgcat 20

<210> 3

<211> 442

<212> DNA

<213> Artificial sequence

<220>

<223> 5 'joining 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 'joining 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 primers

<400> 15

acgatggact ccagagcggc cgcvnvnnng gaa 33

<210> 16

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> TAIL-PCR primers

<400> 16

acgatggact ccagagcggc cgcbnbnnng gtt 33

<210> 17

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> TAIL-PCR primers

<400> 17

acgatggact ccagagcggc cgcvvnvnnn ccaa 34

<210> 18

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> TAIL-PCR primers

<400> 18

acgatggact ccagagcggc cgcbdnbnnn cggt 34

<210> 19

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> TAIL-PCR primers

<400> 19

acgatggact ccagag 16

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> TAIL-PCR primers

<400> 20

ctgttgccgg tcttgcgatg attat 25

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> TAIL-PCR primers

<400> 21

ttctgttgaa ttacgttaag catgt 25

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> TAIL-PCR primers

<400> 22

ggtttttatg attagagtcc cgcaa 25

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> TAIL-PCR primers

<400> 23

ctgcccgtca ccgagatttg 20

<210> 24

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> TAIL-PCR primers

<400> 24

tcctataggg tttcgctcat gtgtt 25

<210> 25

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> TAIL-PCR primers

<400> 25

gtactaaaat ccagatcccc cgaat 25

Claims

1. A maize plant or a part, seed, cell or progeny thereof having an exogenous nucleic acid molecule incorporated into its genome, said exogenous nucleic acid molecule comprising Cry1Ab and Cry2Ab genes, wherein said exogenous nucleic acid molecule is flanked by a 5' flanking region having the amino acid sequence of SEQ ID NO:5 or positions 1-432 or positions 300-432, and the exogenous nucleic acid molecule is flanked by a 3' flanking region having the nucleotide sequence of SEQ ID NO:5 at position 8532-9031 or 8532-8800.

2. A maize plant or a part, seed, cell or progeny thereof having an exogenous nucleic acid molecule incorporated into its genome, said exogenous nucleic acid molecule comprising a Cry1Ab and a Cry2Ab gene, wherein said exogenous nucleic acid molecule is flanked by a 5' flanking region and a 3' flanking region, and the sequence of the portion comprising the 5' flanking region and the exogenous nucleic acid molecule adjacent thereto is as set forth in SEQ ID NO:1, the sequence of the portion comprising the 3' flanking region and the foreign nucleic acid molecule adjacent thereto is set forth in SEQ ID NO:2, respectively.

3. The maize plant, or part, seed, cell or progeny thereof of claim 1 or 2, wherein the location of said exogenous nucleic acid molecule in the genome corresponds to Chr3:179141694bp-179141724 bp.

4. The maize plant, or a part, seed, cell or progeny thereof of any one of claims 1 to 3, wherein the nucleotide sequences of the Cry1Ab and Cry2Ab genes are codon optimized for expression in a monocot (e.g., maize) cell;

preferably, the Cry1Ab gene is as set forth in SEQ ID NO:12 is shown in the specification;

preferably, the Cry2Ab gene is as set forth in SEQ ID NO: shown at 13.

5. The maize plant, or part, seed, cell, or progeny thereof of any one of claims 1-4, wherein said exogenous nucleic acid molecule further comprises a Bar gene;

preferably, 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;

preferably, the exogenous nucleic acid molecule comprises SEQ ID NO:5, 483-8524 nucleotide sequence or a complementary sequence thereof.

6. The maize plant, or part, seed, cell, or progeny thereof of any one of claims 1-5, wherein the genome of said maize plant, or part, seed, cell, or progeny thereof comprises the amino acid sequence of SEQ ID NOs:1-5 or a complement thereof.

7. The maize plant, or part, seed, cell or progeny thereof of any one of claims 1 to 6, wherein said plant, when expressed as SEQ ID NO:6 and SEQ ID NO:7 as forward and reverse primers, respectively, on amplification of the genomic DNA of said maize plant or part, seed, cell or progeny thereof, an amplicon having a length of about 200 and 300bp (e.g. about 258bp) is produced; and/or, when expressed as SEQ ID NO:8 and SEQ ID NO:9 as forward and reverse primers, respectively, to generate an amplicon having a length of about 250-350bp (e.g., about 310 bp).

8. The corn plant, or a part, seed, cell or progeny thereof, of any one of claims 1 to 7, wherein the corn seed from which the corn plant, or part, seed, cell or progeny thereof is produced is deposited at the China general microbiological culture Collection center (CGMCC) and has a accession number of CGMCC No. 17848.

9. The maize plant of any one of claims 1-8 or part, seed, cell, or progeny thereof that is resistant to insect infestation;

preferably, the insects are selected from lepidopteran insects;

preferably, the insects are selected from one or more of myxoplasma, ostrinia nubilalis (e.g. ostrinia furnacalis), cotton bollworm, dichocrocis punctiferalis, spodoptera frugiperda.

10. The corn seed is preserved in China general microbiological culture Collection center (CGMCC) and has the preservation number of CGMCC NO. 17848.

11. A maize plant or part, seed, cell or progeny thereof produced from the maize seed of claim 10;

preferably, the maize plant or part, seed, cell or progeny thereof is resistant to insect infestation;

preferably, the insects are selected from lepidopteran insects;

12. A preparation comprising the maize plant or part, seed, cell or progeny thereof of any one of claims 1-9, 11, or the maize seed of claim 9;

preferably, the preparation comprises the genomic DNA of the maize plant or part, seed, cell or progeny thereof, or maize seed.

13. The article of manufacture of claim 12, wherein the article of manufacture comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:1-5 or a complement thereof.

14. The article of manufacture of claim 12, wherein the polypeptide sequence as set forth in SEQ ID NO:6 and SEQ ID NO:7 as a forward primer and a reverse primer, respectively, to produce an amplicon having a length of about 200 and 300bp (e.g., about 258 bp); and/or, when expressed as SEQ ID NO:8 and SEQ ID NO:9 as forward and reverse primers, respectively, to amplify the nucleic acid contained in the preparation, an amplicon having a length of about 250-350bp (e.g., about 310bp) is generated.

15. The article of any one of claims 12-14, wherein the article is selected from the group consisting of corn ear, degummed corn, corn ear silk, corn pollen, corn grits, corn flour, crushed corn, corn flour, corn oil, corn starch, corn steep liquor, 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 corn fermentation (DDGS), animal feed from corn, cosmetics, and bulking agents.

16. The maize plant, or part, seed, cell, or progeny thereof of any one of claims 1-9, 11, or the article of manufacture of any one of claims 11-14, wherein said part of said maize plant is selected from the group consisting of grain, pollen, ovule, flower, shoot, root, stem, tassel, inflorescence, ear, and leaf.

17. A primer pair comprising a first primer and a second primer, wherein the first primer comprises a primer consisting of SEQ ID NO:5 (e.g., nucleotides 200-432) or a sequence of at least 15 consecutive nucleotides (e.g., 15-30 consecutive nucleotides) of its complement or a sequence having at least 80% identity to said nucleotide sequence, said second primer comprising a sequence consisting of SEQ ID NO:5 (e.g., 483-8524 th nucleotide) or a sequence consisting of at least 15 consecutive nucleotides (e.g., 15-30 consecutive nucleotides) of the complementary sequence thereof or a sequence having at least 80% identity to said nucleotide sequence.

18. The primer pair of claim 17, wherein when the first and second primers are used to amplify the sequence of SEQ ID NO:5, generating an amplicon with the length of 100-500 bp;

preferably, the amplicon has a length of 200-400bp, such as 200-300bp, such as about 258 bp.

19. The primer pair of claim 17 or 18, wherein the first primer comprises a primer consisting of SEQ ID NO:5 or a sequence consisting of at least 15 consecutive nucleotides (e.g., 15-30 consecutive nucleotides) of the sequence of nucleotides 300-400 or the complement thereof or a sequence having at least 80% identity to said nucleotide sequence, said second primer comprising a sequence consisting of SEQ ID NO:5 or a sequence consisting of at least 15 consecutive nucleotides (e.g., 15-30 consecutive nucleotides) of the sequence of nucleotides 500-600 or the complement thereof or a sequence having at least 80% identity compared to the nucleotide sequence;

preferably, the first primer comprises a primer consisting of SEQ ID NO:5 or a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 300-350 or the complement thereof or a sequence having at least 80% identity compared to said nucleotide sequence;

preferably, the second primer comprises a primer consisting of SEQ ID NO:5 or the complement thereof or a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 520-570 or a sequence having at least 80% identity with said nucleotide sequence;

preferably, the first primer comprises SEQ ID NO:6 or a sequence having at least 80% identity thereto, and the second primer comprises the sequence set forth in SEQ ID NO:7 or a sequence having at least 80% identity thereto.

20. A primer pair comprising a first primer and a second primer, wherein the first primer comprises a primer consisting of SEQ ID NO:5 (e.g., 8000-8524 nucleotides, e.g., 8300-8524 nucleotides) or a sequence consisting of at least 15 consecutive nucleotides (e.g., 15-30 consecutive nucleotides) of the sequence of nucleotides 483-8524 (e.g., 8000-8524 nucleotides) or the complement thereof or a sequence having at least 80% identity to the nucleotide sequence, said second primer comprising a sequence consisting of SEQ ID NO:5 (e.g., 8532-position 9031 nucleotide) or a sequence complementary thereto, or a sequence consisting of at least 15 consecutive nucleotides (e.g., 15-30 consecutive nucleotides) of the sequence or the sequence having at least 80% identity to the nucleotide sequence.

21. The primer pair of claim 20, wherein when the first and second primers are used to amplify the sequence of SEQ ID NO:5, generating an amplicon with the length of 100-500 bp;

preferably, the amplicon has a length of 200-400bp, such as 250-350bp, such as about 310 bp.

22. The primer pair of claim 20 or 21, wherein the first primer comprises a primer consisting of SEQ ID NO:5 or a sequence consisting of at least 15 consecutive nucleotides (e.g., 15 to 30 consecutive nucleotides) of the sequence of nucleotides 8400-8500 of the complement thereof or a sequence having at least 80% identity to said nucleotide sequence, said second primer comprising a sequence consisting of SEQ ID NO:5 or a sequence consisting of at least 15 consecutive nucleotides (e.g., 15 to 30 consecutive nucleotides) of the sequence of nucleotide 8700-8800 or the complement thereof or a sequence having at least 80% identity thereto;

preferably, the first primer comprises a primer consisting of SEQ ID NO:5 or a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 8450-8500 of the sequence or the complement thereof or a sequence having at least 80% identity with the nucleotide sequence;

preferably, the second primer comprises a primer consisting of SEQ ID NO:5 or the complement thereof or a nucleotide sequence consisting of at least 15 consecutive nucleotides of the sequence of nucleotides 8750-8800 or a sequence having at least 80% identity compared to said nucleotide sequence;

preferably, the first primer comprises SEQ ID NO:8 or a sequence having at least 80% identity thereto, and the second primer comprises the sequence set forth in SEQ ID NO:9 or a sequence having at least 80% identity thereto.

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

(1) contacting the sample with a primer pair of any one of claims 17-22;

(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 the maize plant or part, seed, cell, or progeny thereof of any one of claims 1-9;

preferably, the maize event 2A-7 comprises an exogenous nucleic acid molecule encoding mcry1Ab and mcry2Ab incorporated into its genome, the exogenous nucleic acid molecule 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 being as set forth in SEQ ID NO:1, the sequence of the portion comprising the 3' flanking region and the foreign nucleic acid molecule adjacent thereto is set forth in SEQ ID NO:2 is shown in the specification;

preferably, said maize event 2A-7 comprises in its genome the amino acid sequence of SEQ ID NOs:1-5 or a complement thereof;

preferably, when the expected amplicon is detected in step (3), it is indicative that the sample comprises a nucleic acid molecule unique to maize event 2A-7.

24. The method of claim 23, comprising:

(1) contacting the sample with a primer pair of any one of claims 17-19;

(2) performing a nucleic acid amplification reaction; and

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

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

preferably, the primer pair comprises: comprises the amino acid sequence shown in SEQ ID NO:6 or a sequence having at least 80% identity thereto and a first primer comprising the sequence set forth in SEQ ID NO:7 or a sequence having at least 80% identity thereto.

25. The method of claim 23, comprising:

(1) contacting the sample with a primer pair of any one of claims 20-22;

(2) performing a nucleic acid amplification reaction; and

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

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

preferably, the primer pair comprises: comprises the amino acid sequence shown in SEQ ID NO:8 or a sequence having at least 80% identity thereto and a first primer comprising the sequence set forth in SEQ ID NO:9 or a sequence having at least 80% identity thereto.

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

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

(3) detecting the amplicon;

preferably, said maize event 2A-7 comprises in its genome the amino acid sequence of SEQ ID NOs:1-5 or a complement thereof.

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

(1) contacting said sample with a nucleic acid probe specific for a target sequence comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOs:1-5 or a complement thereof;

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

preferably, when hybridization is detected, it indicates the presence of a nucleic acid molecule unique to maize event 2A-7 in the sample;

preferably, the nucleic acid probe comprises: a sequence comprising the 5' flanking region and a portion of the exogenous nucleic acid molecule adjacent thereto, or a sequence having at least 80% sequence identity compared to said sequence;

preferably, the nucleic acid probe hybridizes to SEQ ID NO:1 or SEQ ID NO:2 or the complement thereof or a fragment of either has at least 80% sequence identity;

preferably, the nucleic acid probe comprises SEQ ID NO:1 or 2 or a complement thereof;

preferably, the nucleic acid probe carries a fluorescent label.

28. The method of any one of claims 23-27, wherein the sample is selected from the group consisting of corn ear, degummed corn, corn ear silk, corn pollen, corn grits, corn flour, crushed corn, corn flour, corn oil, corn starch, corn steep liquor, 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 corn fermentation (DDGS), animal feed from corn, cosmetics, and bulking agents.

29. A kit, comprising: (i) the primer pair of any one of claims 17-22, and/or (ii) a nucleic acid probe specific for a nucleic acid sequence as set forth in any one of SEQ ID NOs:1-5 or a complement thereof;

preferably, the nucleic acid probe carries a fluorescent label.

30. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOs:1-5 or a complement thereof.

31. 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 maize plant is as defined in any one of claims 1-9, 11;

(2) obtaining a first generation progeny plant from the cross of (1); and

(3a) the progeny plant contains in its genome SEQ ID NOs:1-5 or a complement thereof;

(3b) when expressed as SEQ ID NO:6 and SEQ ID NO:7 as a forward primer and a reverse primer, respectively, to generate an amplicon having a length of about 200-300bp (e.g., about 258 bp); and/or the presence of a gas in the gas,

(3c) when expressed as SEQ ID NO:8 and SEQ ID NO:9 as forward primer and reverse primer, respectively, to generate an amplicon having a length of about 250-350bp (e.g., about 310 bp); or the like, or, alternatively,

(3d) when using the sequences specific for SEQ ID NOs:1-5 or a sequence complementary thereto (e.g., a nucleic acid probe comprising the sequence shown in SEQ ID No.1 or 2 or a sequence complementary thereto) is capable of detecting hybridization when detecting genomic DNA of a progeny plant;

preferably, the first or second parent maize plant comprises incorporated into its genome an exogenous nucleic acid molecule encoding mcry1Ab and mcry2Ab, said exogenous nucleic acid molecule being flanked by 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 being as set forth in SEQ ID NO:1, the sequence of the portion comprising the 3' flanking region and the foreign nucleic acid molecule adjacent thereto is set forth in SEQ ID NO:2 is shown in the specification;

preferably, the first or second parent maize plant comprises in its genome the amino acid sequence of SEQ ID NOs:1-5 or a complement thereof;

preferably, the insects are selected from lepidopteran insects, such as one or more of myxozoa, ostrinia nubilalis (e.g., ostrinia furnacalis), cotton bollworms, dichocrocis punctiferalis, spodoptera frugiperda, and the like.

32. The method of claim 31, wherein the method further comprises the steps of:

33. A method of producing hybrid corn seed that can be grown as an insect resistant corn plant comprising: crossing a first parent maize plant with a second parent maize plant and harvesting the resulting hybrid seed, wherein the first parent maize plant and/or the second parent maize plant are as defined in any one of claims 1-9, 11;

preferably, the first and/or second parent maize plant comprises incorporated into its genome an exogenous nucleic acid molecule encoding mcry1Ab and mcry2Ab flanked by 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 being as set forth in SEQ ID NO:1, the sequence of the portion comprising the 3' flanking region and the foreign nucleic acid molecule adjacent thereto is set forth in SEQ ID NO:2 is shown in the specification;

preferably, the first and/or second parent maize plant comprises in its genome the amino acid sequence of SEQ ID NOs:1-5 or a complement thereof;

34. The method of claim 33, comprising:

(1) planting seed of a first inbred corn line, which is a corn plant as defined in any one of claims 1-9, 11; and planting seeds of a second inbred line having a different genotype;

(3) emasculating a flower of a plant of one of the maize inbred lines;

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

(5) harvesting the hybrid seed produced thereby;

preferably, the first inbred maize line comprises incorporated into its genome an exogenous nucleic acid molecule encoding mcry1Ab and mcry2Ab, 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 being as set forth in SEQ ID NO:1, the sequence of the portion comprising the 3' flanking region and the foreign nucleic acid molecule adjacent thereto is set forth in SEQ ID NO:2 is shown in the specification;

preferably, the first inbred maize line comprises in its genome the amino acid sequence of SEQ ID NOs:1-5 or a complement thereof.

35. Use of the corn plant or part, seed, cell or progeny thereof of any one of claims 1 to 9, 11, or the corn seed of claim 10 for the production of food, feed, cosmetic, pharmaceutical or industrial products.