CN109868273B - Nucleic acid sequence and detection method for detecting corn plant DBN9501 - Google Patents

Abstract

The present invention relates to a nucleic acid sequence for detecting corn plant DBN9501 and a detection method thereof, wherein the nucleic acid sequence includes SEQ ID NO: 1 or its complementary sequence, or SEQ ID NO: 2 or its complementary sequence. The corn plant DBN9501 of the present invention has better resistance to Lepidoptera insects and better tolerance to glufosinate-ammonium herbicide, has no effect on yield, and the detection method can accurately and quickly identify whether a biological sample contains a transgene DNA molecule of maize event DBN9501.

Description

Nucleic acid sequence and detection method for detecting corn plant DBN9501

technical field

The invention relates to the field of plant molecular biology, especially the field of transgenic crop breeding in agricultural biotechnology research. Specifically, the present invention relates to insect-resistant and glufosinate-ammonium herbicide-tolerant transgenic corn event DBN9501 and a nucleic acid sequence for detecting whether a specific transgenic corn event DBN9501 is contained in a biological sample and a detection method thereof.

Background technique

Maize (Zea mays L.) is a major food crop in many parts of the world. Biotechnology has been applied to corn to improve its agronomic traits and quality. Insect resistance is an important agronomic trait in maize production, especially resistance to Lepidoptera insects, such as corn borer, cotton bollworm, and cutworm. The resistance of maize to Lepidoptera insects can be obtained by expressing the resistance genes of Lepidoptera insects in maize plants through transgenic methods. Another important agronomic trait is herbicide tolerance. For example, there have been successful maize transformation events NK603, GA21, etc., and maize has been widely planted in major planting areas such as the United States. It is worth mentioning that the mechanism of action of glufosinate-ammonium herbicide is different from that of glyphosate herbicide. It is a contact-killing herbicide and can be used as an effective means of managing glyphosate-resistant weeds. The tolerance of corn to glufosinate-ammonium herbicides can be obtained by expressing glufosinate-ammonium herbicide-tolerant genes (such as pat) in corn plants through transgenic methods.

The expression of exogenous genes in plants is known to be influenced by their chromosomal location, possibly due to the proximity of chromatin structure (e.g. heterochromatin) or transcriptional regulatory elements (e.g. enhancers) to the integration site. For this reason, it is usually necessary to screen a large number of events before it is possible to identify commercially viable events (ie, events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the amount of expression of an introduced gene can vary considerably between events; there may also be differences in the spatial or temporal pattern of expression, such as the relative expression of the transgene between different plant tissues There are differences in which the actual expression pattern may not correspond to that expected based on the transcriptional regulatory elements in the introduced gene construct. Therefore, it is often necessary to generate hundreds to thousands of different events and select from these a single event that has the expected amount and pattern of transgene expression for commercialization purposes. Events with the expected amount and pattern of transgene expression can be used to introgress the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. The progeny produced by this crossing method maintained the transgene expression characteristics of the original transformant. Applying this strategic model can ensure reliable gene expression in many varieties that are well adapted to local growing conditions.

It would be beneficial to be able to detect the presence of specific events to determine whether the progeny of a sexual cross contain the gene of interest. In addition, methods to detect specific events will help to comply with regulations, such as the need for formal approval and labeling of food derived from recombinant crops before being placed on the market. Detection of the presence of the transgene is possible by any of the well-known polynucleotide detection methods, such as polymerase chain reaction (PCR) or DNA hybridization using polynucleotide probes. These assays typically focus on commonly used genetic elements such as promoters, terminators, marker genes, etc. Therefore, unless the sequence of the chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, this approach cannot be used to distinguish between different events, especially those produced with the same DNA construct. event. Therefore, transgene-specific events are now commonly identified by PCR using a pair of primers spanning the junction of the inserted transgene and flanking DNA, specifically a first primer contained within the insert and a second primer contained within the insert.

Contents of the invention

The object of the present invention is to provide a nucleic acid sequence and detection method for detecting corn plant DBN9501, the transgenic corn event DBN9501 has better resistance to insects and better tolerance to glufosinate-ammonium herbicide, and The detection method can accurately and quickly identify whether the DNA molecule of the transgenic corn event DBN9501 is contained in the biological sample.

To achieve the above object, the present invention provides a nucleic acid sequence having at least 11 consecutive nucleotides in positions 1-384 of SEQ ID NO: 3 or its complementary sequence, and SEQ ID NO: 3 or its complementary sequence at position 1-384. At least 11 consecutive nucleotides in positions 385-768; and/or at least 11 consecutive nucleotides in positions 1-564 of SEQ ID NO: 4 or its complement, and SEQ ID NO: 4 or its complement At least 11 consecutive nucleotides in the 565th-1339th position of the sequence.

Preferably, the nucleic acid sequence has 22-25 consecutive nucleotides in SEQ ID NO: 3 or its complementary sequence 1-384, and 22 consecutive nucleotides in SEQ ID NO: 3 or its complementary sequence 385-768. -25 consecutive nucleotides; and/or 22-25 consecutive nucleotides in SEQ ID NO:4 or its complementary sequence 1-564, and SEQ ID NO:4 or its complementary sequence 565-1339 22-25 consecutive nucleotides in position.

Preferably, the nucleic acid sequence comprises SEQ ID NO: 1 or its complement, and/or SEQ ID NO: 2 or its complement.

Said SEQ ID NO: 1 or its complementary sequence is a sequence of 22 nucleotides in length near the insertion junction at the 5' end of the inserted sequence in the transgenic maize event DBN9501, said SEQ ID NO: 1 or its complementary sequence The complementary sequence spans the flanking genomic DNA sequence of the maize insertion site and the DNA sequence at the 5' end of the insertion sequence, and the presence of the transgenic maize event DBN9501 can be identified by including said SEQ ID NO: 1 or its complementary sequence. Said SEQ ID NO: 2 or its complementary sequence is a sequence of 22 nucleotides in length near the insertion junction at the 3' end of the inserted sequence in the transgenic maize event DBN9501, said SEQ ID NO: 2 or its complementary sequence Complementary sequences spanning the DNA sequence at the 3' end of the insertion sequence and the flanking genomic DNA sequence of the maize insertion site, including said SEQ ID NO: 2 or its complement can be identified as the presence of transgenic maize event DBN9501.

Preferably, the nucleic acid sequence comprises SEQ ID NO: 3 or its complement, and/or SEQ ID NO: 4 or its complement.

In the present invention, the nucleic acid sequence may be at least 11 or more continuous polynucleotides (first nucleic acid sequence) of any part of the T-DNA insertion sequence in the SEQ ID NO: 3 or its complementary sequence, or It is at least 11 or more contiguous polynucleotides (second nucleic acid sequence) of any part of the 5' flanking maize genomic DNA region in said SEQ ID NO: 3 or its complementary sequence. Said nucleic acid sequence may further be homologous or complementary to a part of said SEQ ID NO:3 comprising the entirety of said SEQ ID NO:1. When a first nucleic acid sequence and a second nucleic acid sequence are used together, these nucleic acid sequences can be used as a DNA primer pair in a DNA amplification method to generate an amplification product. The presence of transgenic maize event DBN9501 or progeny thereof can be diagnosed when the amplification product generated in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO: 1. The SEQ ID NO: 3 or its complementary sequence is a sequence of 768 nucleotides in length near the insertion junction at the 5' end of the T-DNA insertion sequence in the transgenic maize event DBN9501, the SEQ ID NO: 3 or its complementary sequence consists of 384 nucleotides of the maize genome 5' flanking sequence (the 1st-384th nucleotide sequence of SEQ ID NO:3), the DBN10707 construct DNA sequence of 168 nucleotides (SEQ ID NO 385-552 nucleotides of 3) and the DNA sequence of the tNos (nopaline synthase) transcription terminator of 216 nucleotides (nucleotides 553-768 of SEQ ID NO: 3), The presence of the transgenic maize event DBN9501 can be identified by the inclusion of said SEQ ID NO: 3 or its complementary sequence.

The nucleic acid sequence can be at least 11 or more continuous polynucleotides (the third nucleic acid sequence) of any part of the T-DNA insertion sequence in the SEQ ID NO: 4 or its complementary sequence, or the SEQ ID NO: 4 or its complementary sequence. At least 11 or more contiguous polynucleotides (fourth nucleic acid sequence) of any part of the 3' flanking maize genomic DNA region in ID NO: 4 or its complement. The nucleic acid sequence may further be homologous or complementary to a portion of said SEQ ID NO:4 comprising the entirety of said SEQ ID NO:2. When the third nucleic acid sequence and the fourth nucleic acid sequence are used together, these nucleic acid sequences can be used as a DNA primer pair in a DNA amplification method to generate an amplification product. The presence of transgenic maize event DBN9501 or progeny thereof can be diagnosed when the amplification product generated in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO:2. The SEQ ID NO: 4 or its complementary sequence is a sequence of 1339 nucleotides in length near the T-DNA insertion junction site at the 3' end of the insertion sequence in the transgenic maize event DBN9501, the SEQ ID NO: 4 or its complementary sequence consists of 271 nucleotides of the pr35S transcription initiation sequence (the 1st-271st nucleotide of SEQ ID NO: 4), the DBN10707 construct DNA sequence of 293 nucleotides (SEQ ID NO: 4 nucleotides 272-564) and 775 nucleotides of the corn genome 3' flanking sequence (SEQ ID NO: 4 nucleotides 565-1339), comprising said SEQ ID NO: 4 or Its complementary sequence can be identified as the existence of transgenic maize event DBN9501.

Further, the nucleic acid sequence comprises SEQ ID NO: 5 or its complementary sequence.

The SEQ ID NO: 5 or its complementary sequence is a sequence of 8559 nucleotides in length characterizing the transgenic maize event DBN9501, and its specific genome and genetic elements are shown in Table 1. The presence of transgenic maize event DBN9501 can be identified by the inclusion of said SEQ ID NO: 5 or its complementary sequence.

Table 1, the genome and genetic elements contained in SEQ ID NO:5

As is well known to those skilled in the art, the first, second, third and fourth nucleic acid sequences need not consist solely of DNA, but may also include RNA, a mixture of DNA and RNA, or DNA, RNA or other sequences that do not serve as one or more Combinations of nucleotides or analogs thereof for the polymerase template. In addition, the probes or primers of the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which can be selected from Nucleotides described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. When selected from the nucleotides set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, the probes and primers can be at least about 21 to about 50 or more contiguous nuclei in length glycosides.

The nucleic acid sequence or its complement can be used in a DNA amplification method to generate amplicons for detecting the presence of transgenic maize event DBN9501 or its progeny in a biological sample; the nucleic acid sequence or its complement Can be used in nucleotide detection methods to detect the presence of transgenic maize event DBN9501 or its progeny in biological samples.

To achieve the above object, the present invention also provides a method for detecting the presence of DNA of transgenic corn event DBN9501 in a sample, comprising:

contacting the sample to be detected with at least two primers for amplifying the target amplification product in a nucleic acid amplification reaction;

performing a nucleic acid amplification reaction; and

detecting the presence of said target amplification product;

The target amplification product comprises the nucleic acid sequence.

Preferably, the target amplification product comprises SEQ ID NO:1 or its complementary sequence, SEQ ID NO:2 or its complementary sequence, SEQ ID NO:6 or its complementary sequence, and/or SEQ ID NO:7 or its complementary sequence complementary sequence.

Specifically, the primers include a first primer and a second primer, the first primer is selected from SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:10; the second primer is selected from SEQ ID NO: 2. SEQ ID NO:9 and SEQ ID NO:11.

To achieve the above object, the present invention also provides a method for detecting the presence of DNA of transgenic corn event DBN9501 in a sample, comprising:

contacting the sample to be detected with a probe comprising the nucleic acid sequence;

Hybridizing the sample to be detected and the probe under stringent hybridization conditions; and

Detecting the hybridization between the sample to be detected and the probe.

The stringent conditions can be in 6×SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution, hybridization at 65° C., and then use 2×SSC, 0.1% SDS and 1×SSC, Wash each membrane once with 0.1% SDS.

Preferably, the probe comprises SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, and/or SEQ ID NO: 7 or its complement .

Optionally, at least one of said probes is labeled with at least one fluorophore.

To achieve the above object, the present invention also provides a method for detecting the presence of DNA of transgenic corn event DBN9501 in a sample, comprising:

contacting the sample to be detected with a marker nucleic acid molecule comprising said nucleic acid sequence;

Hybridizing the sample to be detected and the marker nucleic acid molecule under stringent hybridization conditions;

Detect the hybridization between the sample to be detected and the marker nucleic acid molecule, and then analyze through marker-assisted breeding to determine that insect resistance and/or herbicide tolerance are genetically linked with the marker nucleic acid molecule.

Preferably, the marker nucleic acid molecule comprises at least one selected from the group consisting of SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, and/or SEQ ID NO: 6-11 or its complement complementary sequence.

In order to achieve the above object, the present invention also provides a DNA detection kit, comprising at least one DNA molecule comprising the nucleic acid sequence, which can be used as a DNA primer specific for the transgenic corn event DBN9501 or its progeny One or probe.

Preferably, the DNA molecule comprises SEQ ID NO: 1 or its complement, SEQ ID NO: 2 or its complement, SEQ ID NO: 6 or its complement, and/or SEQ ID NO: 7 or its complement .

To achieve the above object, the present invention also provides a plant cell, comprising a nucleic acid sequence encoding an insect-resistant Vip3Aa protein, a nucleic acid sequence encoding a glufosinate-ammonium herbicide-tolerant PAT protein, and a nucleic acid sequence in a specific region, the specific The nucleic acid sequence of the region includes the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 and/or SEQ ID NO:7.

Preferably, the plant cell comprises a nucleic acid sequence encoding an insect-resistant Vip3Aa protein, a nucleic acid sequence encoding a glufosinate-ammonium herbicide-tolerant PAT protein, and a nucleic acid sequence of a specific region, and the nucleic acid sequence of the specific region includes SEQ ID NO :3 and/or the sequence shown in SEQ ID NO:4.

Preferably, the plant cell comprises SEQ ID NO:1, the 553-7491 nucleic acid sequence of SEQ ID NO:5 and SEQ ID NO:2 in sequence, or comprises the sequence shown in SEQ ID NO:5.

To achieve the above object, the present invention also provides a method for protecting corn plants from insect attack, comprising providing at least one transgenic corn plant cell in the diet of the target insect, said transgenic corn plant cell comprising in its genome SEQ ID NO:1 and/or the sequence shown in SEQ ID NO:2, target insects that feed on the transgenic corn plant cells are inhibited from further feeding on the transgenic corn plant.

Preferably, the transgenic maize plant cell comprises the sequence shown in SEQ ID NO:3 and/or SEQ ID NO:4 in its genome.

Preferably, the transgenic maize plant cell sequentially comprises SEQ ID NO:1, the 553-7491 nucleotide sequence of SEQ ID NO:5 and SEQ ID NO:2 in its genome, or comprises SEQ ID NO:5.

To achieve the above object, the present invention also provides a method for protecting corn plants from damage caused by herbicides or controlling the weeds in the field of planting corn plants, comprising applying an effective dose of glufosinate-ammonium herbicide to the planting at least In the field of a kind of transgenic corn plant, described transgenic corn plant comprises the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2 in its genome, described transgenic corn plant has tolerance to glufosinate-ammonium herbicide sex.

Preferably, the transgenic maize plant comprises the sequence shown in SEQ ID NO:3 and/or SEQ ID NO:4 in its genome.

Preferably, the transgenic maize plant sequentially comprises SEQ ID NO:1, SEQ ID NO:5 553-7491 nucleic acid sequence and SEQ ID NO:2 in its genome, or comprises the sequence shown in SEQ ID NO:5 .

To achieve the above object, the present invention also provides a method of cultivating insects with resistance and/or tolerance to the corn plant of glufosinate-ammonium herbicide, comprising:

Planting at least one corn seed, the genome of the corn seed comprises a nucleic acid sequence encoding an insect resistance Vip3Aa protein and/or a nucleic acid sequence encoding a glufosinate-ammonium herbicide tolerance PAT protein, and a nucleic acid sequence of a specific region, or The genome of the corn seed comprises the nucleotide sequence shown in SEQ ID NO:5;

growing the corn seeds into corn plants;

Invading the corn plant with target insects and/or spraying the corn plant with an effective dose of glufosinate-ammonium herbicide, harvesting plants with reduced plant damage compared with other plants that do not have the nucleic acid sequence of the specific region;

The nucleic acid sequence of the specific region is the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2; preferably, the nucleic acid sequence of the specific region is SEQ ID NO:3 and/or SEQ ID NO:4 sequence shown.

To achieve the above object, the present invention also provides a method for producing a corn plant that is resistant to insects and/or glufosinate-ammonium herbicide tolerant, comprising encoding the insect-resistant gene contained in the first corn plant genome. The nucleic acid sequence of Vip3Aa protein and/or the nucleic acid sequence of coding glufosinate-ammonium tolerance PAT protein, and the nucleic acid sequence of the specific region, or the nucleic acid shown in SEQ ID NO:5 contained in the first corn plant genome sequence, introduced into a second corn plant, thereby producing a large number of progeny plants; selecting the progeny plants having the nucleic acid sequence of the specific region, and the progeny plants are resistant to insects and/or herbicidal to glufosinate Drug tolerance; The nucleic acid sequence of the specific region is the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2; Preferably, the nucleic acid sequence of the specific region is SEQ ID NO:3 and/or Or the sequence shown in SEQ ID NO:4.

Preferably, the method comprises sexually crossing the transgenic maize event DBN9501 with maize plants lacking insect resistance and/or glufosinate-ammonium tolerance, thereby producing a large number of progeny plants, selecting nucleic acid sequences having said specific region The progeny plant of said specific region; the nucleic acid sequence of said specific region comprises the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2; Preferably, the nucleic acid sequence of said specific region comprises SEQ ID NO:3 and /or the sequence shown in SEQ ID NO:4.

To achieve the above object, the present invention also provides an agricultural product or commodity produced from the transgenic corn event DBN9501, the agricultural product or commodity is corn meal, corn flour, corn oil, corn silk, corn starch, corn gluten, corn cake , cosmetics or fillers.

In the nucleic acid sequence and its detection method for detecting corn plants in the present invention, the following definitions and methods can better define the present invention and guide those of ordinary skill in the art to implement the present invention, unless otherwise specified, according to the ordinary skills in the art common usage by people to understand the term.

The term "maize" refers to Zea mays and includes all plant species that can be crossed with maize, including wild maize species.

The "comprising", "including" or "comprising" means "including but not limited to".

The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps and whole plants in plants or plant parts Cells, said plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, and the like. Parts of transgenic plants that are understood to be within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, callus, embryos, as well as flowers, stems, fruits, leaves and roots, the above plant parts being derived from the A transgenic plant transformed with a DNA molecule and thus consisting at least in part of a transgenic cell, or its progeny.

The term "gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding the coding sequence (5' non-coding sequence) and regulatory sequences following the coding sequence (3' non-coding sequence). "Native gene" refers to a gene that is found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences not found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. "Exogenous gene" refers to a foreign gene that exists in the genome of an organism and does not exist originally, and also refers to a gene that is introduced into a recipient cell through a transgenic procedure. Foreign genes may comprise native or chimeric genes inserted into a non-native organism. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The site in the plant genome where the recombinant DNA has been inserted may be referred to as the "insertion site" or "target site".

"Flanking DNA" may comprise the genome naturally present in an organism such as a plant or foreign (heterologous) DNA introduced by a transformation process, such as a fragment associated with a transformation event. Thus, flanking DNA can include a combination of native and foreign DNA. In the present invention, "flank DNA" is also called "flank region" or "flank sequence" or "flank genomic sequence" or "flank genomic DNA", which means at least 3, 5, 10, 11, 15, 20, 50, A sequence of 100, 200, 300, 400, 1000, 1500, 2000, 2500 or 5000 base pairs or longer that is immediately upstream or downstream of and adjacent to the original exogenously inserted DNA molecule. When this flanking region is located downstream, it may also be referred to as a "3' flank" or a "right border flank" or the like. When this flanking region is located upstream, it may also be referred to as a "5' flank" or a "left border flank" or the like.

Transformation procedures that result in random integration of foreign DNA will result in transformants containing different flanking regions that each transformant specifically contains. When recombinant DNA is introduced into plants by conventional crossing, its flanking regions are usually not altered. Transformants will also contain unique junctions between the heterologous insert DNA and the segment of genomic DNA or between two segments of genomic DNA or between two segments of heterologous DNA. A "junction" is the point at which two specific DNA segments join. For example, junctions exist where insert DNA joins flanking DNA. Junctions also exist in transformed organisms where two segments of DNA join together in a manner modified from that found in natural organisms. "Junction region" or "junction sequence" refers to the DNA comprising the junction.

The present invention provides a transgenic maize event designated DBN9501, also known as maize plant DBN9501, including plants and seeds of transgenic maize event DBN9501 and plant cells or regenerable parts thereof, and progeny thereof, Plant parts of transgenic maize event DBN9501, including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, tassels, inflorescences, ears, leaves, and products from maize plant DBN9501, such as corn meal, cornmeal, Corn oil, corn steep liquor, corn silk, corn starch, and biomass left in the field of corn crops.

The transgenic maize event DBN9501 of the present invention comprises a DNA construct that, when expressed in a plant cell, confers insect resistance and tolerance to the glufosinate herbicide. The DNA construct comprises two expression cassettes in tandem, the first expression cassette comprising a suitable promoter for expression in plants and a suitable polyadenylation signal sequence, said promoter being operably linked to Vip3Aa19 The nucleic acid sequence of the protein, the nucleic acid sequence of the Vip3Aa19 protein is mainly resistant to Lepidoptera insects. The second expression cassette comprises a suitable promoter for expression in plants operably linked to a suitable polyadenylation signal sequence encoding phosphinothricin N-acetyltransferase (phosphinothricin N-acetyltransferase). -acetyltransferase, PAT) gene, the nucleic acid sequence of the PAT protein has tolerance to glufosinate-ammonium herbicide. Further, the promoter may be a suitable promoter isolated from a plant, including a constitutive, inducible and/or tissue-specific promoter, and the suitable promoter includes, but is not limited to, cauliflower mosaic virus (CaMV) 35S Promoter, Scrophulariaceae mosaic virus (FMV) 35S promoter, Ubiquitin promoter, Actin promoter, Agrobacterium tumefaciens nopaline synthase (NOS) promoter, octopus Alkali synthase (OCS) promoter, Cestrum yellow leaf curl virus promoter, potato tuber storage protein (Patatin) promoter, ribulose-1,5-bisphosphate carboxylase/oxygenase ( RuBisCO) promoter, glutathione sulfur transferase (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes (Agrobacterium rhizogenes) RolD promoter and Arabidopsis thaliana (Arabidopsis thaliana ) Suc2 promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence that functions in plants, and the suitable polyadenylation signal sequence includes, but is not limited to, derived from Agrobacterium tumefaciens The polyadenylation signal sequence of the nopaline synthase (NOS) gene, the 35S terminator from the cauliflower mosaic virus (CaMV), the polyadenylation signal sequence and the source of the protease inhibitor Ⅱ (PINⅡ) gene The polyadenylation signal sequence of the α-tubulin gene.

In addition, the expression cassette may also include other genetic elements including, but not limited to, enhancers and signal/transit peptides. The enhancer can strengthen the expression level of the gene, and the enhancer includes, but is not limited to, tobacco etch virus (TEV) translation activator, CaMV35S enhancer and FMV35S enhancer. The signal peptide/transit peptide can guide the Vip3Aa19 protein and/or PAT protein to be transported to a specific organelle or compartment outside the cell or within the cell, for example, using the sequence encoding the chloroplast transit peptide to target the chloroplast, or using the 'KDEL' reserved sequence target To the endoplasmic reticulum.

The Vip3Aa19 gene can be isolated from Bacillus thuringiensis (Bt for short), and the nucleotide sequence of the Vip3Aa19 gene can be changed by optimizing codons or in other ways to increase the expression of transcripts in transformed cells. for stability and availability purposes.

The "Lepidoptera (Lepidoptera)" includes two types of insects, moths and butterflies.

The phosphinothricin N-acetyltransferase (PAT) gene can be an enzyme isolated from a Streptomyces viridochromogenes strain, which converts L-phosphinothricin into its inactive form through acetylation, so as to impart plant resistance to Tolerance to glufosinate-ammonium herbicide. Phosphinothricin (PTC, 2-amino-4-methylphosphonobutyric acid) is an inhibitor of glutamine synthetase. PTC is the structural unit of the antibiotic 2-amino-4-methylphosphonyl-alanyl-alanine. This tripeptide (PTT) has anti-gram-positive and gram-negative bacteria and anti-fungal Botrytis cinerea (Botrytis cinerea). cinerea) activity. The phosphinothricin N-acetyltransferase (PAT) gene can also serve as a selectable marker gene.

The "glufosinate-ammonium", also known as glufosinate, refers to 2-amino-4-[hydroxyl (methyl) phosphono] ammonium butyrate, and the treatment with "glufosinate-ammonium herbicide" refers to the use of any one containing Glufosinate-ammonium herbicide formulations. The selection of the application rate of a glufosinate-ammonium formulation to achieve a biologically effective dose is within the skill of the average agronomist. Treatment of a field containing plant material derived from GM maize event DBN9501 with any of the herbicide formulations containing glufosinate will control weed growth in said field without affecting the plant material derived from GM maize event DBN9501 growth or yield.

The DNA construct is introduced into the plant using a transformation method including, but not limited to, Agrobacterium-mediated transformation, biolistic transformation, and pollen tube passage transformation.

The Agrobacterium-mediated transformation method is a common method for plant transformation. The foreign DNA to be introduced into the plant is cloned between the left and right border consensus sequences of the vector, i.e. the T-DNA region. The vector is transformed into Agrobacterium cells, which are then used to infect plant tissue, and the T-DNA region of the vector containing foreign DNA is inserted into the plant genome.

The gene bombardment transformation method is to bombard plant cells with a vector containing foreign DNA (particle-mediated biolistic transformation).

The pollen tube channel transformation method utilizes the natural pollen tube channel (also known as the pollen tube guiding tissue) formed after plant pollination to carry exogenous DNA into the embryo sac through the nucellus channel.

Following transformation, transgenic plants must be regenerated from transformed plant tissue, and appropriate markers used to select for progeny bearing the exogenous DNA.

A DNA construct is an interconnected assembly of DNA molecules that provides one or more expression cassettes. The DNA construct is preferably a plasmid capable of self-replicating in bacterial cells and containing various restriction endonuclease sites for introduction to provide a functional genetic element, i.e. a promoter , introns, leader sequences, coding sequences, 3' terminator regions and other sequences of DNA molecules. The expression cassette contained in the DNA construct includes the genetic elements necessary to provide transcription of the messenger RNA and can be designed for expression in prokaryotic or eukaryotic cells. The expression cassettes of the invention are designed for expression most preferably in plant cells.

A transgenic "event" is obtained by transforming a plant cell with a heterologous DNA construct, i.e., comprising at least one nucleic acid expression cassette containing a gene of interest, inserted transgenically into the plant genome to produce a plant population, which is regenerated , and select specific plants with features inserted into specific genomic loci. The term "event" refers to the original transformant containing heterologous DNA and the progeny of that transformant. The term "event" also refers to the progeny of a sexual cross between an original transformant and an individual of another breed that contains heterologous DNA, even if, after repeated backcrosses with the backcross parent, the inserted DNA from the original transformant parent and flanking genomic DNA are also present at the same chromosomal location in the hybrid offspring. The term "event" also refers to a DNA sequence from an original transformant comprising the insert DNA and flanking genomic sequences in close proximity to the insert DNA, which DNA sequence is expected to be transferred to the progeny consisting of the insert DNA containing The parental line (eg, the original transformant and its progeny produced by selfing) is sexually crossed with a parental line that does not contain the inserted DNA, and the progeny receive the inserted DNA containing the gene of interest.

"Recombinant" in the present invention refers to a form of DNA and/or protein and/or organism that is not normally found in nature and thus produced by human intervention. Such human intervention can produce recombinant DNA molecules and/or recombinant plants. Said "recombinant DNA molecule" is obtained by the artificial combination of two otherwise separate sequence segments, such as by chemical synthesis or by the manipulation of separate nucleic acid segments by genetic engineering techniques. The techniques for performing nucleic acid manipulations are well known.

The term "transgenic" includes any cell, cell line, callus, tissue, plant part or plant, the genotype of which is altered by the presence of heterologous nucleic acid, said "transgenic" including transgenics originally so altered as well as those derived from The offspring individuals produced by the original transgenic body through sexual crossing or asexual reproduction. In the present invention, the term "transgenic" does not include alterations of the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or naturally occurring events such as random heterozygous fertilization, non-recombinant viral infection, non-recombinant Bacterial transformation, non-recombinant transposition, or spontaneous mutation.

"Heterologous" in the context of the present invention means that a first molecule is not normally found in combination with a second molecule in nature. For example, a molecule can be derived from a first species and inserted into the genome of a second species. Such molecules are thus heterologous to the host and are artificially introduced into the genome of the host cell.

Transgenic maize event DBN9501, which is resistant to lepidopteran insects and glufosinate-ammonium herbicide, was developed by first sexually crossing a first parental maize plant with a second parental maize plant to produce A diverse first generation of progeny plants consisting of first parent maize plants consisting of maize plants grown from transgenic maize event DBN9501 and its progeny obtained by using the present invention for Lepidoptera insects Transformation of a resistant and glufosinate herbicide tolerant expression cassette, second parent maize plant lacking lepidopteran insect resistance and/or glufosinate herbicide tolerance Then select the progeny plants that have resistance to the attack of Lepidoptera insects and/or have tolerance to glufosinate-ammonium herbicide, can breed Lepidoptera insects and have resistance to glufosinate-ammonium herbicide. Tolerant corn plants. These steps may further comprise backcrossing the lepidopteran-resistant and/or glufosinate-tolerant progeny plants to a second parent corn plant or a third parent corn plant, followed by infestation with lepidopteran insects, glufosinate herbicide application or selection of progeny by identification of trait-associated molecular markers such as DNA molecules containing junction sites identified at the 5' and 3' ends of the insert sequence in transgenic maize event DBN9501, This results in maize plants that are resistant to Lepidoptera insects and tolerant to the glufosinate herbicide.

It should also be understood that two different transgenic plants can also be mated to produce offspring containing two independent, segregated additions of the exogenous gene. Selfing of suitable progeny can result in progeny plants that are homozygous for both added exogenous genes. Backcrossing to parental plants and outcrossing to non-transgenic plants are also contemplated, as previously described, as is vegetative propagation.

The term "probe" is an isolated nucleic acid molecule to which is bound a conventional detectable label or reporter molecule, for example, a radioisotope, ligand, chemiluminescent agent or enzyme. Such probes are complementary to one strand of the target nucleic acid, and in the present invention, the probe is complementary to one strand of DNA from the genome of transgenic maize event DBN9501, whether the genomic DNA is from transgenic maize event DBN9501 or the seed or derived from the transgene Plant or seed or extract of maize event DBN9501. Probes of the present invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.

The term "primer" is an isolated nucleic acid molecule that, by nucleic acid hybridization, anneals to a complementary target DNA strand, forms a hybrid between the primer and target DNA strand, and then reacts with a polymerase (eg, DNA polymerase) Bottom, stretches along the target DNA strand. The primer pairs of the invention relate to their use in the amplification of target nucleic acid sequences, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

Probes and primers are generally 11 polynucleotides or more in length, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, most preferably 30 polynucleotides in length sour or more. Such probes and primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although probes that are different from the target DNA sequence and maintain the ability to hybridize to the target DNA sequence can be designed by conventional methods, preferably, the probes and primers in the present invention have complete DNA sequences with the continuous nucleic acid of the target sequence identity.

The primers and probes based on the flanking genomic DNA and the insert sequence of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from the plant material derived from the transgenic maize event DBN9501, and determining the nucleic acid sequence of the DNA molecule. The DNA molecule comprises a transgene insert sequence and a maize genome flanking sequence, and fragments of the DNA molecule can be used as primers or probes.

The nucleic acid probes and primers of the invention hybridize to target DNA sequences under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from transgenic maize event DBN9501 in a sample. Nucleic acid molecules or fragments thereof can specifically hybridize to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be capable of specifically hybridizing to each other if the two nucleic acid molecules are capable of forming an antiparallel double-stranded nucleic acid structure. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if two nucleic acid molecules exhibit perfect complementarity. As used herein, two nucleic acid molecules are said to exhibit "perfect complementarity" when every nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to be "complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "high stringency" conditions. Deviations from perfect complementarity are permissible as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to allow the formation of a stable double-stranded structure under the particular solvent and salt concentration employed.

As used herein, a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing to a matching complementary strand of another nucleic acid molecule under highly stringent conditions. Suitable stringent conditions to promote DNA hybridization, for example, treatment with 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by washing with 2.0× SSC at 50° C., are known to those skilled in the art. is well known. For example, the salt concentration in the washing step can be selected from about 2.0×SSC, 50°C for low stringency conditions to about 0.2×SSC, 50°C for high stringency conditions. In addition, the temperature conditions in the washing steps can be increased from room temperature of about 22°C for low stringency conditions to about 65°C for high stringency conditions. Both the temperature condition and the salt concentration can be changed, or one can be held constant while the other variable is changed. Preferably, a nucleic acid molecule of the present invention can be combined with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 under moderately stringent conditions, for example at about 2.0×SSC and about 65° C. , SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, one or more nucleic acid molecules or their complementary sequences, or any fragment of the above sequences specifically hybridizes. More preferably, a nucleic acid molecule of the present invention is combined with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 under highly stringent conditions Specific hybridization occurs with one or more nucleic acid molecules in SEQ ID NO: 7 or its complementary sequence, or any fragment of the above sequence. In the present invention, a preferred marker nucleic acid molecule has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 7 or its complementary sequence, or any fragment of the above sequence. Another preferred marker nucleic acid molecule of the present invention has 80% to 100% of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:7 or its complementary sequence, or any fragment of the above sequence. % or 90% to 100% sequence identity. SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7 can be used as markers in plant breeding methods to identify progeny of genetic crosses. Hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including but not limited to, fluorescent labeling, radioactive labeling, antibody-based labeling, and chemiluminescent labeling.

With respect to amplification of a target nucleic acid sequence using specific amplification primers (for example, by PCR), "stringent conditions" refer to conditions that allow only primers to hybridize to a target nucleic acid sequence in a DNA thermal amplification reaction, with the same The primer corresponding to the wild-type sequence (or its complementary sequence) of the target nucleic acid sequence is capable of binding to the target nucleic acid sequence, and preferably produces a unique amplification product, the amplification product being an amplicon.

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

As used herein, "amplicon" refers to the product of nucleic acid amplification of a nucleic acid sequence of interest that is part of a nucleic acid template. For example, in order to determine whether corn plants were produced by sexual crossing containing the transgenic corn event DBN9501 of the present invention, or whether a corn sample collected from a field contained the transgenic corn event DBN9501, or whether a corn extract, such as meal, flour or oil contained Transgenic corn event DBN9501, DNA extracted from corn plant tissue samples or extracts can be subjected to nucleic acid amplification methods using primer pairs to generate amplicons that are diagnostic for the presence of DNA from transgenic corn event DBN9501. The pair of primers includes a first primer derived from a flanking sequence in the plant genome adjacent to the insertion site of the inserted foreign DNA, and a second primer derived from the inserted foreign DNA. The amplicon has a length and sequence that is also diagnostic for the transgenic maize event DBN9501. The length of the amplicon may range from 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, Most preferably plus about 450 nucleotide base pairs or more.

Alternatively, primer pairs can be derived from flanking genomic sequences flanking the insert DNA to generate amplicons that include the entire insert nucleotide sequence. One of the primer pairs derived from the plant genomic sequence can be located at a distance from the insert DNA sequence, which distance can range from one nucleotide base pair to about twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer-dimers formed in DNA thermal amplification reactions.

Nucleic acid amplification reactions can be accomplished by any nucleic acid amplification reaction method known in the art, including polymerase chain reaction (PCR). Various nucleic acid amplification methods are well known to those skilled in the art. PCR amplification methods have been developed to amplify up to 22kb of genomic DNA and up to 42kb of phage DNA. These methods, as well as other DNA amplification methods known in the art, can be used in the present invention. The inserted exogenous DNA sequence and the flanking DNA sequence from the transgenic maize event DBN9501 can be amplified from the genome of the transgenic maize event DBN9501 using the primer sequences provided, followed by standard analysis of PCR amplicons or cloned DNA. DNA sequencing.

DNA detection kits based on DNA amplification methods contain DNA molecules used as primers that, under appropriate reaction conditions, hybridize specifically to target DNA and amplify diagnostic amplicons. Kits may provide agarose gel-based detection methods or a number of methods known in the art to detect diagnostic amplicons. A kit containing DNA primers that are homologous or complementary to any part of the maize genome of SEQ ID NO: 3 or SEQ ID NO: 4 and any part of the transgene insertion region of SEQ ID NO: 5 is the present invention provided by the invention. A primer pair specifically identified as useful in DNA amplification methods is SEQ ID NO: 8 and SEQ ID NO: 9, which amplifies diagnostic amplification of homology to a portion of the 5' transgene/genomic region of transgenic maize event DBN9501 sub, wherein the amplicon comprises SEQ ID NO: 1. Other DNA molecules used as DNA primers may be selected from SEQ ID NO:5.

Amplicons generated by these methods can be detected by a variety of techniques. One such method is Genetic Bit Analysis, which designs a DNA oligonucleotide strand spanning the insert DNA sequence and the adjacent flanking genomic DNA sequence. The oligonucleotide strands are immobilized in the microwells of a microplate, and after PCR amplification of the region of interest (using one primer each within the insert sequence and adjacent flanking genomic sequences), the single-stranded PCR product Can hybridize to immobilized oligonucleotide strands and serve as templates for single-base extension reactions using DNA polymerase and ddNTPs specifically labeled for the next expected base. Results can be obtained by fluorescent or ELISA-like methods. The signal represents the presence of the insertion/flanking sequence, which indicates that the amplification, hybridization and single base extension reactions were successful.

Another method is pyrosequencing technology (Pyrosequencing). This method designs an oligonucleotide strand that spans the insert DNA sequence and the adjacent genomic DNA binding site. This oligonucleotide strand is hybridized to a single-stranded PCR product of the region of interest (using one primer each within the insert and adjacent flanking genomic sequences), followed by DNA polymerase, ATP, sulfurylase, luciferin The enzyme, apyrase, adenosine-5'-phosphosulfate and luciferin are incubated together. The dNTPs were added separately, and the resulting light signals were measured. The light signal represents the presence of the insertion/flanking sequence, which indicates that the amplification, hybridization, and single- or multi-base extension reactions were successful.

The fluorescence polarization phenomenon described by Chen et al. (Genome Res. 9:492-498, 1999) is also a method that can be used to detect amplicons of the invention. Using this method requires the design of an oligonucleotide strand that spans the insertion DNA sequence and the adjacent genomic DNA binding site. The oligonucleotide strand is hybridized to a single-stranded PCR product of the region of interest (using one primer each within the insert and adjacent flanking genomic sequence) with DNA polymerase and a fluorescently labeled ddNTP Incubation. Single base extensions result in the insertion of ddNTPs. This insertion can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of insertion/flanking sequences, which indicates that the amplification, hybridization and single base extension reactions were successful.

Taqman is described as a method for the detection and quantification of the presence of DNA sequences as detailed in the instructions for use provided by the manufacturer. Briefly described below, a FRET oligonucleotide probe is designed to span the insertion DNA sequence and the adjacent genomic flanking junction site. The FRET probe and PCR primers (one each within the insert and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage of the fluorescent and quencher moieties on the FRET probe and release of the fluorescent moiety. The generation of a fluorescent signal represents the presence of the insertion/flanking sequence, which indicates that the amplification and hybridization were successful.

Based on hybridization principles, suitable techniques for detecting plant material derived from transgenic maize event DBN9501 may also include Southern blot, Northern blot, and in situ hybridization. In particular, such suitable techniques include incubating the probe and sample, washing to remove unbound probe and detecting whether the probe has hybridized. The method of detection depends on the type of label attached to the probe, e.g., radiolabeled probes can be detected by X-ray film exposure and visualization, or enzyme-labeled probes can be detected by substrate conversion to achieve a color change.

Tyangi et al. (Nature Biotech. 14:303-308, 1996) describe the use of molecular markers in sequence detection. Briefly described below, a FRET oligonucleotide probe is designed to span the insertion DNA sequence and the adjacent genomic flanking junction site. The unique structure of this FRET probe results in it containing a secondary structure that is capable of maintaining fluorescent and quencher moieties in close proximity. The FRET probe and PCR primers (one each within the insert and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, the hybridization of the FRET probe to the target sequence results in the loss of the secondary structure of the probe, thereby spatially separating the fluorescent and quenching moieties, resulting in a fluorescent signal. The generation of a fluorescent signal represents the presence of the insertion/flanking sequence, which indicates that the amplification and hybridization were successful.

Other described methods such as microfluidics provide methods and devices for isolating and amplifying DNA samples. Optical dyes are used to detect and measure specific DNA molecules. Nanotube devices comprising electronic sensors for the detection of DNA molecules or nanobeads which bind specific DNA molecules and thus can be detected are useful for the detection of the DNA molecules of the present invention.

DNA detection kits can be developed using the compositions described herein and methods described or known in the field of DNA detection. The kit facilitates the identification of the presence of DNA from transgenic maize event DBN9501 in a sample and can also be used to grow maize plants containing DNA from transgenic maize event DBN9501. The kit may contain DNA primers or probes homologous to or complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4 or 5, or other DNA primers or probes homologous to or complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4 or 5 The DNA contained in the transgenic genetic element is complementary to the DNA, which DNA sequences can be used in DNA amplification reactions, or as probes in DNA hybridization methods. The DNA structure of the junction of the transgene insert sequence and the maize genome contained in the maize genome and illustrated in Figure 1 and Table 1 contains: a region of the maize plant DBN9501 flanking genome located at the 5' end of the transgene insert sequence, from the left side of the Agrobacterium Part of the insert in the border region (LB), the first expression cassette consists of the maize ubiquitin gene 1 promoter (prZmUbi1) operably linked to the glufosinate-tolerant phosphinothricin N-acetyl cPAT and operably linked to the transcription terminator (tNos) of nopaline synthase, the second expression cassette consists of a tandem repeat of the cauliflower mosaic virus 35S promoter containing an enhancer region (pr35S), operably linked to the maize heat shock 70kDa intein (iZmHSP70), operably linked to the insect-resistant Vip3Aa19 protein (cVip3Aa19) of Bacillus thuringiensis, and operably linked to nopaline The transcription terminator (tNos) of the synthase, part of the insert sequence from the right border region (RB) of Agrobacterium, and the flanking genomic region of maize plant DBN9501 located at the 3' end of the transgene insert sequence (SEQ ID NO:5 ). In the DNA amplification method, the DNA molecules used as primers can be derived from any part of the transgene insert sequence in the transgenic maize event DBN9501, or any part of the flanking DNA sequence of the maize genome derived from the transgenic maize event DBN9501.

The transgenic maize event DBN9501 can be combined with other transgenic maize varieties, such as herbicide (such as glyphosate, dicamba, etc.) tolerant transgenic maize varieties, or transgenic maize varieties carrying other insect resistance genes. Various combinations of all these different transgenic events, bred together with the transgenic maize event DBN9501 of the present invention, can provide improved hybrid transgenic maize varieties that are resistant to multiple pests and herbicides. Compared with non-transgenic varieties and single-trait transgenic varieties, these varieties can show more excellent characteristics such as yield improvement.

The transgenic maize event DBN9501 of the present invention is resistant to feeding damage by Lepidoptera pests and is resistant to the phytotoxic effects of agricultural herbicides containing glufosinate-ammonium. This dual-trait maize plant expresses the Vip3Aa19 protein from Bacillus thuringiensis, which confers resistance to feeding damage from lepidopteran pests such as cutworms, and the glufosinate-resistant phosphinothricin N from Streptomyces - Acetyltransferase (PAT) proteins which confer tolerance to glufosinate in plants. The dual-character corn has the following advantages: 1) it is free from economic losses caused by Lepidoptera pests (such as cutworms, cotton bollworms, etc.), which are the main pests in corn planting areas; The agricultural herbicide glufosinate-ammonium gives corn crops the ability to be used for broad spectrum weed control; 3) No reduction in corn yield. Furthermore, the transgenes encoding insect resistance and glufosinate-ammonium tolerance traits are linked on the same DNA segment and are present at a single locus in the genome of transgenic maize event DBN9501, which provides enhanced breeding efficiency and enables the use of Molecular markers to track transgene insertions in breeding populations and their progeny. Meanwhile, in the detection method of the present invention, SEQ ID NO:1 or its complementary sequence, SEQ ID NO:2 or its complementary sequence, SEQ ID NO:6 or its complementary sequence, or SEQ ID NO:7 or its complementary sequence can be used as a DNA primer Or a probe to produce an amplification product that is diagnosed as the transgenic corn event DBN9501 or its progeny, and can quickly, accurately and stably identify the presence of plant material derived from the transgenic corn event DBN9501.

sequence description

A sequence of 22 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in the transgenic maize event DBN9501 of SEQ ID NO: 1, wherein the 1st-11th nucleotides and the 12th-22nd are nucleosides Acids are located on both sides of the insertion site on the maize genome;

A sequence of 22 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in the transgenic maize event DBN9501 of SEQ ID NO: 2, wherein the 1st-11th nucleotides and the 12th-22nd are nucleosides Acids are located on both sides of the insertion site on the maize genome;

SEQ ID NO:3 A sequence of 768 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in transgenic maize event DBN9501;

A sequence of 1339 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in SEQ ID NO: 4 transgenic maize event DBN9501;

The whole T-DNA sequence of SEQ ID NO:5, the maize genome flanking sequence of 5' and 3' ends;

SEQ ID NO:6 is located on the sequence of SEQ ID NO:3, spanning the left border region (LB) and the tNos transcription termination sequence;

SEQ ID NO:7 is located at the sequence on SEQ ID NO:4, spanning the pr35S transcription initiation sequence and the right border region (RB);

SEQ ID NO:8 amplifies the first primer of SEQ ID NO:3;

SEQ ID NO:9 amplifies the second primer of SEQ ID NO:3;

SEQ ID NO:10 amplifies the first primer of SEQ ID NO:4;

SEQ ID NO:11 amplifies the second primer of SEQ ID NO:4;

Primer on SEQ ID NO:12 5' flanking genomic sequence;

Primers on T-DNA paired with SEQ ID NO:13 and SEQ ID NO:12;

The primer on the genomic sequence of SEQ ID NO:14 3 'flanks, and its pairing with SEQ ID NO:12 can detect whether the transgene is homozygous or heterozygous;

Primers on T-DNA paired with SEQ ID NO:15 and SEQ ID NO:14;

SEQ ID NO:16 Taqman detects the first primer of Vip3Aa19 gene;

SEQ ID NO:17 Taqman detects the second primer of Vip3Aa19 gene;

SEQ ID NO:18 Taqman detects the probe of Vip3Aa19 gene;

SEQ ID NO:19 Taqman detects the first primer of pat gene;

SEQ ID NO:20 Taqman detects the second primer of pat gene;

SEQ ID NO:21 Taqman detects the probe of pat gene;

The first primer of SEQ ID NO:22 maize endogenous gene SSIIb;

The second primer of SEQ ID NO:23 maize endogenous gene SSIIb;

The probe of the Vip3Aa19 gene in SEQ ID NO:24 Southern hybridization detection;

The probe of pat gene in SEQ ID NO:25 Southern hybridization detection;

The primer of SEQ ID NO:26 located on the T-DNA is consistent with the direction of SEQ ID NO:13;

SEQ ID NO:27 is positioned at the primer on T-DNA, and SEQ ID NO:13 direction is opposite, is used as obtaining flanking sequence;

SEQ ID NO:28 is positioned at the primer on T-DNA, and SEQ ID NO:13 direction is opposite, is used as obtaining flanking sequence;

The primer of SEQ ID NO: 29 located on the T-DNA is consistent with the direction of SEQ ID NO: 15;

SEQ ID NO:30 is positioned at the primer on T-DNA, and SEQ ID NO:15 direction is opposite, is used as obtaining flanking sequence;

The primer of SEQ ID NO: 31 located on the T-DNA, opposite to that of SEQ ID NO: 15, was used to obtain flanking sequences.

The technical solutions of the present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

Description of drawings

Fig. 1 is a schematic diagram of the structure of the transgene insertion sequence and the junction site of the corn genome used to detect the nucleic acid sequence of corn plant DBN9501 and the detection method of the present invention, and a schematic diagram of the relative position of the nucleic acid sequence used to detect the corn plant DBN9501 (relative position schematic diagram refers to B73RefGen v3);

Fig. 2 is the structural representation of the recombinant expression vector DBN10707 that the present invention is used to detect the nucleotide sequence of corn plant DBN9501 and detection method thereof;

Fig. 3 is the field effect diagram of the transgenic corn event DBN9501 used to detect the nucleic acid sequence of the corn plant DBN9501 and the detection method thereof of the present invention under the natural occurrence condition of the cutworm;

Fig. 4 is the field effect diagram of the transgenic corn event DBN9501 inoculated with cotton bollworm for the nucleic acid sequence of the corn plant DBN9501 and the detection method thereof of the present invention;

Fig. 5 is the field effect diagram of the transgenic corn event DBN9501 used to detect the nucleic acid sequence of the corn plant DBN9501 and the detection method thereof under the natural occurrence condition of Spodoptera litura;

Fig. 6 is a field effect diagram of the transgenic corn event DBN9501 used to detect the nucleic acid sequence of the corn plant DBN9501 and the detection method thereof under natural occurrence conditions of beet armyworm.

Detailed ways

The technical scheme for detecting the nucleic acid sequence of corn plant DBN9501 and the detection method thereof of the present invention is further illustrated by specific examples below.

The first embodiment, cloning and transformation

1.1. Vector cloning

The recombinant expression vector DBN10707 was constructed using standard gene cloning techniques (as shown in Figure 2). The vector DBN10707 contains two tandem transgene expression cassettes, the first expression cassette consists of a tandem repeat cauliflower mosaic virus 35S promoter (pr35S) containing an enhancer region, operably linked to a maize heat shock 70kDa protein containing (iZmHSP70), operably linked to the insect-resistant Vip3Aa19 protein (cVip3Aa19) of Bacillus thuringiensis, and operably linked to the transcription terminator (tNos) of nopaline synthase; the second The expression cassette consists of a maize ubiquitin gene 1 promoter (prZmUbi1), operably linked to the glufosinate-tolerant phosphinothricin N-acetyltransferase (cPAT) of Streptomyces, and operably linked to to the transcription terminator (tNos) of nopaline synthase.

The vector DBN10707 was transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat.No: 18313-015) with liquid nitrogen method, and 4-[hydroxyl (methyl)phosphono]-DL-homoalanine Acid was used as a selectable marker to select transformed cells.

1.2. Plant transformation

The conventional Agrobacterium infection method was used for transformation, and the aseptically cultured corn immature embryos were co-cultured with the Agrobacterium described in Example 1.1 to transfer the T-DNA in the constructed recombinant expression vector DBN10707 into the corn genome to generate the transgenic maize event DBN9501.

For Agrobacterium-mediated transformation of maize, briefly, immature immature embryos were isolated from maize and contacted with a suspension of Agrobacterium capable of converting the nucleotide sequence of the Vip3Aa19 gene and the nucleotide sequence of the pat gene The sequence is transferred to at least one cell of one of the immature embryos (step 1: infection step), during which the immature embryos are preferably immersed in an Agrobacterium suspension (OD ₆₆₀ =0.4-0.6, infection medium (MS salt 4.3 g/L, MS vitamins, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, pH 5.3)) to initiate inoculation. The immature embryos were co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step). Preferably, the immature embryos are cultured on solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/L, AS 100mg/L, 2,4- D 1mg/L, agar 8g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step. In the "recovery" step, recovery medium (MS salt 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, 2,4-D 1mg/L, cephalosporin 250mg/L, phytogel At least one antibiotic (cephalosporin 150-250 mg/L) known to inhibit the growth of Agrobacterium was present in the gum 3 g/L, pH 5.8), and no selection agent for plant transformants was added (step 3: recovery step). Preferably, immature embryos are cultured on solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for infected cells. Next, the inoculated immature embryos were cultured on a medium containing a selection agent (4-[hydroxy(methyl)phosphono]-DL-homoalanine) and the growing transformed callus was selected (step 4: selection step ). Preferably, the immature embryos are cultured in the selection solid medium of selective agent (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, cephalosporin 250mg/L, 4-[hydroxyl (methyl ) Phosphono]-DL-homoalanine 10 mg/L, 2,4-D 1 mg/L, Phytogel 3 g/L, pH 5.8), resulting in selective growth of transformed cells. Then, the callus regenerates into plants (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on solid medium (MS differentiation medium and MS rooting medium) to regenerated plants.

The resistant callus obtained by screening was transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyl adenine 2mg/L, cephalosporin 250 mg/L 4-[hydroxy(methyl)phosphono]-DL-homoalanine 5 mg/L, 3 g/L phytogel, pH 5.8), cultured and differentiated at 25°C. Differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, cephalosporin 250mg/L, indole-3-acetic acid 1 mg/L L, plant gel 3g/L, pH 5.8), cultivate at a temperature of 25°C to a height of about 10cm, move to the greenhouse and cultivate until firm. In the greenhouse, cultivate at a temperature of 28° C. for 16 hours every day, and then cultivate at a temperature of 20° C. for 8 hours.

1.3. Identification and screening of transgenic events

A total of 200 independent transgenic T ₀ individual plants were generated.

Since genetic transformation and gene insertion may affect the agronomic traits of maize plants (such as short and thick, clustered leaves, mosaic leaves, opposite leaves, abnormal spinning and loose powder, or poor seed setting, etc.), the above 200 independent The transgenic T ₀ individual plants were sent to the greenhouse for transplanting and cultured to identify the agronomic performance of the transgenic T ₀ individual plants at different stages (seedling stage-jointing stage, jointing stage-loose powder stage and filling stage-mature stage). 136 transgenic T ₀ individual plants with normal agronomic traits.

Through TaqMan ^TM analysis, it was detected whether there were single copies of Vip3Aa19 and pat genes in the above 136 transgenic maize plants without vector backbone sequences, and a total of 83 transgenic T ₀ individual plants were obtained; a total of 28 transgenic plants were screened through transgene insertion site analysis Transgenic T ₀ individual plants with complete sequences on both sides of the T-DNA, no T-DNA insertion into important genes of the maize genome, and no new open reading frame (ORF) generated by the gene insertion; , Cotton bollworm, Spodoptera litura or Beet Spodoptera) resistance evaluation and comparison, 13 transgenic T ₀ individual plants with good insect resistance were screened; by evaluation and comparison of glufosinate herbicide tolerance, A total of 12 transgenic T ₀ individual plants with good tolerance to glufosinate-ammonium herbicide were screened; in the case of different generations, different geographical environments and/or different background materials, through the agronomic traits of transgenic maize plants, molecular biology , target insect resistance, glufosinate tolerance, etc. can be stably genetically screened, and the selected transgenic corn event DBN9501 is excellent, which has a single-copy transgene (see the second embodiment), good insect resistance, Glufosinate-ammonium herbicide tolerance and agronomic performance (see fifth and sixth examples).

The second embodiment, detection of transgenic corn event DBN9501 with TaqMan

About 100 mg of the leaves of the transgenic corn event DBN9501 were taken as a sample, and the genomic DNA was extracted with a plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen), and the copy numbers of the Vip3Aa19 gene and the pat gene were detected by the Taqman probe fluorescence quantitative PCR method. At the same time, the wild-type maize plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

The specific method is as follows:

Step 1, take 100 mg of leaves (after pollination) of transgenic corn event DBN9501, grind into a homogenate with liquid nitrogen in a mortar, and get 3 repetitions for each sample;

Step 2, use plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen) to extract the genomic DNA of above-mentioned sample, specific method refers to its product manual;

Step 3, measure the genomic DNA concentration of above-mentioned sample with ultra-micro spectrophotometer (NanoDrop 2000, Thermo Scientific);

Step 4, adjusting the genomic DNA concentration of the above sample to the same concentration value, the range of the concentration value is 80-100ng/μL;

Step 5, using the Taqman probe fluorescent quantitative PCR method to identify the copy number of the sample, using the sample with known copy number after identification as a standard, and using the sample of the wild-type corn plant as a control, each sample was repeated 3 times, and the average Value; Fluorescence quantitative PCR primer and probe sequences are respectively:

The following primers and probes were used to detect the Vip3Aa19 gene sequence:

Primer 1: cgaatacagaaccctgtcggc as shown in SEQ ID NO: 16 in the sequence listing;

Primer 2: cgtgaggaaggtctcagaaatgac as shown in SEQ ID NO: 17 in the sequence listing;

Probe 1: cgacgatggcgtgtatatgcctcttgg as shown in SEQ ID NO: 18 in the sequence listing;

The following primers and probes were used to detect the pat gene sequence:

Primer 3: gagggtgttgtggctggtattg as shown in SEQ ID NO: 19 in the sequence listing;

Primer 4: tctcaactgtccaatcgtaagcg as shown in SEQ ID NO: 20 in the sequence listing;

Probe 2: ccttacgctgggccctggaaggctag is shown in SEQ ID NO: 21 in the sequence listing;

The PCR reaction system is:

The 50× primer/probe mixture contains 45 μL of each primer at 1 mM concentration, 50 μL of probe at 100 μM concentration and 860 μL of 1×TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and at 4° C. , stored in amber test tubes.

The PCR reaction conditions are:

Utilize fast real-time fluorescent quantitative PCR system software (Applied Biosystems 7900HT Fast Real-Time PCR System SDS v2.3, Applied Biosystems) to analyze data, the result shows that the obtained transgenic maize event DBN9501 is a single copy.

The third embodiment, analysis of the insertion site of the transgenic corn event DBN9501

3.1. Genomic DNA extraction

The DNA was extracted according to the conventional CTAB (cetyltrimethylammonium bromide) method: take 2g of the young leaves of the transgenic corn event DBN9501 and grind them into powder in liquid nitrogen, add 0.5mL of DNA extraction CTAB buffer (20g/L CTAB, 1.4M NaCl, 100mM Tris-HCl, 20mM EDTA (ethylenediaminetetraacetic acid), adjust the pH to 8.0 with NaOH), mix thoroughly, and extract at 65°C for 90min ; Add 0.5 times the volume of phenol and 0.5 times the volume of chloroform, mix upside down; centrifuge at 12000 rpm (revolutions per minute) for 10 minutes; absorb the supernatant, add 2 times the volume of absolute ethanol, shake the centrifuge tube gently, and put it at a temperature of 4 Centrifuge at 12000rpm for 10min; collect the DNA to the bottom of the tube; discard the supernatant and wash the precipitate with 1mL of 70% ethanol; centrifuge at 12000rpm for 5min; Blow dry on the bench; dissolve the DNA precipitate in an appropriate amount of TE buffer and store at -20°C.

3.2. Analysis of flanking DNA sequences

The concentration of the DNA sample extracted above was determined so that the concentration of the sample to be tested was between 80-100 ng/μL. Genomic DNA was digested with restriction endonucleases Kpn I (5' end analysis) and Spe I (3' end analysis). Add 26.5 μL of genomic DNA, 0.5 μL of the aforementioned restriction enzymes, and 3 μL of restriction enzyme digestion buffer to each enzyme digestion system (the restriction enzymes used are all enzymes from NEB Company and their supporting buffers or universal buffers, now called NEBCutSmart), digested for 1h. After enzyme digestion, add 70 μL of absolute ethanol to the enzyme digestion system, ice bath for 30 minutes, centrifuge at 12000 rpm for 7 minutes, discard the supernatant, blow dry, then add 8.5 μL double distilled water, 1 μL 10×T ₄ -DNA ligation Enzyme buffer (NEB T4 DNA LigaseReaction Buffer, its specific formula can visit NEB website or refer to https://www.neb.com/products/restriction-endonucleases, https://www.neb.com/products/b0202-t4 -dna-ligase-reaction-buffer) and 0.5 μL T ₄ -DNA ligase were ligated overnight at a temperature of 4°C. PCR amplification using a series of nested primers isolates 5' and 3' genomic DNA. Specifically, the primer combination for isolating 5' end genomic DNA includes SEQ ID NO: 13 and SEQ ID NO: 26 as the first primer, SEQ ID NO: 27 and SEQ ID NO: 28 as the second primer, and SEQ ID NO: 13 as the sequencing primers. Isolating the 3' end genomic DNA primer combination includes SEQ ID NO: 15 and SEQ ID NO: 29 as the first primer, SEQ ID NO: 30 and SEQ ID NO: 31 as the second primer, SEQ ID NO: 15 as the sequencing primer, The PCR reaction conditions are shown in Table 3.

The amplified products obtained by the above PCR amplification reaction were electrophoresed on a 2.0% agarose gel to separate the PCR amplified products, and then the gel extraction kit (QIAquick Gel Extraction Kit, catalog #_28704, Qiagen Inc., Valencia, CA) to isolate the fragment of interest from the agarose matrix. Purified PCR amplification products were then sequenced (e.g., using ABI Prism™ 377, PE Biosystems, Foster City, CA) and analyzed (e.g., using DNASTAR sequence analysis software, DNASTAR Inc., Madison, WI).

5' and 3' flanking and junction sequences were confirmed using standard PCR methods. The 5' flanking and junction sequences can be identified using SEQ ID NO:8 or SEQ ID NO:12 in combination with SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:26. 3' flanking and junction sequences can be identified using SEQ ID NO: 11 or SEQ ID NO: 14 in combination with SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO:29. The PCR reaction system and amplification conditions are shown in Table 2 and Table 3. Those skilled in the art will understand that other primer sequences can also be used to identify flanking and junction sequences.

DNA sequencing of the PCR amplification products provided DNA that could be used to design other DNA molecules that could be used as primers and probes to identify maize plants or seeds derived from transgenic maize event DBN9501.

It is found that the nucleotides 1-384 of SEQ ID NO: 5 show the maize genome sequence at the left border flank (5' flanking sequence) of the transgenic maize event DBN9501 insertion sequence, and the nucleotides in SEQ ID NO: 5 Positions 7785-8559 show the maize genome sequence flanking the right border of the transgenic maize event DBN9501 insert (3' flanking sequence). The 5' junction sequence is set forth in SEQ ID NO:1 and the 3' junction sequence is set forth in SEQ ID NO:2.

3.3. PCR zygosity determination

Junction sequences are relatively short polynucleotide molecules that are novel DNA sequences that are diagnostic for the DNA of transgenic maize event DBN9501 when detected in a polynucleotide detection assay. The junction sequence in SEQ ID NO: 1 and SEQ ID NO: 2 is the insertion site of the transgene fragment in transgenic maize event DBN9501 and 11 polynucleotides on each side of the maize genomic DNA. Longer or shorter polynucleotide junction sequences can be selected from SEQ ID NO:3 or SEQ ID NO:4. Junction sequences (5' junction region SEQ ID NO: 1, and 3' junction region SEQ ID NO: 2) are useful in DNA detection methods as DNA probes or as DNA primer molecules. Junction sequences SEQ ID NO: 6 and SEQ ID NO: 7 are also novel DNA sequences in transgenic maize event DBN9501, which can also be used as DNA probes or as DNA primer molecules to detect the presence of transgenic maize event DBN9501 DNA. Said SEQ ID NO:6 (nucleotides 385-574 of SEQ ID NO:3) spanned the DBN10707 construct DNA sequence and the tNos transcription termination sequence, said SEQ ID NO:7 (the core of SEQ ID NO:4 Nucleotides 252-451) spanned the pr35S transcription initiation sequence and the DNA sequence of the DBN10707 construct.

In addition, an amplicon is generated by using at least one primer from SEQ ID NO:3 or SEQ ID NO:4, which when used in a PCR method generates a diagnostic amplicon of transgenic maize event DBN9501.

Specifically, a PCR amplification product comprising genomic DNA flanking the 5' end of the T-DNA insert sequence in the genome of plant material derived from transgenic maize event DBN9501 was generated from the 5' end of the transgene insert sequence. part. This PCR amplification product comprises SEQ ID NO:3. For PCR amplification, primer 5 (SEQ ID NO:8) was designed to hybridize to the genomic DNA sequence flanking the 5' end of the transgene insert, and its paired primer located at the tNos transcription termination sequence in the T-DNA insert 6 (SEQ ID NO: 9).

A PCR amplification product comprising a portion of the genomic DNA flanking the 3' end of the T-DNA insert in the genome of plant material derived from transgenic maize event DBN9501 was generated from the 3' end of the transgene insert. This PCR amplification product comprises SEQ ID NO:4. For PCR amplification, primer 7 (SEQ ID NO: 10) located at the pr35S transcription initiation sequence in the T-DNA insert was designed, and paired with it, hybridizes to the genomic DNA sequence flanking the 3' end of the transgene insert Primer 8 (SEQ ID NO: 11).

The DNA amplification conditions described in Tables 2 and 3 can be used in the PCR zygosity assay described above to generate diagnostic amplicons for transgenic maize event DBN9501. The detection of the amplicon can be carried out by using Stratagene Robocycler, MJEngine, Perkin-Elmer 9700 or Eppendorf Mastercycler Gradient thermocycler, etc., or by methods and equipment known to those skilled in the art.

Table 2. PCR steps and reaction mixture conditions for identification of the 5' transgene insert/genomic junction region of transgenic maize event DBN9501

Table 3. Thermal cycler amplification conditions

Mix gently, adding 1-2 drops of mineral oil on top of each reaction if there is no thermal cap on the thermal cycler. Cycling parameters in Table 3 were used in Stratagene Robocycler (Stratagene, La Jolla, CA), MJEngine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA) or Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) thermal cycler for PCR reactions. The MJ Engine or Eppendorf Mastercycler Gradient thermal cycler should be run in calculation mode. The Perkin-Elmer 9700 Thermal Cycler was run with the ramp speed set to the maximum value.

Experimental results show that: primers 5 and 6 (SEQ ID NO:8 and 9), when used in the PCR reaction of transgenic maize event DBN9501 genomic DNA, produce an amplification product of 768bp fragment, when it is used in non-transformed maize genome DNA and non-DBN9501 maize genomic DNA in the PCR reaction, no fragment was amplified; primers 7 and 8 (SEQ ID NO: 10 and 11), when used in the PCR reaction of transgenic maize event DBN9501 genomic DNA, produced The amplified product of the 1339 bp fragment, when it was used in a PCR reaction of non-transformed maize genomic DNA and non-DBN9501 maize genomic DNA, no fragment was amplified.

PCR zygosity assays can also be used to identify whether material derived from transgenic maize event DBN9501 is homozygous or heterozygous. Primer 9 (SEQ ID NO: 12), Primer 10 (SEQ ID NO: 13) and Primer 11 (SEQ ID NO: 14) were used in amplification reactions to generate diagnostic amplicons for transgenic maize event DBN9501. The DNA amplification conditions described in Tables 4 and 5 can be used in the zygosity assay described above to generate diagnostic amplicons for transgenic maize event DBN9501.

Table 4. Reaction solution for zygosity determination

Table 5. Thermal cycler amplification conditions for zygosity determination

Cycling parameters in Table 5 were used in Stratagene Robocycler (Stratagene, La Jolla, CA), MJEngine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA) or Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) thermal cycler for PCR reactions. The MJ Engine or Eppendorf Mastercycler Gradient thermal cycler should be run in calculation mode. The Perkin-Elmer 9700 Thermal Cycler was run with the ramp speed set to the maximum value.

In the amplification reaction, the biological sample containing template DNA contains DNA diagnostic for the presence of transgenic maize event DBN9501 in the sample. Or the amplification reaction will produce two different DNA amplicons from a biological sample containing DNA derived from the maize genome that corresponds to the allele corresponding to the inserted DNA present in transgenic maize event DBN9501 is heterozygous. These two distinct amplicons would correspond to the first amplicon (SEQ ID NO: 12 and SEQ ID NO: 14) derived from the wild-type maize genomic locus and the second amplicon diagnostic of the presence of transgenic maize event DBN9501 DNA. Two amplicons (SEQ ID NO: 12 and SEQ ID NO: 13). Generating only a single amplicon corresponding to the second amplicon described for the heterozygous genome, a maize DNA sample in which the presence of transgenic maize event DBN9501 can be diagnostically determined is generated relative to the presence of DBN9501 in transgenic maize plants The alleles corresponding to the inserted DNA are produced in maize seeds that are homozygous.

It should be noted that the primer pair for transgenic maize event DBN9501 was used to generate amplicons that were diagnostic for the genomic DNA of transgenic maize event DBN9501. These primer pairs include, but are not limited to, primers 5 and 6 (SEQ ID NO: 8 and 9), and primers 7 and 8 (SEQ ID NO: 10 and 11), for use in the DNA amplification method described. Additionally, a control primer 12 and 13 (SEQ ID NO: 22 and 23) for amplification of maize endogenous genes was included as an internal standard for reaction conditions. Analysis of DNA extract samples from GM corn event DBN9501 should include a positive tissue DNA extract control from GM corn event DBN9501, a negative DNA extract control from non-GM corn event DBN9501 and a corn DNA extract containing no template. Extract negative control. In addition to these primer pairs, it is also possible to use any primer pair from SEQ ID NO:3 or its complement, or SEQ ID NO:4 or its complement, which when used in a DNA amplification reaction yields a Tissue from the transgenic event maize plant DBN9501 was diagnostic for comprising the amplicon of SEQ ID NO: 1 or SEQ ID NO:2. The DNA amplification conditions described in Tables 2-5 can be used to generate diagnostic amplicons for transgenic maize event DBN9501 using appropriate primer pairs. Extracts of maize plant or seed DNA putatively containing transgenic maize event DBN9501, or products derived from transgenic maize event DBN9501, that yield a diagnostic amplicon for transgenic maize event DBN9501 when tested in a DNA amplification method may Used as a template for amplification to determine the presence or absence of transgenic maize event DBN9501.

The fourth embodiment, detection of transgenic maize event DBN9501 by Southern blot hybridization

4.1. DNA extraction for Southern blot hybridization

Using a mortar and pestle, grind approximately 5-10 g of leaf tissue in liquid nitrogen. Resuspend 4-5 g of ground leaf tissue in 20 mL of CTAB Lysis Buffer (100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 0.2% v/v β-mercaptoethanol, 2% w/v CTAB) , and incubated at a temperature of 65°C for 60 min. During the incubation period, the samples were mixed by inversion every 10 min. After incubation, an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added, mixed gently by inversion for extraction, and centrifuged at 4000 rpm for 20 min. The aqueous phase was repeatedly extracted once with an equal volume of chloroform/isoamyl alcohol (24:1). After collecting the aqueous phase again, add an equal volume of isopropanol, mix well, and place at -20°C for 1 h to precipitate DNA, then centrifuge at 4000 rpm for 5 min to obtain the DNA precipitate, and then in 1 mL TE buffer (10 mM Tris-HCl, 1 mM EDTA , pH 8.0) to resuspend the DNA pellet. To degrade any RNA present, DNA was incubated with 40 μL of 10 mg/mL RNase A for 30 min at a temperature of 37 ° C, centrifuged at 4000 rpm for 5 min, and 3 M sodium acetate (pH 5.2) at a concentration of 0.1 times volume and 2 times volume In the presence of absolute ethanol, centrifuge at 12,000 rpm for 10 min to precipitate DNA. After discarding the supernatant, the pellet was washed with 1 mL of 70% (v/v) ethanol, dried at room temperature, and the DNA was redissolved in 1 mL of TE buffer.

4.2. Restriction enzyme digestion

The genomic DNA concentration of the above samples was measured with an ultramicro spectrophotometer (NanoDrop 2000, Thermo Scientific).

In a 100 μL reaction system, 5 μg of DNA was digested each time, genomic DNA was digested with restriction endonucleases Nco I and Nde I respectively, and partial sequences of Vip3Aa19 gene and pat gene on T-DNA were used as probes. For each enzyme, digests were incubated overnight at the appropriate temperature. The samples were spun down to reduce the volume to 20 μL using a speed vacuum, ThermoScientific.

4.3. Gel electrophoresis

Bromophenol blue loading buffer was added to each sample derived from Example 4.2, and each sample was added to a 0.7% TAE agarose gel containing ethidium bromide, and the TAE electrophoresis buffer ( 40mM Tris-acetic acid, 2mM EDTA, pH 8.5) were separated by electrophoresis, and the gel was electrophoresed overnight at a voltage of 20V.

After electrophoresis, treat the gel with 0.25M HCl for 10min to depurinate the DNA, and then use denaturing solution (1.5M NaCl, 0.5M NaOH) and neutralizing solution (1.5M NaCl, 0.5M Tris-HCl, pH 7.2) respectively Gels were processed for 30 min each. Pour 5×SSC (3M NaCl, 0.3M sodium citrate, pH 7.0) into a porcelain dish, put a glass plate on it, and then put the soaked filter paper bridge, gel, positively charged nylon membrane (Roche, Cat.No. 11417240001), three filter papers, paper tower, weight. After transferring the membrane overnight at room temperature, the nylon membrane was rinsed twice in deionized water, and the DNA was immobilized on the membrane by an ultraviolet crosslinker (UVP, UVCrosslinker CL-1000).

4.4 Hybridization

Appropriate DNA sequences were amplified by PCR for probe preparation. The DNA probe is SEQ ID NO: 24 or SEQ ID NO: 25, or is partially homologous or complementary to the above sequence. Use the DNA Labeling and Detection Starter Kit II kit (Roche, Cat. No. 11585614910) for DIG labeling of probes, Southern blot hybridization, membrane washing and other operations. For specific methods, refer to its product manual. Finally, X-ray film (Roche, Cat. No. 11666916001) was used to detect the binding position of the probe.

Two control samples were included on each Southern: (1) DNA from negative (untransformed) segregants, which was used to identify any endogenous maize sequences hybridizable to element-specific probes; (2) DNA from negative (untransformed) segregants; The DNA of the segregant, into which the Nco I-digested DBN10707 plasmid was introduced, in an amount equivalent to one copy number based on the probe length, served as a positive control for hybridization and was used to illustrate the sensitivity of the experiment.

Hybridization data provided corroborating evidence in support of TaqMan ^™ PCR analysis that maize plant DBN9501 contained a single copy of the Vip3Aa19 gene and the pat gene. Using the Vip3Aa19 gene probe, Nco I and Ned I enzymolysis produced single bands of about 11 kb and 9 kb in size; A single band, indicating that one copy each of the Vip3Aa19 gene and the pat gene is present in maize plant DBN9501. In addition, for the backbone probe, no hybridization band was obtained, indicating that no backbone sequence of the DBN10707 vector entered the genome of the maize plant DBN9501 during the transformation process.

Insect resistance detection of the fifth embodiment, event

5.1. Bioassay of corn plant DBN9501

The transgenic corn event DBN9501 and wild-type corn plants (non-transgenic, NGM) 2 planting plants were respectively resistant to small cutworm (Agrotis ypsilon Rottemberg, BCW), Spodoptera litura (TCW), beet armyworm (Spodoptera exigua, BAW ), Sesamia inferens (PSB), sorghum borer (Chilosacchariphagus, SGB) and millet moth (Chilo infuscatellus, MSB) were bioassayed as follows:

The fresh leaves (V3-V4 period) of transgenic corn event DBN9501 and wild-type corn plants (non-transgenic, NGM) were taken respectively, rinsed with sterile water, and the water on the leaves was blotted dry with gauze, and then the corn leaves were Remove the veins, cut into strips of about 1cm × 3cm at the same time, get 1-3 pieces (determining the number of leaves according to the food intake of insects) and put the cut long strip leaves on the filter paper at the bottom of the circular plastic culture dish. Wet with distilled water, put 5-10 artificially reared newly hatched larvae in each petri dish, after the worm test petri dish is covered, at temperature 26-28 ℃, relative humidity 70%-80%, photoperiod (light/ Dark) under the condition of 16:8, the results were counted after 3 days. The three indicators of larval development progress, mortality rate and leaf damage rate were counted to obtain the total resistance score (full score: 300 points): total resistance score=100×mortality rate+[100×mortality rate+90×(number of newly hatched worms/ number of inoculated insects)+60×(initial hatch-negative control insects/inoculated insects)+10×(negative control insects/inoculated insects)]+100×(1-leaf damage rate). Among them, the number of inoculated insects refers to the number of inoculated insects, that is, 5 or 10 per dish (depending on the feeding amount of pests); the progress of larval development has been reflected by the total score formula of resistance; the leaf damage rate refers to the number of insects taken by pests. The ratio of the leaf area eaten to the total leaf area. Five plants were selected from transgenic corn event DBN9501 and wild-type corn plants (non-transgenic, NGM) for testing, and each plant was repeated 6 times. The results are shown in Table 6 and Table 7.

Table 6. Bioassay results of insect resistance in transgenic corn event DBN9501-death rate (%)

Table 7. Insect resistance bioassay results of transgenic corn event DBN9501 - total resistance score (points)

The results showed that the transgenic maize event DBN9501 had good resistance to cutworms, Spodoptera litura, Spodoptera litura, Sarmaea moth, Sorghum moth and Millet arborescens, and the mortality and resistance of the transgenic maize event DBN9501 were relatively high. The total score of sex was significantly higher than that of NGM.

5.2 Field effects of the transgenic corn event DBN9501

The seeds of the transgenic corn event DBN9501 and wild-type corn plants (non-transgenic, NGM) were set as 2 treatments, each treatment was designed as a random block, repeated 3 times, and the area of the plot was 30m ² (5m×6m) , the row spacing is 60cm, the plant spacing is 25cm, conventional cultivation management, and no pesticides are sprayed during the whole growth period. There is a distance of 2m between different insect inoculation test plots to avoid the spread of insects between different plots. (1) Small cutworm

Only in areas where the natural occurrence of cutworms is relatively serious (natural pest occurrence conditions: the first generation of larvae are the first generation of cutworms, and it is easy to occur when the environmental conditions are suitable, such as the temperature is 16-26 ℃, relatively The humidity is 80-90%, and the soil moisture content is 15-20%; in addition, when the first-generation adults are lured to a certain amount, such as more than 20). At the corn seedling stage, when the material grows to about V2-V3, the corn plant develops to the 2-3 leaf stage, and begins to track and investigate whether plant wilting occurs in NGM. When wilting occurs near the rhizosphere of NGM plants, it is mostly 4-6 years old. When larvae were infested, the damage rate of cutworms to corn plants was investigated plant by plant (damage rate=number of corn plants eaten by pests/total number of plants×100%). The resistance results of the transgenic corn event DBN9501 to the cutworm are shown in Table 8.

Table 8. Results of resistance to cutworms of transgenic corn event DBN9501 under natural insect-susceptible conditions

The results showed that under the condition of natural occurrence of cutworms, compared with NGM, the damage rate of cutworms to transgenic corn event DBN9501 was significantly reduced, which indicated that the transgenic corn event DBN9501 had better resistance to cutworms, and the transgenic corn event DBN9501 had better resistance to cutworms. The field effect of the corn event DBN9501 under the condition of natural occurrence of cutworms is shown in Figure 3.

(2) Cotton bollworm (Helicoverpa armigera Hubner, CBW)

Carry out artificial inoculation at the corn silking stage, inoculate 2 times, artificially inoculate no less than 40 plants in each plot, and inoculate about 20 artificially raised newly hatched larvae in each corn silk, after 3 days after inoculation, the second The number of infestation is the same as the first time. After 14-21 days of inoculation, the ear damage rate, the number of surviving larvae per ear, and the length of ear damage were investigated plant by plant. Usually, the investigation starts 14 days after inoculation. If the damage level of NGM reaches a certain or high level, it is considered effective. If it does not reach the level, the investigation can be postponed appropriately. deemed invalid. According to the female ear damage rate, surviving larvae number, female ear damage length (cm), calculate the average value of the damage level of the ear stage cotton bollworm in each plot, the judgment standard is as shown in table 9, and then judge by the standard of table 10 The level of resistance to cotton bollworm at ear stage of corn. Table 11 shows the results of transgenic corn event DBN9501 resistance to cotton bollworm at silking stage.

Table 9. Grading standards for the degree of damage to corn ears by cotton bollworms

ear damage level symptom description 0 ear not injured 1 Only the filaments are killed 2 Spike top killed 1cm 3+ For every 1cm increase in the damage under the spike top, the corresponding damage level will increase by 1 …N

Table 10. Evaluation criteria for the resistance of corn ears to cotton bollworm

The average level of ear damage resistance level 0-1.0 High resistance (HR) 1.1-3.0 Anti (R) 3.1-5.0 Moderate resistance (MR) 5.1-7.0 Sense (S) ≥7.1 High Sensitivity (HS)

Table 11. Resistance results of transgenic corn event DBN9501 to cotton bollworm at silking stage

The results showed that under the condition of artificial inoculation, the ear damage rate, number of surviving larvae, length of ear damage and ear damage level of the transgenic maize event DBN9501 were significantly lower than those of NGM, which indicated that the transgenic maize event DBN9501 was resistant to cotton bollworm. The resistance level is at the level of resistance (R), and the field effect of the transgenic corn event DBN9501 inoculated with cotton bollworm is shown in Figure 4.

(3) Spodoptera litura

Only in the areas where Spodoptera litura occurs naturally are relatively serious (natural pest occurrence conditions: the peak period of damage is from July to September, and the optimum temperature for pest development is 28-30°C). 10-15 days after the first occurrence of insect infestation, and when NGM is mostly 4-6 instar larvae, the damage rate of Spodoptera litura to corn plants was investigated plant by plant (damage rate=the number of corn plants eaten by pests/total plant number ×100%). Table 12 shows the resistance results of the transgenic maize event DBN9501 to Spodoptera litura.

Table 12. The results of resistance to Spodoptera litura of transgenic corn event DBN9501 under natural insect-susceptible conditions

The results showed that under the condition of natural occurrence of Spodoptera litura, compared with NGM, the damage rate of Spodoptera litura to transgenic corn event DBN9501 was significantly reduced, which indicated that the transgenic corn event DBN9501 had better resistance to Spodoptera litura, and the transgenic corn event DBN9501 had better resistance to Spodoptera litura. The field effect of maize event DBN9501 under the condition of natural occurrence of Spodoptera litura is shown in Figure 5.

(4) Beet armyworm

The experimental design and experimental method were essentially the same as described above for the evaluation of Spodoptera litura resistance. The difference is that after 10-15 days of the first discovery of insect damage, and when NGM is mostly 4-5 instar larvae, the damage rate of beet armyworm to corn plants is investigated plant by plant (damage rate=the number of corn plants eaten by pests) /total number of plants×100%). Table 13 shows the resistance results of the transgenic corn event DBN9501 to beet armyworm.

Table 13. The resistance results of the transgenic corn event DBN9501 to beet armyworm under natural insect-susceptible conditions

The results showed that under the natural occurrence conditions of beet armyworm, compared with NGM, the damage rate of beet armyworm to transgenic corn event DBN9501 was significantly reduced, which indicated that the transgenic corn event DBN9501 had better resistance to beet armyworm. The field effect of corn event DBN9501 under natural occurrence conditions of beet armyworm is shown in Figure 6.

It is particularly worth mentioning that, according to the contents recorded in Chinese Patent (Application) Nos. 201310289848.6, 201310573441.6, 201410806573.3, 201510259396.6, 201610006375.8, as well as the field efficacy and biological assay results of the transgenic corn event DBN9501 of this application, it shows that The transgenic corn event DBN9501 realizes the method and/or use of controlling pests, specifically the large borer, Spodoptera litura, Pepper borer, Sorghum barborer and Peach borer; that is, any transgenic corn plant expressing Vip3Aa19 protein can be controlled A method and/or use of pests of S. chinensis, Spodoptera litura, D. sorghum borer, Sorghum sliver borer and/or Peach borer.

Sixth embodiment, detection of herbicide tolerance of events

In this experiment, Basta herbicide (glufosinate-ammonium saline solution with 18% active ingredient) was selected for spraying. A random block design was adopted with 3 repetitions. The area of the plot is 15m ² (5m×3m), the row spacing is 60cm, the plant spacing is 25cm, conventional cultivation management, and there is a 1m wide isolation zone between the plots. The transgenic corn event DBN9501 was subjected to the following two treatments: (1) no spraying, and (2) spraying herbicides while spraying an equal volume of water; (2) 800g ai/ha (ai/ha refers to " Spray Baoshida herbicide at the V2-V3 leaf stage at the active ingredient per hectare") dosage. It should be noted that glufosinate-ammonium herbicides (such as Basta) are contact-killing herbicides. If they are used improperly in the field, or if there is too much liquid accumulated locally, phytotoxicity may appear, and it is not a problem with the tolerance of the genetically modified corn event DBN9501. The conversion of glufosinate-ammonium herbicides with different contents and formulations into the above-mentioned equivalent active ingredient glufosinate-ammonium is applicable to the following conclusions.

Investigate the symptoms of phytotoxicity 1 week and 2 weeks after the application, and measure the output of the plot when harvesting; the grading of phytotoxicity symptoms is shown in Table 14. The herbicide damage rate is used as an index to evaluate the herbicide tolerance of the transformation event, specifically, the herbicide damage rate (%)=∑(number of injured plants at the same level×number of levels)/(total number of plants×highest level); The injury rate of glufosinate refers to the injury rate of glufosinate-ammonium. The injury rate of glufosinate-ammonium is determined according to the results of the phytotoxicity survey 2 weeks after the treatment of glufosinate-ammonium. tolerance level. The corn yield of each plot is the total yield (weight) of corn kernels in the middle 3 rows of each plot, and the yield difference between different treatments is measured in the form of yield percentage, yield percentage (%)=sprayed yield/no spraying Yield. The results of transgenic corn event DBN9501 on herbicide tolerance and corn yield are shown in Table 15.

Table 14. Grading standards for glufosinate-ammonium herbicide damage to corn

Phytotoxicity level symptom description 1 Normal growth without any symptoms of injury 2 Slight phytotoxicity, phytotoxicity less than 10% 3 Moderate phytotoxicity, can recover later, does not affect yield 4 The drug damage is heavy, it is difficult to recover, resulting in reduced production 5 The phytotoxicity is serious and cannot be recovered, resulting in obvious production reduction or cessation of production

Table 15. Results of transgenic corn event DBN9501 tolerance to glufosinate-ammonium herbicide and corn yield results

The results show that, in terms of the damage rate of glufosinate-ammonium herbicide: the damage rate of transgenic corn event DBN9501 is 0 under the treatment of glufosinate-ammonium herbicide (800g a.i./ha); drug tolerance.

In terms of yield: the yield of transgenic corn event DBN9501 had no significant difference between no spraying and spraying 800g a.i./ha glufosinate-ammonium treatments, and the yield of transgenic corn event DBN9501 increased slightly after spraying glufosinate-ammonium herbicide , thus further indicating that the transgenic maize event DBN9501 has good tolerance to glufosinate-ammonium herbicide.

Seventh embodiment

Agricultural products or commodities such as can be produced from transgenic maize event DBN9501. If a sufficient amount of expression is detected in said agricultural product or commodity, said agricultural product or commodity is expected to contain a nucleotide sequence capable of diagnosing the presence of transgenic maize event DBN9501 material in said agricultural product or commodity. Such agricultural products or commodities include, but are not limited to, corn oil, corn meal, cornmeal, corn gluten, corn tortillas, cornstarch, and any other food product that is to be consumed by an animal as a food source, or otherwise as a bulking agent or in a cosmetic composition The ingredients are used for cosmetic purposes etc. Nucleic acid detection methods and/or kits based on probes or primer pairs can be developed to detect the nucleotide sequence derived from transgenic maize event DBN9501 such as shown in SEQ ID NO: 1 or SEQ ID NO: 2 in biological samples, wherein Probe sequence or primer sequence is selected from the sequence shown in as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 or part thereof, to diagnose transgenic maize The existence of event DBN9501.

To sum up, the transgenic corn event DBN9501 of the present invention has good resistance to Lepidoptera insects, and has high tolerance to glufosinate-ammonium herbicide, has no effect on yield, and the detection method can be accurately and quickly Identification of DNA molecules from transgenic maize event DBN9501 in biological samples.

The seeds corresponding to the genetically modified corn event DBN9501 have been deposited in the General Microbiology Center of the China Committee for Microbial Culture Collection (CGMCC for short) according to the Budapest Treaty on January 23, 2019. Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip code 100101), classification name: corn (Zea mays), preservation number is CGMCC No.17099. The deposit will be kept in the depository for 30 years.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation, although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be The scheme shall be modified or equivalently replaced without departing from the spirit and scope of the technical scheme of the present invention.

sequence listing

<110> Beijing Dabeinong Biotechnology Co., Ltd.

<120> Nucleic acid sequence and detection method for detecting corn plant DBN9501

<130> DBNBC145

<160> 31

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212>DNA

<213> A sequence of 22 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in DBN9501 (Artificial Sequence)

<400> 1

ggggaaaagt taataacaca tt 22

<210> 2

<211> 22

<212>DNA

<213> A sequence of 22 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in DBN9501 (Artificial Sequence)

<400> 2

gtttaaacta tcagtgtaac ta 22

<210> 3

<211> 768

<212>DNA

<213> A sequence of 768 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in DBN9501 (Artificial Sequence)

<400> 3

tgtccggcat acctgttcat catttaaata atctaacacg gagtgtcctt tcttgagtga 60

cgcgccaaac ggtgtacttc tgttgtttct tgagtgacat aaaatggtgt acttctatta 120

cctaaggcat aattctaatt tataaagaga aggtaataca gccacaaatt agtaacatct 180

aagcaaatac agcactgctt ctggaaattt tatcatctat attcagtaag ctaaacccag 240

ccactattaa gtttcatgat tttatcatat gaaaaaaaaa tccaaagaaa ccatacggtt 300

atcaaaaact tagaaaaaca ggatgccttt ttgccaaagg gatttgatag acctttattt 360

ttcaggaaaaacaggggaaaagttaataacattgcgga tacggccagg cgcgtccctg 420

ttaacgtcct aactagctaa actaggtaca gattgcgagg ctcacgaggc gatcctggcc 480

gcgtgacagt cgcgtgcgag gctcttgact aagtaggcgg ccgcgtgcac ttaattaaga 540

attccctgca gggatctagt aacatagatg acaccgcgcg cgataattta tcctagtttg 600

cgcgctatat tttgttttct atcgcgtatt aaatgtataa ttgcgggact ctaatcataa 660

aaaccccatct cataaataac gtcatgcatt acatgttaat tattacatgc ttaacgtaat 720

tcaacagaaa ttatatgata atcatcgcaa gaccggcaac aggattca 768

<210> 4

<211> 1339

<212>DNA

<213> A sequence of 1339 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in DBN9501 (Artificial Sequence)

<400> 4

caatcggacc atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt 60

ccacgatgct cctcgtgggt ggggtccat ctttgggacc actgtcggca gaggcatctt 120

caacgatggc ctttccttta tcgcaatgat ggcatttgta ggagccacct tccttttcca 180

ctatcttcac aataaagtga cagatagctg ggcaatggaa tccgaggagg tttccggata 240

ttaccctttg ttgaaaagtc tcaatcggac caagcttatt taaatggtac cttaattaag 300

tgcacgttta aactacctag tcagtgccgt tgagagcgta gctgcgactt agcggcctcg 360

tctgcgaagt cggtgaggct agtgccacta attagtcatt agtttaatac aaatccacct 420

gcggccaatt cctgcagcgt tgcggttctg tcagttccaa acgtaaaacg gcttgtcccg 480

cgtcatcggc gggggtcata acgtgactcc cttaattctc cgctcatgat cagattgtcg 540

tttcccgcct tcagtttaaa ctatcagtgt aactataaat ggtatagtag tccggatttt 600

gtttccgaat aaagaaatgt tcaatgtatt tgaaatgatc aatagatact caaattatat 660

gattagcaag cttaaataga gttttttttt caggtccatt tcttttcctt agattacaga 720

agcaccataa cacattccaa aagtaattgc atgcacccaa gaatctgcta atgttccaag 780

tgttgaaaac aaccaaaact gtacttatca cttaatgaaa tacatggcac aaacaaaaaa 840

aaaaactgaa tatgcctgcg aacatagcag aagtggttgt catgtactag aaacattatt 900

gtgtcttcga acaagctgct catataatgg aagatggttc tttgcaacaa aaaccctgaa 960

cctgtaaagc aaatgagaag gacaattaag cataaataaa ggtgcaataa cacaagggaa 1020

caaaccacta aaccaatgtt ttaaagttgc ccgactaatc gtgattactc aaccttattg 1080

cagcacgatt aggattaggc tgtacgacac gactagggtg attagaactc ggatcgtctg 1140

actaatcatg attaaacggt gattagtcat cggactagga gttcaaccca ctaacctgag 1200

ccgcctgact ttttttttgg gccggaccac agtgggtaag gtttttttat tttctcttga 1260

caaaaccctg tctgccctag gatacgaacg tatcatgtac ctacagccga cggctccagt 1320

tcgcggcttt ccctcctatc 1339

<210> 5

<211> 8559

<212>DNA

<213> Entire T-DNA sequence, 5' and 3' end maize genome flanking sequence (Artificial Sequence)

<400> 5

tgtccggcat acctgttcat catttaaata atctaacacg gagtgtcctt tcttgagtga 60

cgcgccaaac ggtgtacttc tgttgtttct tgagtgacat aaaatggtgt acttctatta 120

cctaaggcat aattctaatt tataaagaga aggtaataca gccacaaatt agtaacatct 180

aagcaaatac agcactgctt ctggaaattt tatcatctat attcagtaag ctaaacccag 240

ccactattaa gtttcatgat tttatcatat gaaaaaaaaa tccaaagaaa ccatacggtt 300

atcaaaaact tagaaaaaca ggatgccttt ttgccaaagg gatttgatag acctttattt 360

ttcaggaaaaacaggggaaaagttaataacattgcgga tacggccagg cgcgtccctg 420

ttaacgtcct aactagctaa actaggtaca gattgcgagg ctcacgaggc gatcctggcc 480

gcgtgacagt cgcgtgcgag gctcttgact aagtaggcgg ccgcgtgcac ttaattaaga 540

attccctgca gggatctagt aacatagatg acaccgcgcg cgataattta tcctagtttg 600

cgcgctatat tttgttttct atcgcgtatt aaatgtataa ttgcgggact ctaatcataa 660

aaaccccatct cataaataac gtcatgcatt acatgttaat tattacatgc ttaacgtaat 720

tcaacagaaa ttatatgata atcatcgcaa gaccggcaac aggattcaat cttaagaaac 780

tttattgcca aatgtttgaa cgatcactag ttcagatctg ggtaactggc ctaactggcc 840

ttggagggagc tggcaactca aaatcccttt gccaaaaacc aacatcatgc catccaccat 900

gcttgtatcc agctgcgcgc aatgtaccccc gggctgtgta tcccaaagcc tcatgcaacc 960

taacagatgg atcgtttgga aggcctataa cagcaaccac agacttaaaa ccttgcgcct 1020

ccatagactt aagcaaatgt gtgtacaatg tggatcctag gcccaacctt tgatgcctat 1080

gtgacacgta aacagtactc tcaactgtcc aatcgtaagc gttcctagcc ttccagggcc 1140

cagcgtaagc aataccagcc acaacaccct caacctcagc aaccaaccaa gggtatctat 1200

cttgcaacct ctctagatca tcaatccact cttgtggtgt ttgtggctct gtcctaaagt 1260

tcactgtaga cgtctcaatg taatggttaa cgatatcaca aaccgcggcc atatcagctg 1320

ctgtagctgg cctaatctca actggtctcc tctccggaga catggtaccc tgcagaagta 1380

acaccaaaca acagggtgag catcgacaaa agaaacagta ccaagcaaat aaatagcgta 1440

tgaaggcagg gctaaaaaaa tccacatata gctgctgcat atgccatcat ccaagtatat 1500

caagatcaaa ataattataa aacatacttg tttattataa tagataggta ctcaaggtta 1560

gagcatatga atagatgctg catatgccat catgtatatg catcagtaaa acccacatca 1620

acatgtatac ctatcctaga tcgatatttc catccatctt aaactcgtaa ctatgaagat 1680

gtatgacaca cacatacagt tccaaaatta ataaatacac caggtagttt gaaacagtat 1740

tctactccga tctagaacga atgaacgacc gcccaaccac accacatcat cacaaccaag 1800

cgaacaaaaa gcatctctgt atatgcatca gtaaaacccg catcaacatg tatacctatc 1860

ctagatcgat atttccatcc atcatcttca attcgtaact atgaatatgt atggcacaca 1920

catacagatc caaaattaat aaatccacca ggtagtttga aacagaattc tactccgatc 1980

tagaacgacc gcccaaccag accacatcat cacaaccaag acaaaaaaaa gcatgaaaag 2040

atgacccgac aaacaagtgc acggcatata ttgaaataaa ggaaaagggc aaaccaaacc 2100

ctatgcaacg aaacaaaaaa aatcatgaaa tcgatcccgt ctgcggaacg gctagagcca 2160

tcccaggatt ccccaaagag aaacactggc aagttagcaa tcagaacgtg tctgacgtac 2220

aggtcgcatc cgtgtacgaa cgctagcagc acggatctaa cacaaacacg gatctaacac 2280

aaacatgaac agaagtagaa ctaccgggcc ctaaccatgg accggaacgc cgatctagag 2340

aaggtagaga gggggggggg gggaggacga gcggcgtacc ttgaagcgga ggtgccgacg 2400

ggtggatttg ggggagatct ggttgtgtgtgtgtgtgcgctc cgaacaacac gaggttgggg 2460

aaagagggtg tggagggggt gtctatttat tacggcgggc gaggaaggga aagcgaagga 2520

gcggtgggaa aggaatcccc cgtagctgcc ggtgccgtga gaggaggagg aggccgcctg 2580

ccgtgccggc tcacgtctgc cgctccgcca cgcaatttct ggatgccgac agcggagcaa 2640

gtccaacggt ggagcggaac tctcgagagg ggtccagagg cagcgacaga gatgccgtgc 2700

cgtctgcttc gcttggcccg acgcgacgct gctggttcgc tggttggtgt ccgttagact 2760

cgtcgacggc gtttaacagg ctggcattat ctactcgaaa caagaaaaat gtttccttag 2820

tttttttaat ttcttaaagg gtatttgttt aatttttagt cactttattttattctattt 2880

tatatctaaa ttattaaata aaaaaactaa aatagagttt tagttttctt aatttagagg 2940

ctaaaataga ataaaataga tgtactaaaa aaattagtct ataaaaacca ttaaccctaa 3000

accctaaatg gatgtactaa taaaatggat gaagtattat ataggtgaag ctatttgcaa 3060

aaaaaaagga gaacacatgc acactaaaaa gataaaactg tagagtcctg ttgtcaaaat 3120

actcaattgt cctttagacc atgtctaact gttcatttat atgattctct aaaacactga 3180

tattattgta gtactataga ttatattatt cgtagagtaa agtttaaata tatgtataaa 3240

gatagataaa ctgcacttca aacaagtgtg acaaaaaaaa tatgtggtaa ttttttataa 3300

cttagacatg caatgctcat tatctctaga gaggggcacg accgggtcac gctgcactgc 3360

aggccctagg atttaagtga ctagggtcac gtgactctag tcacttactg gcgcgccgat 3420

ctagtaacat agatgacacc gcgcgcgata atttatccta gtttgcgcgc tatattttgt 3480

tttctatcgc gtattaaatg tataattgcg ggactctaat cataaaaacc catctcataa 3540

ataacgtcat gcattacatg ttaattatta catgcttaac gtaattcaac agaaattata 3600

tgataatcat cgcaagaccg gcaacaggat tcaatcttaa gaaactttat tgccaaatgt 3660

ttgaacgatc tcacttgatg ctcacgtcgt aaaaatgaac aatggggccg ccgtagaggt 3720

tattcccttg ggacagttcg atataaaagt tgtccttctc aaacttcgtg gtgaacattt 3780

ctgacacatc cttagcgcca gacatgtatc tcttctcgaa gaggacttcc cgcgagttcc 3840

tgatgcgcac attcgcgtcg ccggaaactg aaaaatagac tctgtaggta gagaacgaat 3900

ccagttggag gttctgcttc agaatgcctc tcccgccctg gtaaagcgtc agggtgttcc 3960

ccgagatatt cgtgctgcca gtggatgtcc agttattggt gttaatcagc tcggggctca 4020

ggagcttctc gctaggggag atttcgagaa tgatgaagtt gtcgccccag gcctcatcgc 4080

cattctggga cttgagaata agatagaccc ccttcaggtc agtgccagtt gtgaacctct 4140

tattgattgt ctggtagtcc tccaggttgt tgttagtatc ttcgtaatgg atgtaccctg 4200

tgttctcatc cttgaggtgg atgcttggct tgcccttcac agtatattga atcacgtatt 4260

ctgtcttcgg cttcagcttg tcgccaatga actgggagat gccaccatcc ttatgcacat 4320

agagcgcctt agtgccattc accccgcctg tgtggtcaac gtatgcgttc ttatgttag 4380

ccttccacgg ttccagatta tcttcctcaa ttgacccgtt ctccacaata ttggagatga 4440

agcctgatgg cggaacgatc agcttcgttt ccttgttaga gaggtcggtg gcaagcagca 4500

gctccctgag gtagctcttg cacgtaaggg tgatcaggcg ggagttctca tctgcctgga 4560

gcccaaagcc attgatgggc gtgaggaagg tctcagaaat gaccccaaga ggcatataca 4620

cgccatcgtc gttcgccgac agggttctgt attcggcctc ggatgattcg accttcttct 4680

tgttgaggtc gatctcgccc gtagacgaat catagaagtt agccgtcacc tcgtaccgga 4740

gtgtcttcat cttcttcgta aagtcaatct tggtgatcac gtattcgttg gggaagacaa 4800

tattgttcgt atagtagatt tgctcggact gatcagggca aagcagctta tccatgtcgc 4860

cgtagataac ctctgacaga gaatccttgt ccacttgata attctgcttg agcttcgcct 4920

cgtagacctt cagcacggta attgagtcgt tagaaatctc gaacccgatg agcgcatggc 4980

caggcttggc ctcaacgatc atctttgcat cttcgtcgga gcccttgacc ttagcgtagt 5040

ttggattaga aaaagtgttc gagagtgtcg gaaggatatt cacgcggaac tcctccttct 5100

ccttgttcag gtgctcgttc atgatggagg tgtaatcgat gtctgcgagg ccgaggagct 5160

tgcggcaggt ggtcagcgtg agaaacgctt gggcctggag tgcggtgagg acgataagga 5220

agttgtaaac attcccgacc tccgagccgc tcgtcttaac gttctccttg gtgatcagtt 5280

ccgatgcagt cttgagagcg ctccgcccaa aaagattgtt gcccaccata acgtcgtgga 5340

acgtgttcag gtaaaactcg aagccatcga cgtcattctt ggtcacggac ttcgccagct 5400

cagtgagttc tgtaagctca tccaggatgt cggctggtga gccatccttc ttgaccttgc 5460

tggaagtttc tgtagcaaaa gtgagttcct cgaacttctc attcacatac ttgatcctct 5520

ggtacgccgg tgtaatctcc gtcagggtgc tgttgatgag gacgttcaca ttaatgatat 5580

ccagcttgtc cgagatttct tgaagctgct tgctcagata ctcaatttga agggacagcg 5640

cgtagttctg cttcatgacg tccgagagca tgctagtaat ctttgggagg tacacgcgaa 5700

gcattgtgtt gatggcgtcg agcttattgt tcacatcatt aagaacttgg ttctgctcat 5760

ttgcaatctt gaggatctcc ttagacagtt ctgtgttgag attcccctga gcgatgaggt 5820

cgttaagtga cccattcacg ccgtccagct tgccagagat atcgttcagg agttgctgat 5880

tcttaagaat ctcgtcgagc gtaagatccc cgccagtgtc tgtcttgaag atcatgttca 5940

taatgtcctt gatccccgta gcaaacccat agatgccatt aaagtagtca ataaaggagg 6000

gaagtgcccg tgtggagagc ttggtgttgt tcttgttcat actagtggcc gcttggtatc 6060

tgcattacaa tgaaatgagc aaagactatg tgagtaacac tggtcaacac tagggagaag 6120

gcatcgagca agatacgtat gtaaagagaa gcaatatagt gtcagttggt agatactaga 6180

taccatcagg aggtaaggag agcaacaaaa aggaaactct ttaatttttaa attttgttac 6240

aacaaacaag cagatcaatg catcaaaata ctgtcagtac ttatttcttc agacaacaat 6300

atttaaaaca agtgcatctg atcttgactt atggtcacaa taaaggagca gagataaaca 6360

tcaaaatttc gtcatttata tttatcctt caggcgttaa caatttaaca gcacacaaac 6420

aaaaacagaa taggaatatc taattttggc aaataataag ctctgcagac gaacaaatta 6480

ttatagtatc gcctataata tgaatcccta tactattgac ccatgtagta tgaagcctgt 6540

gcctaaatta acagcaaact tctgaatcca agtgccctat aacaccaaca tgtgcttaaa 6600

taaataccgc taagcaccaa attacacatt tctcgtattg ctgtgtaggt tctatcttcg 6660

tttcgtacta ccatgtccct atattttgct gctacaaagg acggcaagta atcagcacag 6720

gcagaacacg atttcagagt gtaattctag atccagctaa accactctca gcaatcacca 6780

cacaagagag cattcagaga aacgtggcag taacaaaggc agagggcgga gtgagcgcgt 6840

accgaagacg gttctgctag agtcagcttg tcagcgtgtc ctctccaaat gaaatgaact 6900

tccttatata gaggaagggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc 6960

agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 7020

cacgatgctc ctcgtgggtgggggtccatc tttgggacca ctgtcggcag aggcatcttc 7080

aacgatggcc tttcctttat cgcaatgatg gcatttgtag gagccacctt ccttttccac 7140

tatcttcaca ataaagtgac agatagctgg gcaatggaat ccgaggaggt ttccggatat 7200

taccctttgt tgaaaagtct caatcggacc atcacatcaa tccacttgct ttgaagacgt 7260

ggttggaacg tcttcttttt ccacgatgct cctcgtgggt gggggtccat ctttgggacc 7320

actgtcggca gaggcatctt caacgatggc ctttccttta tcgcaatgat ggcatttgta 7380

ggagccacct tccttttcca ctatcttcac aataaagtga cagatagctg ggcaatggaa 7440

tccgaggagg tttccggata ttaccctttg ttgaaaagtc tcaatcggac caagcttatt 7500

taaatggtac cttaattaag tgcacgttta aactacctag tcagtgccgt tgagagcgta 7560

gctgcgactt agcggcctcg tctgcgaagt cggtgaggct agtgccacta attagtcatt 7620

agtttaatac aaatccacct gcggccaatt cctgcagcgt tgcggttctg tcagttccaa 7680

acgtaaaacg gcttgtcccg cgtcatcggc gggggtcata acgtgactcc cttaattctc 7740

cgctcatgat cagattgtcg tttcccgcct tcagtttaaa ctatcagtgt aactataaat 7800

ggtatagtag tccggatttt gtttccgaat aaagaaatgt tcaatgtatt tgaaatgatc 7860

aatagatact caaattatat gattagcaag cttaaataga gttttttttt caggtccatt 7920

tcttttccttagattacaga agcaccataa cacattccaa aagtaattgc atgcacccaa 7980

gaatctgcta atgttccaag tgttgaaaac aaccaaaact gtacttatca cttaatgaaa 8040

tacatggcac aaacaaaaaa aaaaactgaa tatgcctgcg aacatagcag aagtggttgt 8100

catgtactag aaacattatt gtgtcttcga acaagctgct catataatgg aagatggttc 8160

tttgcaacaa aaaccctgaa cctgtaaagc aaatgagaag gacaattaag cataaataaa 8220

ggtgcaataa cacaagggaa caaaccacta aaccaatgtt ttaaagttgc ccgactaatc 8280

gtgattactc aaccttattg cagcacgatt aggattaggc tgtacgacac gactagggtg 8340

attagaactc ggatcgtctg actaatcatg attaaacggt gattagtcat cggactagga 8400

gttcaaccca ctaacctgag ccgcctgact ttttttttgg gccggaccac agtgggtaag 8460

gtttttttat tttctcttga caaaaccctg tctgccctag gatacgaacg tatcatgtac 8520

ctacagccga cggctccagt tcgcggcttt cctcctatc 8559

<210> 6

<211> 190

<212>DNA

<213> The sequence located on SEQ ID NO:3, spanning the left border region (LB and tNos transcription termination sequence Artificial Sequence)

<400> 6

aataacacat tgcggatacg gccaggcgcg tccctgttaa cgtcctaact agctaaacta 60

ggtacagatt gcgaggctca cgaggcgatc ctggccgcgt gacagtcgcg tgcgaggctc 120

ttgactaagt aggcggccgc gtgcacttaa ttaagaattc cctgcaggga tctagtaaca 180

tagatgacac 190

<210> 7

<211> 200

<212>DNA

<213> The sequence located on SEQ ID NO:4, spanning the pr35S transcription initiation sequence and the right border region (RBArtificial Sequence)

<400> 7

tgaaaagtct caatcggacc aagcttattt aaatggtacc ttaattaagt gcacgtttaa 60

actacctagt cagtgccgtt gagagcgtag ctgcgactta gcggcctcgt ctgcgaagtc 120

ggtgaggcta gtgccactaa ttagtcatta gtttaataca aatccacctg cggccaattc 180

ctgcagcgtt gcggttctgt 200

<210> 8

<211> 21

<212>DNA

<213> The first primer for amplifying SEQ ID NO:3 (Artificial Sequence)

<400> 8

tgtccggcat acctgttcat c 21

<210> 9

<211> 20

<212>DNA

<213> The second primer for amplifying SEQ ID NO:3 (Artificial Sequence)

<400> 9

tgaatcctgt tgccggtctt 20

<210> 10

<211> 20

<212>DNA

<213> First primer for amplifying SEQ ID NO:4 (Artificial Sequence)

<400> 10

gataggagga aagccgcgaa 20

<210> 11

<211> 21

<212>DNA

<213> The second primer (Artificial Sequence) for amplifying SEQ ID NO:4

<400> 11

caatcggacc atcacatcaa t 21

<210> 12

<211> 22

<212>DNA

<213> Primers on the 5' flanking genomic sequence (Artificial Sequence)

<400> 12

tgccaaaggg atttgataga cc 22

<210> 13

<211> 20

<212>DNA

<213> Primer (Artificial Sequence) on T-DNA paired with SEQ ID NO:12

<400> 13

gccgtatccg caatgtgtta 20

<210> 14

<211> 21

<212>DNA

<213> Primers on the 3' flanking genomic sequence, which paired with SEQ ID NO: 12 can detect whether the transgene is homozygous or heterozygous (Artificial Sequence)

<400> 14

ttttggaatg tgttatggtg c 21

<210> 15

<211> 20

<212>DNA

<213> Primer (Artificial Sequence) on T-DNA paired with SEQ ID NO:14

<400> 15

cgttgagagc gtagctgcga 20

<210> 16

<211> 21

<212>DNA

<213> Taqman detects the first primer of Vip3Aa19 gene (Artificial Sequence)

<400> 16

cgaatacaga accctgtcgg c 21

<210> 17

<211> 24

<212>DNA

<213> Taqman detects the second primer of Vip3Aa19 gene (Artificial Sequence)

<400> 17

cgtgaggaag gtctcagaaa tgac 24

<210> 18

<211> 27

<212>DNA

<213> Taqman probe for detection of Vip3Aa19 gene (Artificial Sequence)

<400> 18

cgacgatggc gtgtatatgc ctcttgg 27

<210> 19

<211> 22

<212>DNA

<213> Taqman detects the first primer of pat gene (Artificial Sequence)

<400> 19

gagggtgttg tggctggtat tg 22

<210> 20

<211> 23

<212>DNA

<213> Taqman detects the second primer of pat gene (Artificial Sequence)

<400> 20

tctcaactgt ccaatcgtaa gcg 23

<210> 21

<211> 25

<212>DNA

<213> Taqman probe for detection of pat gene (Artificial Sequence)

<400> 21

ccttacgctgg gccctggaag gctag 25

<210> 22

<211> 21

<212>DNA

<213> The first primer of maize endogenous gene SSIIb (Artificial Sequence)

<400> 22

cggtggatgc taaggctgat g 21

<210> 23

<211> 23

<212>DNA

<213> The second primer of maize endogenous gene SSIIb (Artificial Sequence)

<400> 23

aaagggccag gttcattatc ctc 23

<210> 24

<211> 348

<212>DNA

<213> Probe for Vip3Aa19 gene in Southern hybridization detection (Artificial Sequence)

<400> 24

tctcaagtcc cagaatggcg atgaggcctg gggcgacaac ttcatcattc tcgaaatctc 60

ccctagcgag aagctcctga gccccgagct gattaacaccc aataactgga catccactgg 120

cagcacgaat atctcgggga acaccctgac gctttaccag ggcggggagag gcattctgaa 180

gcagaacctc caactggatt cgttctctac ctacagagtc tatttttcag tttccggcga 240

cgcgaatgtg cgcatcagga actcgcggga agtcctcttc gagaagagat acatgtctgg 300

cgctaaggat gtgtcagaaa tgttcaccac gaagtttgag aaggacaa 348

<210> 25

<211> 310

<212>DNA

<213> Probe for pat gene in Southern hybridization detection (Artificial Sequence)

<400> 25

cagacttaaa accttgcgcc tccatagact taagcaaatg tgtgtacaat gtggatccta 60

ggcccaacct ttgatgccta tgtgacacgt aaacagtact ctcaactgtc caatcgtaag 120

cgttcctagc cttccagggc ccagcgtaag caataccagc cacaacaccc tcaacctcag 180

caaccaacca agggtatcta tcttgcaacc tctctagatc atcaatccac tcttgtggtg 240

tttgtggctc tgtcctaaag ttcactgtag acgtctcaat gtaatggtta acgatatcac 300

aaaccgcggc 310

<210> 26

<211> 20

<212>DNA

<213> Primer located on T-DNA, in the same direction as SEQ ID NO:13 (Artificial Sequence)

<400> 26

gcgcgcaaac taggataaat 20

<210> 27

<211> 20

<212>DNA

<213> Primer located on T-DNA, opposite to SEQ ID NO:13, used to obtain flanking sequence (ArtificialSequence)

<400> 27

ccagccacaa caccctcaac 20

<210> 28

<211> 22

<212>DNA

<213> Primer located on T-DNA, opposite to SEQ ID NO:13, used to obtain flanking sequence (ArtificialSequence)

<400> 28

gtggtgtttg tggctctgtc ct 22

<210> 29

<211> 22

<212>DNA

<213> Primer located on T-DNA, in the same direction as SEQ ID NO:15 (Artificial Sequence)

<400> 29

tgctttgaag acgtggttgg aa 22

<210> 30

<211> 20

<212>DNA

<213> Primer located on T-DNA, opposite to SEQ ID NO:15, used to obtain flanking sequence (ArtificialSequence)

<400> 30

ctgctccttt attgtgacca 20

<210> 31

<211> 20

<212>DNA

<213> Primer located on T-DNA, opposite to SEQ ID NO:15, used to obtain flanking sequence (ArtificialSequence)

<400> 31

tatcttgctc gatgccttct 20

Claims (21)

1. A nucleic acid molecule having the following nucleic acid sequence, characterized in that, the nucleic acid sequence comprises SEQ ID NO: 1 or its complementary sequence and/or SEQ ID NO: 2 or its complementary sequence, and the nucleic acid molecule is derived from The transgenic corn event DBN9501 was deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms in the form of seeds and under the deposit number CGMCC No. 17099. 2. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence comprises SEQ ID NO: 3 or its complement, and/or SEQ ID NO: 4 or its complement. 3. The nucleic acid molecule according to claim 2, wherein the nucleic acid sequence comprises SEQ ID NO: 5 or its complementary sequence. 4. A method for detecting the presence of DNA of transgenic corn event DBN9501 in a sample, characterized in that, comprising: contacting the sample to be detected with at least two primers for amplifying the target amplification product in a nucleic acid amplification reaction; performing a nucleic acid amplification reaction; and detecting the presence of said target amplification product; The target amplification product comprises the nucleic acid molecule according to any one of claims 1-3; The transgenic corn event DBN9501 is deposited in the General Microorganism Center of China Committee for the Collection of Microbial Cultures in the form of seeds with the deposit number CGMCC No. 17099. 5. the method for the DNA existence of transgenic corn event DBN9501 in the detection sample according to claim 4, is characterized in that, described target amplification product also comprises SEQ ID NO:6 or its complementary sequence, and/or SEQ ID NO :7 or its complement. 6. according to the method for the DNA existence of transgenic corn event DBN9501 in the detection sample described in claim 4 or 5, it is characterized in that, described two kinds of primers comprise SEQ ID NO:8 and SEQ ID NO:9 or SEQ ID NO: 10 and SEQ ID NO:11. 7. A method for detecting the presence of DNA of the transgenic corn event DBN9501 in a sample, comprising: contacting the sample to be detected with a probe comprising the nucleic acid molecule of any one of claims 1-3; Hybridizing the sample to be detected and the probe under stringent hybridization conditions; and Detecting the hybridization between the sample to be detected and the probe; The transgenic corn event DBN9501 is deposited in the General Microorganism Center of China Committee for the Collection of Microbial Cultures in the form of seeds with the deposit number CGMCC No. 17099. 8. the method for the DNA existence of transgenic maize event DBN9501 in the detection sample according to claim 7, is characterized in that, described probe also comprises SEQ ID NO:6 or its complementary sequence, and/or SEQ ID NO:7 or its complementary sequence. 9. The method for detecting the presence of DNA of transgenic maize event DBN9501 in a sample according to claim 7 or 8, characterized in that at least one of said probes is labeled with at least one fluorescent group. 10. A method for detecting the presence of DNA of the transgenic corn event DBN9501 in a sample, comprising: contacting the sample to be detected with a marker nucleic acid molecule comprising the nucleic acid molecule of any one of claims 1-3; Hybridizing the sample to be detected and the marker nucleic acid molecule under stringent hybridization conditions; Detecting the hybridization between the sample to be detected and the marker nucleic acid molecule, and then performing marker-assisted breeding analysis to determine that insect resistance and/or herbicide tolerance are genetically linked to the marker nucleic acid molecule; The transgenic corn event DBN9501 is deposited in the General Microorganism Center of China Committee for the Collection of Microbial Cultures in the form of seeds with the deposit number CGMCC No. 17099. 11. The method for detecting the presence of DNA of the transgenic maize event DBN9501 in a sample according to claim 10, wherein the marker nucleic acid molecule further comprises SEQ ID NO: 6-11 or its complementary sequence. 12. A DNA detection kit, characterized in that it comprises at least one DNA molecule, said DNA molecule comprising the nucleic acid molecule according to any one of claims 1-3, which can be used as a specific gene for the transgenic corn event DBN9501 or its progeny One of the DNA primers or probes; the transgenic maize event DBN9501 is deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee in the form of seeds and with the deposit number CGMCC No. 17099. 13. The DNA detection kit according to claim 12, wherein the DNA molecule further comprises SEQ ID NO: 6 or its complementary sequence, and/or SEQ ID NO: 7 or its complementary sequence. 14. A method of protecting a maize plant from attack by a target insect inhibited by a Vip3Aa protein, comprising providing in the diet of the target insect at least one transgenic maize plant cell, said transgenic maize plant cell sequentially in its genome Comprising SEQ ID NO:1, the 553-7491 nucleotide sequence of SEQ ID NO:5 and SEQ ID NO:2, or comprising the sequence shown in SEQ ID NO:5, the target insects that ingest the transgenic corn plant cells are inhibited The transgenic maize plants were further ingested. 15. A method for protecting corn plants from damage caused by herbicides or controlling weeds in a field where corn plants are grown, comprising applying an effective dose of glufosinate-ammonium herbicide to at least one transgenic corn In the field of plants, the transgenic maize plant sequentially comprises SEQ ID NO:1, the 553-7491 nucleotide sequence of SEQ ID NO:5 and SEQ ID NO:2 in its genome, or comprises the sequence shown in SEQ ID NO:5 The sequence of the transgenic maize plant is tolerant to glufosinate-ammonium herbicide. 16. A method of cultivating a corn plant resistant and/or tolerant to glufosinate-ammonium herbicides to the target insects suppressed by Vip3Aa protein, characterized in that, comprising: Plant at least one corn seed, the genome of the corn seed comprises sequentially SEQ ID NO:1, SEQ ID NO:5 553-7491 nucleotide sequence and SEQ ID NO:2, or the genome of the corn seed comprises The nucleic acid sequence shown in SEQ ID NO:5; growing the corn seeds into corn plants; Invading the corn plant with target insects and/or spraying the corn plant with an effective dose of glufosinate-ammonium herbicide, harvesting plants with reduced plant damage compared with other plants that do not have the nucleic acid sequence of the specific region; The nucleic acid sequence of the specific region is the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2. 17. the method for cultivating the target insect that Vip3Aa protein suppresses according to claim 16 has resistance and/or the method for the maize plant of tolerance glufosinate-ammonium herbicide, it is characterized in that, the nucleotide sequence of described specific region is SEQ ID NO:3 and/or the sequence shown in SEQ ID NO:4. 18. A method for producing a corn plant resistant to the target insects inhibited by the Vip3Aa protein and/or glufosinate-ammonium herbicide tolerant, comprising the steps of sequentially comprising SEQ in the first corn plant genome ID NO:1, the 553-7491 nucleotide sequence of SEQ ID NO:5 and the nucleotide sequence of SEQ ID NO:2, or the nucleotide sequence shown in SEQ ID NO:5 contained in the first corn plant genome, Introducing a second maize plant to generate a large number of progeny plants; selecting the progeny plants with the nucleic acid sequence of a specific region, and the progeny plants are resistant to the target insects inhibited by the Vip3Aa protein and/or to glufosinate-ammonium The herbicide is tolerant; the nucleic acid sequence of the specific region is the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2. 19. The method for producing a corn plant that is resistant to target insects inhibited by Vip3Aa protein and/or tolerant to glufosinate-ammonium herbicide according to claim 18, characterized in that the nucleic acid sequence of the specific region It is the sequence shown in SEQ ID NO:3 and/or SEQ ID NO:4. 20. according to claim 18 or 19, produce the method for the corn plant that has resistance to the target insect that Vip3Aa protein suppresses and/or has tolerance to glufosinate-ammonium herbicide, it is characterized in that, described method comprises Transgenic corn event DBN9501 is sexually crossed with corn plants lacking target insect resistance and/or glufosinate tolerance, thereby producing a large number of progeny plants, and selecting the progeny plants having the nucleic acid sequence of the specific region; Invading with target insects and/or treating said progeny plants with glufosinate; selecting said progeny plants that are resistant to the target insect and/or tolerant to the glufosinate-ammonium herbicide; The transgenic corn event DBN9501 is deposited in the General Microorganism Center of China Committee for the Collection of Microbial Cultures in the form of seeds with the deposit number CGMCC No. 17099. 21. An agricultural product or commodity produced from the transgenic corn event DBN9501, characterized in that the agricultural product or commodity is corn meal, corn flour, corn oil, corn silk, corn starch, corn gluten, Corn cakes, cosmetics or fillers; the transgenic corn event DBN9501 is deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms under the deposit number CGMCC No. 17099 in the form of seeds.

Images