BR112021000302A2 - NUCLEIC ACID SEQUENCE, METHOD FOR DETECTING THE PRESENCE OF THE DNA FROM THE DBN9501 TRANSMENING CORN EVENT IN A SAMPLE, DNA DETECTION KIT, METHODS TO PROTECT A CORN PLANT, METHOD OF CREATING A RESISTANT CORN PLANT / LARGE CORN PLANT TO THE HERBICIDE GLUFOSINATE AND AGRICULTURAL PRODUCT OR COMMODITY DERIVED FROM THE GMN CORN EVENT DBN9501 - Google Patents

Abstract

nucleic acid sequence, method for detecting the presence of dbn9501 transgenic corn event dna in a sample, dna detection kit, methods for protecting a maize plant, method for creating an insect resistant and / or tolerant maize plant to the herbicide glufosinate and agricultural product or commodity derived from the dbn9501 transgenic corn event. the present invention relates to a nucleic acid sequence for detecting a genetically modified corn event dbn9501, as well as a method of detecting it, wherein the nucleic acid sequence comprises sequence no .: 1 or a sequence complementary thereto , or seq id no .: 2 or a complementary sequence thereof. there is also provided a method for preparing a corn plant that is insect resistant and / or tolerant to the ammonium-glufosinate herbicide, a method for growing a corn plant that is insect resistant and / or tolerant to the ammonium-glufosinate herbicide, as well as a method to protect a maize plant from herbicide damage or to control weeds in the field where a maize plant is grown, where the maize plant genome comprises the nucleic acid sequences shown in seq id no .: 1, and / or seq id no .: 2, the nucleic acid sequences shown in seq id no .: 3, and / or seq id no .: 4 or a nucleic acid sequence shown in seq id no .: 5.

Description

DESCRIPTION PATENT REPORT NUCLEIC ACID SEQUENCE, METHOD FOR DETECTING THE PRESENCE OF THE DNA OF THE GMN9501 CORN EVENT IN A SAMPLE, DNA DETECTION KIT, METHODS FOR PROTECTING A CORN PLANT, METHOD OF CREATING A MAIN PLANT INSECTS AND / OR HERBICIDE TOLERANT

GLUFOSINATE AND AGRICULTURAL PRODUCT OR COMMODITY DERIVED FROM THE TRANSGENIC CORN EVENT DBN9501 Field of the Invention

[0001] The present invention relates to the field of plant molecular biology, especially the field of the creation of transgenic crops in agricultural biotechnology research. In particular, the present invention relates to an insect-resistant and tolerant to the herbicide glufosinate transgenic corn event DBN9501, and nucleic acid sequences and methods for detecting whether a biological sample contains the specific DBN9501 transgenic corn event. Background of the Invention

[0002] Maize (Zea mays L.) is an important grain crop in many areas of the world. Biotechnology was applied to corn to improve agronomic traits and their quality. Insect resistance, especially resistance to Lepidoptera insects such as Ostrinia nubilalis, Helicoverpa armigera Hubner and Agrotis ypsilon Rottemberg, is an important agronomic trait in maize production. The resistance of corn to Lepidoptera can be conferred by the expression of the gene resistant to Lepidoptera in corn plants through a transgenic method. Another important agronomic trait is herbicide tolerance. For example, there are effectively transformed corn events such as NK603 and GA21, which have been extensively developed in major areas of corn planting such as America. It is important to note that the herbicide glufosinate has a different mode of action than the herbicide glyphosate, since the herbicide glufosinate is a non-selective contact type herbicide and can be used as a means for the effective management of glyphosate resistant weeds. The tolerance of corn to the herbicide glufosinate can be checked by the expression of the gene tolerant to the herbicide glufosinate (eg, pat) in corn plants using a transgenic method.

[0003] The expression of exogenous genes in plants is known to be influenced by their positions on the chromosome, perhaps due to the structure of the chromatin (eg, heterochromatin) or the proximity of the transcriptional regulatory elements (eg, enhancer) to the site of integration. For this reason, it is often necessary to track a large number of events in order to identify the event that can be marketed (that is, the event in which a target gene introduced is optimally expressed). For example, it has been observed in plants and other organisms that there can be a big difference between events in terms of the level of expression of an introduced gene; and there may also be differences in terms of spatial or temporal expression patterns, for example, differences in the relative expression of a transgene in various plant tissues, which are reflected in the discrepancy between the current expression pattern and the expected expression pattern based on in the elements of transcriptional regulation in the construct of the introduced gene. For this reason, it is usually necessary to produce hundreds to thousands of different events and to track those events by a single event that has the desired level of expression and transgene standards for commercial purposes. An event that has desired levels or patterns of transgene expression is useful for the transgene's introgression into other genetic antecedents by sexual cross between species using conventional breeding methods. The progeny reproduced by such a crossing maintains the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in several varieties that are well-adapted to local developmental conditions.

[0004] It would be advantageous to be able to detect the presence of a particular event in order to determine whether the progeny of sexual crossing contain a target gene. In addition, the method for detecting a particular event would be useful to comply with regulations such as regulations requiring approval before marketing and labeling of foods derived from recombinant cultivated plants. It is possible to detect the presence of a transgene by any well-known methods for the detection of polynucleotide, such as polymerase chain reaction (PCR) or DNA hybridization using polynucleotide probes. These detection methods generally focus on the frequently used genetic elements, such as promoters, terminators, marker genes. As a result, such methods may not be useful for distinguishing different events, particularly those produced using the same DNA construct, unless the sequence of the chromosomal DNA adjacent to the inserted transgenic DNA (flanking DNA) is known. Thus, a pair of primers covering the junction between the inserted transgene and the flanking DNA is generally used to identify a particular transgenic event by PCR, in particular, a first primer comprised in the inserted sequence and a second primer comprised in the sequence inserted. Summary of the Invention

[0005] The purpose of the present invention is to provide nucleic acid sequences and methods for the detection of DBN9501 maize plant. The DBN9501 transgenic corn event has good insect resistance as well as good tolerance to the herbicide glufosinate, while detection methods can accurately and quickly identify whether a biological sample contains the DNA molecule from the DBN9501 transgenic corn event.

[0006] To achieve the above objective, the present invention provides a nucleic acid sequence comprising at least 11 consecutive nucleotides at positions 1-384 of SEQ ID No .: 3 or a complementary sequence thereof, and at least 11 consecutive nucleotides at positions

385-768 of SEQ ID No .: 3 or a complementary sequence thereof; and / or at least 11 consecutive nucleotides in positions 1-564 of SEQ ID No .: 4 or a complementary sequence thereof, and at least 11 consecutive nucleotides in positions 565-1339 of SEQ ID No .: 4 or a complementary sequence thereof.

[0007] Preferably, the nucleic acid sequence comprises 22 to 25 consecutive nucleotides in positions 1-384 of SEQ ID No .: 3 or a complementary sequence thereof, and 22 to 25 consecutive nucleotides in positions 385-768 of SEQ ID No .: 3 or a complementary sequence thereof; and / or 22 to 25 consecutive nucleotides in positions 1-564 of SEQ ID No .: 4 or a complementary sequence thereof, and 22 to 25 consecutive nucleotides in positions 565-1339 of SEQ ID No .: 4 or a complementary sequence of this.

[0008] Preferably, the nucleic acid sequence comprises SEQ ID No .: 1 or a complementary sequence thereof, and / or SEQ ID No .: 2 or a complementary sequence thereof.

[0009] SEQ ID No .: 1 or a complementary sequence thereof is a sequence of 22 nucleotides that is located around the insertion junction at the 5 'end of the sequence inserted in the transgenic corn event DBN9501. SEQ ID No .: 1 or a complementary sequence thereof encompasses the genomic DNA sequence that flanks the corn insertion site and the DNA sequence at the 5 'end of the inserted sequence. Therefore, the inclusion of SEQ ID No .: 1 or a complementary sequence of this could indicate the presence of the DBN9501 transgenic corn event. SEQ ID No .: 2 or a complementary sequence thereof is a sequence of 22 nucleotides that is located around the insertion junction at the 3 'end of the sequence inserted in the DBN9501 transgenic corn event. SEQ ID No .: 2 or a complementary sequence thereof encompasses the DNA sequence at the 3 'end of the inserted sequence and the genomic DNA sequence that flanks the maize insertion site. Therefore, the inclusion of SEQ ID No .: 2 or a complementary sequence of this could indicate the presence of the DBN9501 transgenic corn event.

Preferably, the nucleic acid sequence comprises SEQ ID No .: 3 or a complementary sequence thereof, and / or SEQ ID No .: 4 or a complementary sequence thereof.

The nucleic acid sequence of the present invention can be a sequence comprising at least 11 or more consecutive polynucleotides in any portion of the inserted T-DNA sequence in SEQ ID No .: 3 or a complementary sequence thereof (the first sequence nucleic acid), or at least 11 or more consecutive polynucleotides in any portion of the 5 'flanking corn genomic DNA region in SEQ ID No .: 3 or a complementary sequence thereof (the second nucleic acid sequence). In addition, the nucleic acid sequence can be a sequence homologous or complementary to a portion of SEQ ID No .: 3 comprising the entire SEQ ID No .: 1. When used together, the first nucleic acid sequence and the second sequence of nucleic acids can act as a DNA primer pair in a DNA amplification method that generates an amplification product. If the amplification product produced using said DNA primer pair in the DNA amplification method comprises SEQ ID No .: 1, the presence of the DBN9501 transgenic corn event or progeny thereof can be diagnosed. SEQ ID No .: 3 or a complementary sequence thereof is a 768 nucleotide sequence that is located around the insertion junction at the 5 'end of the T-DNA sequence inserted into the DBN9501 transgenic corn event. SEQ ID No .: 3 or a complementary sequence thereof consists of 384 nucleotides of the corn genomic 5 'flanking sequence (nucleotides 1-384 of SEQ ID No .: 3), 168 nucleotides of the construct DNA sequence DBN10707 (nucleotides 385-552 of SEQ ID No .: 3) and 216 nucleotides of the DNA sequence of a tNos transcription terminator (nopaline synthase) (nucleotides 553-768 of SEQ ID No .: 3). Therefore, the inclusion of SEQ ID No .: 3 or a complementary sequence of this could indicate the presence of the DBN9501 transgenic corn event.

The nucleic acid sequence can be a sequence comprising at least 11 or more consecutive polynucleotides in any portion of the inserted T-DNA sequence in SEQ ID No .: 4 or a complementary sequence thereof (the third nucleic acid sequence ), or at least 11 or more consecutive polynucleotides in any portion of the 3 'flanking corn genomic DNA region in SEQ ID No .: 4 or a complementary sequence thereof (the fourth nucleic acid sequence). In addition, the nucleic acid sequence can be a sequence homologous or complementary to a portion of SEQ ID No .: 4 comprising the entire SEQ ID No .: 2. When used together, the third nucleic acid sequence and the fourth sequence of nucleic acids can act as a DNA primer pair in a DNA amplification method that generates an amplification product. If the amplification product produced using said DNA primer pair in the DNA amplification method comprises SEQ ID No .: 2, the presence of the DBN9501 transgenic corn event or progeny thereof can be diagnosed. SEQ ID No .: 4 or a complementary sequence thereof is a 1339 nucleotide sequence that is located around the insertion junction at the 3 'end of the T-DNA sequence inserted into the DBN9501 transgenic corn event. SEQ ID No .: 4 or a complementary sequence thereof consists of 271 nucleotides from a pr35S transcription start sequence (nucleotides 1-271 of SEQ ID No .: 4), 293 nucleotides from the construct DNA sequence DBN10707 (nucleotides 272-564 of SEQ ID No .: 4) and 775 nucleotides of the corn genomic 3 'flanking sequence (nucleotides 565-1339 of SEQ ID No .: 4). Therefore, the inclusion of SEQ ID No .: 4 or a complementary sequence of this could indicate the presence of the DBN9501 transgenic corn event.

[0013] In addition, the nucleic acid sequence comprises SEQ ID No .: 5 or a complementary sequence thereof.

[0014] SEQ ID No .: 5 or a complementary sequence to this is an 8559 nucleotide sequence that characterizes the transgenic corn event DBN9501. The specific genomes and genetic elements contained in SEQ ID No .: 5 are shown in Table 1. Inclusion of SEQ ID No .: 5 or a complementary sequence thereof could indicate the presence of the DBN9501 transgenic corn event.

Table 1: The genomes and genetic elements contained in SEQ ID No .: 5.

Element Length Position in SEQ ID No .: 5 genetic / genome (bp) genome 5 '384 1-384 LB 168 385-552 tNos 253 553-805 cPAT 552 812-1363 prZmUbi1 1993 1370-3362 tNos 253 3418-3670 cVip3Aa19 2370 3671-6040 iZmHSP70 806 6047-6852 pr35S 639 6853-7491 RB 293 7492-7784 genome 3 '775 7785-8559

[0015] As is well known to those skilled in the art, the first, second, third and fourth nucleic acid sequences may not only consist of DNA, but may also comprise RNA, a mixture of DNA and RNA, or a combination of DNA, RNA and other nucleotides or analogues thereof that do not act as models for one or more polymerases. 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 and can be selected from the nucleotides of 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 of SEQ ID No .: 3, SEQ ID No .: 4 and SEQ ID No .: 5, probes and primers can be at least about 21 to about 50 or more consecutive nucleotides in length.

[0016] Nucleic acid sequences or complementary sequences thereof can be used in DNA amplification methods to produce amplicons that are used to detect the presence of the DBN9501 transgenic corn event or progeny thereof in a biological sample. The nucleic acid sequences or complementary sequences thereof can be used in nucleotide detection methods to detect the presence of the DBN9501 transgenic corn event or progeny thereof in a biological sample.

[0017] To achieve the above objective, the present invention also provides a method to detect the presence of the DNA of the transgenic corn event DBN9501 in a sample, comprising: - contact of the sample to be detected with at least two primers to amplify a product target amplification in a nucleic acid amplification reaction; - carrying out the nucleic acid amplification reaction; e - detection of the presence of the target amplification product; wherein the target amplification product comprises the nucleic acid sequence.

[0018] Preferably, the target amplification product comprises SEQ ID No .: 1 or a complementary sequence thereof, SEQ ID No .: 2 or a complementary sequence thereof, SEQ ID No .: 6 or a complementary sequence thereof, and / or SEQ ID No .: 7 or a complementary sequence thereof.

[0019] In particular, primers comprise a first primer and a second primer, where the first primer is selected from SEQ ID

No .: 1, SEQ ID No .: 8 and SEQ ID No .: 10; and the second primer is selected from SEQ ID No .: 2, SEQ ID No .: 9 and SEQ ID No .: 11.

[0020] To achieve the above objective, the present invention also provides a method for detecting the presence of the DNA of the transgenic corn event DBN9501 in a sample, comprising: - contact of the sample to be detected with a probe, in which the probe comprises the nucleic acid sequence; - hybridization of the sample to be detected with the probe under strict hybridization conditions; and - hybridization detection of the sample to be detected with the probe.

[0021] The rigid conditions can be hybridization at 65 ºC in a solution of 6 x SSC (sodium citrate) and 0.5% SDS (sodium dodecyl sulfate), followed by washing the membrane in a solution of 2 x SSC and 0.1% SDS and a solution of 1 x SSC and 0.1% SDS (each at a time).

[0022] Preferably, the probe comprises SEQ ID No .: 1 or a complementary sequence thereof, SEQ ID No .: 2 or a complementary sequence thereof, SEQ ID No .: 6 or a complementary sequence thereof, and / or SEQ ID No .: 7 or a complementary sequence of this.

[0023] Optionally, at least one probe is marked with at least one fluorophore.

[0024] To achieve the above objective, the present invention also provides a method for detecting the presence of the DNA of the transgenic corn event DBN9501 in a sample, comprising: - contact of the sample to be detected with a nucleic acid marker molecule, in that the nucleic acid marker molecule comprises the nucleic acid sequence; - hybridization of the sample to be detected with the nucleic acid marker molecule under strict hybridization conditions; - detection of the hybridization of the sample to be detected with the nucleic acid marker molecule, and, also, a breeding analysis aided by marker to determine whether insect resistance and / or herbicide tolerance is genetically linked to the marker molecule. nucleic acid.

[0025] Preferably, the nucleic acid marker molecule comprises at least one sequence selected from the group consisting of SEQ ID No .: 1 or a complementary sequence thereof, SEQ ID No .: 2 or a complementary sequence thereof, and / or SEQ ID Nos .: 6-11 or sequences complementary to these.

[0026] To achieve the above objective, the present invention also provides a DNA detection kit comprising at least one DNA molecule, in which the DNA molecule comprises the nucleic acid sequence, and the DNA molecule can act as a DNA primer or a probe specific for the DBN9501 transgenic corn event or progeny thereof.

[0027] Preferably, the DNA molecule comprises SEQ ID No .: 1 or a complementary sequence thereof, SEQ ID No .: 2 or a complementary sequence thereof, SEQ ID No .: 6 or a complementary sequence thereof, and / or SEQ ID No .: 7 or a complementary sequence thereof.

[0028] To achieve the above objective, the present invention also provides a plant cell comprising a nucleic acid sequence that encodes the insect resistant Vip3Aa protein, a nucleic acid sequence that encodes the PAT protein tolerant to the herbicide glufosinate and a region-specific nucleic acid sequence, wherein the region-specific nucleic acid sequence comprises the sequence as defined in SEQ ID No .: 1, SEQ ID No .: 2, SEQ ID No .: 6, and / or SEQ ID No .: 7.

[0029] Preferably, the plant cell comprises a nucleic acid sequence encoding the insect resistant Vip3Aa protein, a nucleic acid sequence encoding the PAT protein tolerant to the herbicide glufosinate and a nucleic acid sequence from a specific region, wherein the region-specific nucleic acid sequence comprises the sequence as defined in SEQ ID No .: 3, and / or SEQ ID No .: 4.

[0030] Preferably, the plant cell successively comprises SEQ ID No .: 1, the nucleic acid sequence at positions 553-7491 of SEQ ID No .: 5 and SEQ ID No .: 2, or comprises the sequence as defined in SEQ ID No .: 5.

[0031] To achieve the objective, the present invention also provides a method to protect a corn plant against insect invasion, comprising the provision of at least one transgenic corn plant cell in the target insect's diet, in which the cell The transgenic corn plant comprises, in its genome, the sequence as defined in SEQ ID No .: 1, and / or SEQ ID No .: 2; and the ingestion of the transgenic corn plant cell inhibits the target insect from still feeding on the transgenic corn plant.

[0032] Preferably, the transgenic corn plant cell comprises, in its genome, the sequence as defined in SEQ ID No .: 3, and / or SEQ ID No .: 4.

[0033] Preferably, the transgenic corn plant cell successively comprises, in its genome, SEQ ID No .: 1, the nucleic acid sequence at positions 553-7491 of SEQ ID No .: 5 and SEQ ID No .: 2, or comprises SEQ ID No .: 5.

[0034] To achieve the objective, the present invention also provides a method to protect a corn plant from damage caused by a herbicide or to control weeds in a corn field, comprising the application of an effective amount of glufosinate herbicide. in a planting field of at least one transgenic corn plant, where the transgenic corn plant comprises, in its genome, the sequence as defined in SEQ ID No .: 1, and / or SEQ ID No .: 2, and the transgenic corn plant has tolerance to the herbicide glufosinate.

[0035] Preferably, the transgenic corn plant comprises, in its genome, the sequence as defined in SEQ ID No .: 3, and / or SEQ ID No .: 4.

[0036] Preferably, the transgenic maize plant successively comprises, in its genome, SEQ ID No .: 1, the nucleic acid sequence in positions 553-7491 of SEQ ID No .: 5 and SEQ ID No .: 2, or comprises the sequence as defined in SEQ ID No .: 5.

[0037] To achieve the above objective, the present invention also provides a method for creating an insect resistant corn and / or tolerant to the herbicide glufosinate, comprising: - planting at least one corn seed, in which the seed of corn comprises, in its genome, a nucleic acid sequence that encodes the insect-resistant Vip3Aa protein and / or a nucleic acid sequence that encodes the PAT protein tolerant to the herbicide glufosinate, and a sequence of nucleic acids from a specific region, or the corn seed comprises, in its genome, the nucleic acid sequence as defined in SEQ ID No .: 5; - corn seed growth in a corn plant; e - invasion of the corn plant with a target insect and / or sprinkling the corn plant with an effective amount of herbicide glufosinate, and then harvesting the plant with reduced damage to the plant compared to other plants that do not comprise the nucleic acid sequence the specific region; wherein the region-specific nucleic acid sequence is the sequence as defined in SEQ ID No .: 1, and / or SEQ ID No .: 2; preferably, the region-specific nucleic acid sequence is the sequence as defined in SEQ ID No .: 3, and / or SEQ ID No .: 4.

[0038] To achieve the above objective, the present invention also provides a method for producing an insect resistant and / or tolerant to the herbicide glufosinate corn plant, comprising: - introducing a nucleic acid sequence encoding the Vip3Aa resistant protein insects and / or a nucleic acid sequence encoding the PAT protein tolerant to the herbicide glufosinate, and a nucleic acid sequence from a specific region in the genome of a first corn plant, or the nucleic acid sequence as defined in SEQ ID No..25 in the genome of a first corn plant, in a second corn plant, thus producing a plurality of progeny plants; and - selection of progeny plants comprising the nucleic acid sequence of the specific region, which are also resistant to insects and / or tolerant to the herbicide glufosinate; wherein the region-specific nucleic acid sequence is the sequence as defined in SEQ ID No .: 1, and / or SEQ ID No .: 2; preferably, the region-specific nucleic acid sequence is the sequence as defined in SEQ ID No .: 3, and / or SEQ ID No .: 4.

[0039] Preferably, the method comprises - sexual crossing between species of the DBN9501 transgenic corn event with a corn plant that does not have the characteristic of insect resistance and / or tolerance to the herbicide glufosinate, thus producing a plurality of progeny plants; and - selection of progeny plants comprising the nucleic acid sequence of the specific region; wherein the region-specific nucleic acid sequence comprises the sequence as defined in SEQ ID No .: 1, and / or SEQ ID No .: 2; preferably, the region-specific nucleic acid sequence comprises the sequence as defined in SEQ ID No .: 3, and / or SEQ ID No .: 4.

[0040] To achieve the above objective, the present invention also provides an agricultural product or commodity derived from the DBN9501 transgenic corn event, in which the agricultural product or commodity is corn flake, corn flour, corn oil, corn silk , corn starch, corn gluten, corn cake, a cosmetic or a bulking agent.

[0041] In the nucleic acid sequences and methods for the detection of maize plants according to the present invention, the following definitions and methods are provided to further define the present invention and guide those skilled in the art in implementing the present invention. Unless otherwise defined, the terms must be understood in accordance with conventional usage by those skilled in the art.

[0042] The term "maize" refers to Zea mays and includes all plant varieties that can be crossed with maize, including wild maize species.

[0043] The term "comprising", "understand" or "contain" means "including among others".

[0044] The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, tissue cultures of plant cells whose plants can be regenerated, plant calluses, plant tufts and plant cells that are intact in plants or parts of the plant, in which the parts of the plant may be, for example, embryos, pollen, ova, seeds, leaves, flowers, branches, fruits, stems, roots, root tips or anthers. It should be understood that within the scope of the present invention, parts of transgenic plants include, among other plant cells, protoplasts, tissues, calluses, embryos, flowers, stems, fruits, leaves and roots. The plant parts mentioned above are derived from the transgenic plants or progeny thereof which are previously transformed with the DNA molecule of the present invention and, therefore, at least partially consist of transgenic cells.

[0045] The term "gene" refers to a fragment of nucleic acid that expresses a specific protein, including regulatory sequences preceding (5 'non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as 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 that are not found in nature. "Endogenous gene" refers to a gene native to its natural location in the genome of an organism. "Exogenous gene" is a foreign gene currently present in the genome of an organism that does not naturally contain it, and also refers to a gene introduced into a recipient cell through a transgenic process. Exogenous genes can comprise native genes or chimeric genes inserted into a non-native organism. "Transgene" is a gene that was introduced into a genome by a transformation procedure. "Insertion site" or "target site" refers to the site in a plant genome into which a recombinant DNA has been inserted.

[0046] "Flanking DNA" can comprise either a genome that is naturally present in an organism such as a plant or an exogenous (heterologous) DNA introduced through a transformation process, for example, a fragment associated with an event of transformation. Thus, the flanking DNA can include a combination of native DNA and exogenous DNA. As used here, the term "flanking DNA", also called "flanking region", "flanking sequence", "genomic flanking sequence", or "genomic flanking DNA", refers to a sequence of at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500 or 5000 base pairs or greater, which is located either immediately upstream or downstream of the molecule of Exogenous DNA originally inserted and adjacent to it. When this flanking region is located downstream, it can also be referred to as “flank 3’ ”or“ flank of the right edge ”, and the like. When this flanking region is located upstream, it can also be referred to as “flank 5’ ”or“ flank of the left edge ”, and the like.

[0047] The transformation procedures that lead to the random integration of an exogenous DNA will result in transformants containing different flanking regions that are characteristic of and unique to each transformant. When recombinant DNA is introduced into a plant through traditional breeding, its flanking regions will generally not be changed. Transformants will also contain unique unions between heterologous insert DNA and fragments of genomic DNA, or between two fragments of genomic DNA, or between two fragments of heterologous DNA. The "junction" is a point where two specific DNA fragments are linked. For example, a junction exists at the position where an insert DNA is linked to a flanking DNA. A junction point also exists in a transformed organism in which two fragments of DNA are linked together in a way that is modified from that found in the native organism. The "junction region" or "junction sequence" refers to DNA that comprises a junction point.

[0048] The present invention provides a transgenic corn event called DBN9501 and its progeny. The DBN9501 transgenic corn event is also referred to as a DBN9501 corn plant, including plants and seeds from the DBN9501 transgenic corn event, along with plant cells or renewable plant parts thereof, where the plant parts of the DBN9501 transgenic corn event include among other cells, pollen, ova, flowers, buds, roots, stems, silks, inflorescences, ears, leaves and products derived from the DBN9501 corn plant, such as cornmeal, cornmeal, corn oil, corn syrup, silk of corn, corn starch and biomass remaining in the field of corn plantations.

[0049] The DBN9501 transgenic corn event of the present invention contains a DNA construct that, when expressed in plant cells, gives the DBN9501 transgenic corn event insect resistance and tolerance to the herbicide glufosinate. The DNA construct comprises two expression cassettes arranged in tandem. The first expression cassette comprises a promoter suitable for expression in a plant and a suitable polyadenylation signal sequence, in which the promoter is operably linked to the Vip3Aa19 protein nucleic acid sequence, which is mainly resistant to Lepidoptera insects. The second expression cassette comprises a promoter suitable for expression in a plant and a suitable polyadenylation signal sequence, in which the promoter is operably linked to a gene encoding phosphinothricin N-acetyltransferase (PAT), and the nucleic acid sequence of PAT protein is tolerant to the herbicide glufosinate. In addition, the promoter may be a suitable promoter isolated from plants, including constitutive, inducible and / or tissue-specific promoters. The appropriate promoter includes, but is not limited to, cauliflower mosaic virus (CaMV) 35S promoter, scrofular mosaic virus (FMV) 35S promoter, ubiquitin promoter, actin promoter, nopaline synthase promoter ( NOS) of Agrobacterium tumefaciens, octopine synthase (OCS) promoter, Cestrum yellow leaf ripple virus promoter, patatin promoter, ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) promoter, S- glutathione promoter transferase (GST), E9 promoter, GOS promoter, alcA / alcR promoter, Agrobacterium rhizogenes RoID promoter and Arabidopsis thaliana Suc2 promoter. The polyadenylation signal sequence can be a suitable polyadenylation signal sequence that has plant functions. The appropriate polyadenylation signal sequence includes, but is not limited to, the polyadenylation signal sequence derived from the nopaline synthase (NOS) gene of Agrobacterium tumefaciens, a polyadenylation signal sequence derived from the 35S terminator of the cauliflower mosaic virus (CaMV) , a polyadenylation signal sequence derived from the protease inhibitor II (PIN II) gene and a polyadenylation signal sequence derived from the α-tubulin gene.

[0050] In addition, the expression cassette may comprise other genetic elements, including, among others, an enhancer and a signal peptide / transit peptide. The intensifier, level of expression of the enhancement gene, includes, but is not limited to, the tobacco etch virus (TEV) translation activator, CaMV35S enhancer and FMV35S enhancer. The signal peptide / transit peptide can target the Vip3Aa19 protein and / or PAT protein for transport to the extracellular space or into a specific intracellular organelle or compartment, for example, targeting the chloroplast using a sequence encoding the transit peptide chloroplast, or targeting the endoplasmic reticulum using a 'KDEL' retention sequence.

[0051] The Vip3Aa19 gene can be isolated from Bacillus thuringiensis (Abbr. As Bt), and the nucleotide sequence of the Vip3Aa19 gene can be changed through codonoptimization or other methods, in order to improve the stability and availability of a transcript in cells transformed.

[0052] The term "Lepidoptera", including both moths and butterflies, is the largest order of pests in agriculture and forest. It includes, for example, Agrotis ypsilon Rottemberg, Helicoverpa armigera Hubner, Prodenia litura, Athetis lepigone, and Conogethes punctiferalis.

[0053] The phosphinothricin N-acetyltransferase (PAT) gene can be an enzyme isolated from the strain of Streptomyces viridochromogenes, which catalyzes the conversion of L-phosphinothricin in its inactive form by acetylation in order to confer tolerance to the herbicide glufosinate to the plant. Phosphinothricin (PTC, 2-amino-4-methylphosphinyl-butyric acid) is a glutamine synthetase inhibitor. PTC is a structural unit of the antibiotic 2-amino-4-methylphosphinyl-alanyl-alanine. This tripeptide (PTT) is active against Gram-positive and Gram-negative bacteria and fungi, Botrytis cinerea. The phosphinothricin N-acetyltransferase (PAT) gene can also serve as a selectable gene marker.

[0054] The "glufosinate" (also known as phosphinothricin) refers to ammonium-2-amino-4- [hydroxy (methyl) phosphinyl] butyrate. Treatments with a "glufosinate herbicide" refer to treatments with any herbicide formulation containing glufosinate. It is within the skill of the ordinary agricultural technician to select the application rates for a glufosinate formulation in order to achieve a biologically effective amount. Treating a field containing plant materials from the DBN9501 transgenic corn event with any herbicidal formulation containing glufosinate will control the growth of weeds in the field and will not affect the growth or yield of plant materials derived from the DBN9501 transgenic corn event.

[0055] The DNA construct is introduced into a plant by means of transformation methods including, among others, Agrobacterium-mediated transformation, ballistic transformation and transformation of the pollen tube path.

[0056] Agrobacterium-mediated transformation is a method commonly used for plant transformation. The exogenous DNA to be introduced into a plant is cloned into a vector between left and right border consensus sequences, that is, a region of T-DNA. The vector is transformed into Agrobacterium cells, which are subsequently used to infect plant tissue. The T-DNA region in the vector comprising the exogenous DNA is inserted into the plant genome.

[0057] Ballistic transformation is bombardment of plant cells with a vector comprising exogenous DNA (particle-mediated biological transformation).

[0058] Transformation of the pollen tube pathway is a method for carrying out exogenous DNA in an embryonic sac via a nucellus pathway using a natural pollen tube (also known as the pollen tube transmission tissue) formed after pollination of the plant.

[0059] After transformation, it is necessary to regenerate the transgenic plant from the transformed plant tissue, and select the progeny with exogenous DNA using an appropriate marker.

[0060] A DNA construct is a set of DNA molecules linked together that provides one or more expression cassettes. The DNA construct is preferably a plasmid that can self-replicate in a bacterial cell and contains several restriction sites of the endonuclease enzyme that are useful for introducing DNA molecules that provide functional genetic elements, that is, promoters, introns, sequences leaders,

coding sequences, 3 'termination regions and other sequences. The expression cassettes contained in a DNA construct comprise the genetic elements necessary for the transcription of a messenger RNA and can be designed to express in prokaryotic cells or eukaryotic cells. Most preferably, the expression cassettes of the present invention are designed to express in plant cells.

[0061] A transgenic "event" is produced by transforming plant cells with a heterologous DNA construct, including the steps of inserting a nucleic acid expression cassette comprising at least one target gene into a plant's genome through a transgenic method in order to generate a plant population, regeneration of the plant population, and selection of a particular plant having the characteristics of insertion in a particular genome site. The term “event” refers to the original transformant and progeny thereof, which include heterologous DNA. The term "event" also refers to the progeny produced by sexual crossing between the original transformant and the individual of other varieties that include heterologous DNA. Even after repeated backcrossing with a backcrossing parent, the inserted DNA and flanking genomic DNA from the original transforming parent is still present at the same chromosomal site in the crossing progeny. The term "event" also refers to the DNA sequence of the original transformant comprising the inserted DNA and the flanking genomic sequence immediately adjacent to the inserted DNA, where the DNA sequence is expected to be transferred to a progeny that receives the inserted DNA including the target gene and is produced by sexual crossing between a parental lineage that includes the inserted DNA (for example, the original transformant and the progeny of this resulting from self-pollination) and a parental lineage that does not contain the inserted DNA.

[0062] "Recombination" as used here refers to a form of a DNA and / or a protein and / or an organism that would not normally be found in nature and as such is created by human intervention. Such human intervention can produce a recombinant DNA molecule and / or a recombinant plant. The “recombinant DNA molecule” is made by an artificial combination of two otherwise separated sequence segments, for example, by chemical synthesis or by manipulation of isolated nucleic acid segments by genetic engineering technology. Nucleic acid operating technology is well known in the art.

[0063] The term "transgene" includes any cell, cell line, callus, tissue, part of the plant or plant, whose genotype has been changed due to the presence of heterologous nucleic acid. “Transgene” includes those heterologous organisms initially altered as well as the progeny created from the initial transgenic organism by sexual crossing or asexual propagation. The term “transgene” as used here does not cover altering the genome by conventional plant breeding methods or by naturally occurring events (intrachromosomal or extrachromosomal), such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, mutation by transposition or non-recombinant spontaneous.

[0064] "Heterologist" as used here refers to a first molecule that 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. The molecule would thus be heterologous to the host and artificially introduced into the genome of the host cell.

[0065] The event of transgenic corn DBN9501 having resistance to Lepidoptera insects and tolerance to the herbicide glufosinate can be created by the following steps: first, sexual crossing of a first parent corn plant consisting of a corn plant grown from the corn event transgenic DBN9501 and its progeny, with a second parent corn plant that lacks resistance to insects Lepidoptera and tolerance to the herbicide glufosinate, thus producing a plurality of first progeny plants, in which the event of transgenic corn DBN9501 and its progeny are obtained from transformation using the expression cassette of the present invention which is resistant to Lepidoptera insects and tolerant to the herbicide glufosinate; and then selecting a progeny plant that is resistant to invasion by Lepidoptera insects and / or tolerant to the herbicide glufosinate, thereby creating a corn plant that is resistant to invasion by Lepidoptera insects and tolerant to the herbicide glufosinate. These steps may also include backcrossing the Lepidoptera-resistant and / or glufosinate-tolerant progeny plant with the second parent corn plant or a third parent corn plant, then screening the progeny by Lepidoptera insect invasion, application of the herbicide glufosinate or identification of molecular markers (for example, the DNA molecule comprising the unions identified from the 5 'and 3' ends of the sequence inserted in the DBN9501 transgenic corn event) associated with the trait, thereby producing an insect resistant corn plant Lepidoptera and tolerant to the herbicide glufosinate.

[0066] It should also be understood that two different transgenic plants can also be crossed to produce progeny that contain two independent exogenous genes and added separately. Self-pollination of appropriate progeny can produce plant progenies that are homozygous for both added exogenous genes. Backcrossing with a parental plant and the crossing between species with a non-transgenic plant as described above are also contemplated, since it is a vegetative propagation.

[0067] The term "probe" means an isolated nucleic acid molecule that is attached to a conventional detectable label or reporter molecule, for example, a radioactive isotope, a linker, a chemiluminescent agent or an enzyme. Such a probe is complementary to a strand of a target nucleic acid, in the present invention, to a strand of DNA from the genome of the genome of the transgenic corn event DBN9501, no matter if the DNA of the genome is from the event of transgenic corn DBN9501 or seed or from a plant or seed or extract of these derived from the DBN9501 transgenic corn event. The 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 that target DNA sequence.

[0068] The term "primer" is an isolated nucleic acid molecule that is annealed with a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the strand of target DNA under the action of a polymerase (for example, a DNA polymerase). The primer pairs of the present invention refer to their use in amplifying a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

[0069] 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 or more. Such probes and primers hybridize specifically to a target sequence under stringent hybridization conditions. Preferably, probes and primers of the present invention have complete DNA sequence identity with consecutive nucleic acids in a target sequence, although a probe that differs from the target DNA sequence and retains the ability to hybridize to the target sequences under stringent conditions can be designed by conventional methods.

[0070] Primers and probes, based on the flanking genomic DNA and the inserted sequence of the present invention, can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from a plant material derived from the event of transgenic corn DBN9501, and determination of the nucleic acid sequence of the DNA molecule. The DNA molecule comprises the inserted transgenic sequence and corn genomic flanking sequence, and a fragment of the DNA molecule can be used as a primer or probe.

[0071] The probe and nucleic acid primer of the present invention hybridize to a target DNA sequence under strict conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from the DBN9501 transgenic corn event in a sample. These nucleic acid molecules or fragments are able to specifically hybridize to other nucleic acid molecules under certain circumstances. As used here, two nucleic acid molecules are able to specifically hybridize to each other if the two molecules are capable of forming an antiparallel double-stranded nucleic acid structure. A nucleic acid molecule is said to be the "complementary sequence" of another nucleic acid molecule if they exhibit complete complementarity. As used here, two nucleic acid molecules are said to exhibit "complete complementarity" if the entire 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 can hybridize to each other with sufficient stability such that they can anneal and bond to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to have "complementarity" if they can hybridize to each other with sufficient stability that they can anneal and bond to each other under conventional "high stringency" conditions. Deviations from complete complementarity are permissible, as long as such deviations do not completely prevent the two molecules from forming a double-stranded structure. To serve as a primer or probe, a nucleic acid molecule only needs to have sufficient complementarity in sequence such that it can form a stable double-stranded structure under the particular concentrations of solvent and salt employed.

[0072] As used herein, a substantially homologous sequence is a nucleic acid molecule that will hybridize specifically to the complementary strand of another corresponding nucleic acid molecule under stringent conditions. Rigid conditions that promote DNA hybridization are known to those skilled in the art, for example, treatment with 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by washing with 2.0 x SSC at 50 ºC. For example, the salt concentration in the washing step can be about 2.0 x SSC at 50 ° C at low accuracy to about 0.2 x SSC at 50 ° C at high accuracy. In addition, the temperature in the washing step can be increased from room temperature (about 22 ºC) in conditions of low rigor to about 65 ºC in conditions of high rigor. Both temperature and salt concentration can be varied, or one can be kept constant while the other variable is changed. Preferably, a nucleic acid molecule of the present invention will specifically hybridize to one or more nucleic acid molecules of SEQ ID No .: 1, SEQ ID No .: 2, SEQ ID No .: 3, SEQ ID No .: 4, SEQ ID No .: 5, SEQ ID No .: 6 and SEQ ID No .: 7, or complementary sequences thereof, or any fragment of the above sequences under conditions of moderate stringency, for example at about 2.0 x SSC and about 65 ºC. More preferably, a nucleic acid molecule of the present invention will specifically hybridize to one or more nucleic acid molecules of SEQ ID No .: 1, SEQ ID No .: 2, SEQ ID No .: 3, SEQ ID No .: 4, SEQ ID No .: 5, SEQ ID No .: 6 and SEQ ID No .: 7, or sequences complementary thereto, or any fragment of the above sequences under highly rigid conditions. A preferred nucleic acid marker molecule of the present invention comprises SEQ ID No .: 1, SEQ ID No .: 2, SEQ ID No .: 6 and SEQ ID No .: 7, or complementary sequences thereof, or any fragment of the sequences above. Another preferred nucleic acid marker molecule of the present invention shares 80% to 100% or 90% to 100% sequence identity with SEQ ID No .: 1, SEQ ID No .: 2, SEQ ID No .: 6 and SEQ ID No .: 7, or sequences complementary thereto, or any fragment of the above sequences. 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 the progeny of genetic crossing. Hybridization of a probe to a target DNA molecule can be detected by any method well known to those skilled in the art, including but not limited to fluorescent markers, radioactive markers, antibody-based markers and chemiluminescent markers.

[0073] Regarding the amplification of a target nucleic acid sequence using a pair of particular amplification primers (eg, PCR), "stringent conditions" are conditions that allow a primer pair to hybridize only to a sequence of target nucleic acids in a DNA thermal amplification reaction, in which the primer has a wild-type sequence corresponding to the target nucleic acid sequence (or its complementary sequence) and, thus, could bind to it, preferably, to produce a single amplification product, that is, amplicon.

[0074] The term "specifically binding to (a target sequence)" indicates that under rigid hybridization conditions, a probe or primer hybridizes only to a target sequence in a sample comprising the target sequence.

[0075] As used herein, "amplicon" refers to the nucleic acid amplification product of a target nucleic acid sequence used as a part of the nucleic acid model. For example, in order to determine whether a corn plant is produced by sexually crossing the DBN9501 transgenic corn event, or whether a corn sample collected from a field comprises the DBN9501 transgenic corn event, or whether a corn extract (such as such as semolina, flour or oil) comprises the event of transgenic corn DBN9501, DNA extracted from a sample or extract of corn plant tissue can be subjected to a nucleic acid amplification method using a pair of primers to produce an amplicon which is diagnostic for the presence of the DNA from the DBN9501 transgenic corn event. The primer pair includes a first primer derived from the flanking sequence in the plant genome adjacent to the insertion site of the exogenous inserted DNA, and a second primer derived from the exogenous inserted DNA. Amplicon has a length and a sequence that are also diagnostic for the DBN9501 transgenic corn event. The amplicon may have a length equal to the sum of the combined length of the primer pairs plus one nucleotide base pair, or preferably about 50 more nucleotide base pairs, or more preferably about 250 nucleotide base pairs, or more maximum preference plus about 450 base pairs of nucleotides or more.

Alternatively, a pair of primers can be derived from the genomic flanking sequence on both sides of the inserted DNA in order to produce an amplicon that includes the entire inserted nucleotide sequence. One of the primer pair derived from the plant henomic sequence can be located at a distance from the inserted DNA sequence, and this distance can range from a nucleotide base pair to about twenty thousand nucleotide base pairs. As used here, the term "amplicon" specifically excludes primer dimers formed in a DNA thermal amplification reaction.

The nucleic acid amplification reaction can be performed by any of several nucleic acid amplification methods known in the art, including polymerase chain reaction (PCR). A variety of amplification methods are known to those skilled in the art. PCR amplification methods were developed to amplify up to 22 kb of genomic DNA and up to 42 kb of bacteriophage 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 of the DBN9501 transgenic corn event can be obtained by amplifying the genome of the DBN9501 transgenic corn event using the primer sequences provided, followed by performing standard DNA sequencing methods on the amplicon of PCR or cloned DNA.

[0078] DNA detection kits that are based on DNA amplification methods contain DNA molecules used as primers that specifically hybridize to a target DNA and amplify a diagnostic amplicon under appropriate reaction conditions. The kit can provide a detection method based on an agarose gel or many methods known in the art for the detection of a diagnostic amplicon. A kit is provided by the present invention that contains DNA primers that are homologous or complementary to any portion of the genomic region of corn in SEQ ID No .: 3 or SEQ ID No .: 4 and to any portion of the transgenic inserted region in SEQ ID No .: 5. A pair of primers that is specifically identified as useful in a DNA amplification method comprises SEQ ID No .: 8 and SEQ ID No .: 9, which amplify a diagnostic amplicon homologous to a portion of the region of transgene / genome 5 'of the DBN9501 transgenic corn event, where the amplicon comprises SEQ ID No .: 1. Other DNA molecules useful as DNA primers can be derived from SEQ ID No .: 5.

[0079] The amplicon produced by these methods can be detected by a plurality of techniques. One such method is Genetic Bit Analysis, in which a DNA oligonucleotide strand that comprises the inserted DNA sequence and the adjacent flanking genomic DNA sequence is designed. The oligonucleotide filament is immobilized in the wells of a microwell plate. Following PCR amplification of the target region (using two primers, respectively, for the inserted sequence and the adjacent flanking genomic sequence), a single-stranded PCR product can be hybridized to the immobilized oligonucleotide and serves as a model for a reaction single base extension using a

DNA polymerase and ddNTPs specifically labeled for the next expected base. The result can be obtained by fluorescent methods or based on ELISA. A signal indicates the presence of the inserted / flanking genomic sequence, which demonstrates that amplification, hybridization and single base extension are successful.

[0080] Another method is the pyro-sequencing technique. In this method, an oligonucleotide strand that comprises the inserted DNA sequence and the junction part of the adjacent genomic DNA is designed. The oligonucleotide strand is hybridized with a single strand PCR product from the target region (using two primers, respectively, for the inserted sequence and the adjacent flanking genomic sequence) and incubated with a DNA polymerase, ATP, sulfurylase, luciferase, apyrase. , adenosine 5'-phosphosulfate and luciferin. The dNTPs are added separately and the light signal produced is measured. A light signal indicates the presence of the inserted / flanking sequence, which demonstrates that amplification, hybridization and extension of single or multiple bases are successful.

[0081] The fluorescence polarization phenomenon as described by Chen et al. (Genome Res., 1999, 9: 492-498) is also a method that can be used to detect the amplicon of the present invention. To use this method, an oligonucleotide strand that encompasses the inserted DNA sequence and the adjacent genomic DNA junction part needs to be designed. The oligonucleotide strand is hybridized to the single-stranded PCR product from the target region (using two primers, respectively, for the inserted sequence and the adjacent flanking genomic sequence) and incubated with fluorescently labeled DNA polymerase and ddNTP. The single base extension results in the incorporation of ddNTP. Such incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the inserted / flanking sequence, which demonstrates that amplification, hybridization and single base extension are successful.

[0082] Taqman is described as a method for the detection and quantitative analysis of the presence of a DNA sequence and is fully described in the instructions provided by the manufacturer. Now, it is briefly illustrated as follows. A FRET oligonucleotide probe covering the inserted DNA sequence and the adjacent flanking genomic junction part is designed. The FRET probe and PCR primers (using two primers, respectively, for the inserted sequence and the adjacent flanking genomic sequence) are subjected to reaction cycles in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in the cleavage between the fluorescent portion and the sudden cooling portion in the FRET probe and release of the fluorescent portion. The generation of a fluorescent signal indicates the presence of the inserted / flanking sequence, which demonstrates that amplification and hybridization are successful.

[0083] Based on the hybridization principle, suitable techniques for detecting plant materials from the DBN9501 transgenic corn event also comprise Southern blotting, Northern blotting and in situ hybridization. Particularly, suitable technologies involve incubating a probe with a sample, washing it to remove unbound probe, and detecting whether the probe has been hybridized. The detection method is dependent on the type of the marker attached to the probe, for example, a radioactively labeled probe can be detected by exposure and development of X-ray film, or an enzymatically labeled probe can be detected by converting a substrate to perform a color change.

[0084] The application of molecular markers in sequence detection has been described by Tyangi et al. (Nature. Biotech., 1996, 14: 303-308), which is briefly described as follows. A FRET oligonucleotide probe covering the inserted DNA sequence and the adjacent flanking genomic junction part is designed. Due to the unique structure of the FRET probe, it contains a secondary structure that keeps the fluorescent portion and the rough cooling portion in close proximity. The FRET probe and PCR primers (using two primers, respectively, for the inserted sequence and the flanking genomic sequence) are subjected to reaction cycles in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, hybridization of the FRET probe to the target sequence results in the loss of the secondary structure of the probe, so that the fluorescent portion and the rough cooling portion are spatially separated and a fluorescent signal is generated. The generation of a fluorescent signal indicates the presence of the inserted / flanking sequence, which demonstrates that amplification and hybridization are successful.

[0085] Other methods described, such as microfluidics, provide methods and devices for the separation and amplification of DNA samples. Optical dyes are used to detect and determine specific DNA molecules. Nantube devices that comprise an electronic sensor for the detection of DNA molecules or nanosphere for the binding of specific DNA molecules and thus can be detected are useful for the detection of the DNA molecules of the present invention.

[0086] DNA detection kits can be developed using the compositions described here and the methods described or known in the field of DNA detection. The kits are useful for identifying whether a sample contains DNA from the DBN9501 transgenic corn event and can be applied to create corn plants containing the DNA from the DBN9501 transgenic corn event. The kits may contain primers or DNA probes that are homologous or complementary to at least a portion of SEQ ID No .: 1, 2, 3, 4 or 5, or contain other primers or DNA probes homologous or complementary to the DNA contained in transgenic genetic elements of DNA, and these DNA sequences can be used in DNA amplification reactions or as probes in DNA hybridization methods. The DNA structure of the inserted transgenic sequence and the genomic junction part of the corn contained in the corn genome and illustrated in Figure 1 and Table 1 comprise: the genomic flanking region DBN9501 of the corn adjacent to the 5 'end of the inserted transgenic sequence; a portion of the sequence inserted from the left edge region (LB) of Agrobacterium; a first expression cassette consisting of a corn ubiquitin 1 promoter (prZmUbi1), operably linked to phosphorothricin-N-acetyltransferase (cPAT) tolerant to glufosinate from Streptomyces viridochromogenes, and operably linked to a nopaline synthase (tNos) terminator; a second expression cassette consisting of a cauliflower mosaic virus (pr35S) promoter containing a tandem duplication of an enhancer region, operably linked to a 70 kDa protein intron of Zea mays heat shock (iZmHSP70), operably linked to the insect-resistant protein Vip3Aa19 (cVip3Aa19) from Bacillus thuringiensis, and operably linked to a nopaline synthase (tNos) terminator; a portion of the sequence inserted from the Agrobacterium right edge (RB) region; and DBN9501 flanking genomic region of the corn plant at the 3 'end of the inserted transgenic sequence (SEQ ID No .: 5). In DNA amplification methods, DNA molecules useful as primers can be any portion of the inserted transgene sequence derived from the DBN9501 transgenic corn event, or any portion of the flanking corn genomic DNA sequence derived from the transgenic corn event DBN9501 .

[0087] The DBN9501 transgenic corn event can be used in combination with other transgenic corn varieties, for example, herbicide tolerant transgenic corn varieties (such as glyphosate and dicamba), or transgenic corn varieties carrying other insect resistant genes . Various combinations of these different transgenic events, when used to breed with the DBN9501 transgenic corn event of the present invention, can provide improved varieties of hybrid transgenic corn resistant to various insects and tolerant to various herbicides. These varieties exhibit better performances such as increased production, when compared to non-GM varieties and single-line GM varieties.

[0088] The DBN9501 transgenic corn event of the present invention is resistant to damage from the Lepidoptera pest feed, and tolerant to the phytotoxicity of agricultural herbicides containing glufosinate. The double-streaked maize plant expresses Bacillus thuringiensis Vip3Aa19 protein, providing resistance to damage from Lepidoptera pest feeding (such as Agrotis ypsilon Rottemberg), and expresses phosphorothricin-N-acetyl transferase (PAT) protein resistant to glufosinate from Streptomyces viridochromogenes, giving tolerance to glufosinate to the plant. Dual trait maize has the following advantages: 1) economic losses due to Lepidoptera pests (such as Agrotis ypsilon Rottemberg and Helicoverpa armigera Hubner) are avoided, in which Agrotis ypsilon Rottemberg and Helicoverpa armigera Hubner are the main pests in plantation areas. corn; 2) application of agricultural herbicides containing glufosinate gives corn a capacity to control a broad spectrum of weeds; and 3) corn production is not reduced. In addition, transgenes that encode insect-resistant and glufosinate-tolerant traits are linked to the same segment of DNA, and exist in a single location in the genome of the GMN9501 transgenic corn event, which provides enhanced breeding efficacy and allows tracking of transgenic fragments inserted in the population of propagules and their progeny by the use of molecular markers. Meanwhile, in the detection methods of the present invention, SEQ ID No .: 1 or a complementary sequence thereof, SEQ ID No .: 2 or a complementary sequence thereof, SEQ ID No .: 6 or a complementary sequence thereof, or SEQ ID No .: 7 or a complementary sequence can be used as primers or DNA probes to generate diagnostic amplification products for the DBN9501 transgenic corn event or progeny thereof, and can quickly, accurately and steadily identify the presence of plant materials from the DBN9501 transgenic corn event. Brief Description of the Strings

[0089] SEQ ID No .: 1, a sequence of 22 nucleotides in length that is located around the insertion junction at the 5 'end of the sequence inserted in the transgenic corn event DBN9501, where nucleotides 1-11 and nucleotides 12-22 are respectively located on either side of the maize genome insertion site;

[0090] SEQ ID No .: 2, a sequence of 22 nucleotides in length that is located around the insertion junction at the 3 'end of the sequence inserted in the transgenic corn event DBN9501, where nucleotides 1-11 and nucleotides 12-22 are respectively located on either side of the maize genome insertion site;

[0091] SEQ ID No .: 3, a 768 nucleotide sequence that is located around the insertion junction at the 5 'end of the sequence inserted in the DBN9501 transgenic corn event;

[0092] SEQ ID No .: 4, a 1339 nucleotide sequence that is located around the insertion junction at the 3 'end of the sequence inserted in the DBN9501 transgenic corn event;

[0093] SEQ ID No .: 5, the entire T-DNA sequence, genomic corn flanking sequences at the 5 'and 3' ends;

[0094] SEQ ID No .: 6, a sequence located in SEQ ID No .: 3, covering the left edge region (LB) and a tNos transcription termination sequence;

[0095] SEQ ID No .: 7, a sequence located in SEQ ID No .: 4, comprising a pr35S transcription start sequence and the right edge region (RB);

[0096] SEQ ID No .: 8, a first primer to amplify SEQ ID No .: 3;

[0097] SEQ ID No .: 9, a second primer to amplify SEQ ID No .: 3;

[0098] SEQ ID No .: 10, a first primer to amplify SEQ ID No .: 4;

[0099] SEQ ID No .: 11, a second primer to amplify SEQ ID No .: 4;

[0100] SEQ ID No .: 12, a 5 'flanking genomic sequence primer;

[0101] SEQ ID No .: 13, a T-DNA primer, which is paired with SEQ ID

No .: 12;

[0102] SEQ ID No .: 14, a 3 'flanking genomic sequence primer, which can be used in conjunction with SEQ ID No .: 12 to detect whether a transgene is homozygous or heterozygous;

[0103] SEQ ID No .: 15, a T-DNA primer, which is paired with SEQ ID No .: 14;

[0104] SEQ ID No .: 16, a first primer to detect the Vip3Aa19 gene in Taqman;

[0105] SEQ ID No .: 17, a second primer to detect the Vip3Aa19 gene in Taqman;

[0106] SEQ ID No .: 18, a probe to detect the Vip3Aa19 gene in Taqman;

[0107] SEQ ID No .: 19, a first primer to detect the pat gene in Taqman;

[0108] SEQ ID No .: 20, a second primer to detect pat in Taqman;

[0109] SEQ ID No .: 21, a probe to detect the pat gene in Taqman;

[0110] SEQ ID No .: 22, a first primer for the endogenous corn SSIIb gene;

[0111] SEQ ID No .: 23, a second primer for the endogenous corn SSIIb gene;

[0112] SEQ ID No .: 24, a probe for the Vip3Aa19 gene in the Southern blot assay;

[0113] SEQ ID No .: 25, a probe for the pat gene in the Southern blot assay;

[0114] SEQ ID No .: 26, a T-DNA primer, which has the same direction as SEQ ID No .: 13;

[0115] SEQ ID No .: 27, a T-DNA primer, which has an opposite direction to SEQ ID No .: 13 and is used to obtain a flanking sequence;

[0116] SEQ ID No .: 28, a T-DNA primer, which has an opposite direction to SEQ ID No .: 13 and is used to obtain a flanking sequence;

[0117] SEQ ID No .: 29, a T-DNA primer, which has the same direction as SEQ ID No .: 15;

[0118] SEQ ID No .: 30, a T-DNA primer, which has an opposite direction to SEQ ID No .: 15 and is used to obtain a flanking sequence;

[0119] SEQ ID No .: 31, a T-DNA primer, which has an opposite direction to SEQ ID No .: 15 and is used to obtain a flanking sequence.

[0120] The technical solutions of the present invention will be further described below in detail with reference to the figures and examples. Brief Description of the Figures

[0121] Figure 1 is a structural schematic of the junction sites between the inserted transgenic sequences and the corn genomes in the nucleic acid sequences for the detection of corn plants and methods of detecting them according to the present invention, and a scheme of relative positions of nucleic acid sequences for the detection of corn plant DBN9501 (for the scheme of relative positions, please refer to B73 RefGen v3);

[0122] Figure 2 is a structural schematic of the recombinant expression vector DBN10707 in the nucleic acid sequences for the detection of the DBN9501 corn plant and methods of detection thereof according to the present invention;

[0123] Figure 3 shows the field effect of the DBN9501 transgenic maize event on nucleic acid sequences for the detection of the DBN9501 maize plant and methods of detection according to the present invention, under the naturally occurring conditions of Agrotis ypsilon Rottemberg;

[0124] Figure 4 shows the field effect of the event of transgenic corn DBN9501 inoculated with Helicoverpa armigera Hubner in the nucleic acid sequences for the detection of the corn plant DBN9501 and methods of detection of these according to the present invention;

[0125] Figure 5 shows the field effect of the transgenic corn event

DBN9501 in the nucleic acid sequences for the detection of the DBN9501 maize plant and methods of detecting them according to the present invention, under the conditions of naturally occurring Spodoptera litura;

[0126] Figure 6 shows the field effect of the DBN9501 transgenic maize event on nucleic acid sequences for the detection of the DBN9501 maize plant and methods of detection according to the present invention, under the naturally occurring conditions of Spodoptera demand it. Detailed Description of the Invention

[0127] The technical solutions of the nucleic acid sequences for the detection of the DBN9501 maize plant and the methods of detecting them according to the present invention will be further illustrated below with reference to the specific examples. Example 1: Cloning and Transformation

1.1 Vector Cloning

[0128] A recombinant expression vector DBN10707 (as shown in Figure 2) is constructed using standard gene cloning techniques. The DBN10707 vector comprises two transgenic expression cassettes arranged in tandem: the first expression cassette consists of a cauliflower mosaic virus (pr35S) promoter containing a tandem repeat from an enhancer region, operably linked to a protein intron 70 kDa of Zea mays heat shock (iZmHSP70), operably linked to Bacillus thuringiensis insect resistant protein Vip3Aa19 (cVip3Aa19), and operably linked to a nopaline synthase (tNos) terminator; the second expression cassette consists of a corn ubiquitin 1 promoter (prZmUbi1), operably linked to a phosphorothricin-N-acetyl transferase (cPAT) tolerant to Streptomyces viridochromogenes glufosinate, and operably linked to a nopaline synthase terminator (tNos ).

[0129] The vector DBN10707 is transformed into Agrobacterium LBA4404 (Invitrogen, Chicago, USA; Cat. No .: 18313-015) using liquid nitrogen method, and 4- (hydroxy (methyl) phosphinylphosphinoyl) -DL-homoalanine is used as a selectable marker for screening transformed cells.

1.2 Transformation of Plants

[0130] Transformation is carried out by a conventional Agrobacterium-mediated transformation method, comprising: co-culture of the unripe embryos of sterile cutlived corn with Agrobacterium as described in Example 1.1 to transform T-DNA into the recombinant expression vector DBN10707 of the corn genome, in order to produce the DBN9501 transgenic corn event.

[0131] In summary, Agrobacterium-mediated maize transformation comprises: contact of unripe embryos isolated from maize with a suspension of Agrobacterium, in which Agrobacterium can transfer the nucleotide sequence of Vip3Aa19 and pat genes within at least one cell in one of the unripe embryos (stage 1: infection stage), and in this stage, the unripe embryos were preferably immersed in the Agrobacterium suspension (OD660 = 0.4-0.6, infection medium (4.3 g / L of salts MS, vitamins MS, 300 mg / L of casein, 68.5 g / L of sucrose, 36 g / L of glucose, 40 mg / L of acetosyringone (AS), 1 mg / L of acid 2,4- dichlorophenoxyacetic (2,4-D); pH 5.3)) to initiate inoculation. Unripe embryos were co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-culture step). Preferably, unripe embryos were grown in a solid medium (4.3 g / L of MS salts, vitamins MS, 300 mg / L of casein, 20 g / L of sucrose, 10 g / L of glucose, 100 mg / L of acetosyringone (AS), 1 mg / L of 2,4-dichlorophenoxyacetic acid (2,4-D), 8 g / L of agar; pH 5,8) after the infection stage. After the co-culture step, there may be an optional “recovery” step. In the “recovery” stage, a recovery medium (4.3 g / L of MS salts, vitamins MS, 300 mg / L of casein, 30 g / L of sucrose, 1 mg / L of 2,4-dichlorophenoxyacetic acid (2,4-D), 250 mg / L of cefamycin, 3 g / L of plant gel; pH 5.8) comprises at least one antibiotic (150-250 mg / L of cefamycin) known to inhibit the growth of

Agrobacterium, while it does not comprise any selective agent for plant transformers (step 3: recovery step). Preferably, the unripe embryos were cultured in a solid medium comprising antibiotic, but no selective agent to eliminate Agrobacterium and provide a recovery period for the infected cells. Next, the inoculated unripe embryos were cultured in a medium containing a selective agent 4- (hydroxy (methyl) phosphinyl) -DL-homoalanine and the transformed transformed calluses were selected (step 4: selection step). Preferably, the unripe embryos were grown in a solid selective medium (4.3 g / L of MS salts, vitamins MS, 300 mg / L of casein, 30 g / L of sucrose, 250 mg / L of cephamycin, 10 mg / L of 4- (hydroxy (methyl) phosphinyl) -DL- homoalanine, 1 mg / L of 2,4-dichlorophenoxyacetic acid (2,4-D), 3 g / L of plant gel; pH 5.8) comprising a selective agent, which results in the selective growth of the transformed cells. Then, the calluses regenerated in plants (step 5: regeneration step). Preferably, the calluses that develop in the medium containing a selective agent were grown in a solid medium (MS differential medium and MS rooting medium) to regenerate plants.

[0132] The resistant calluses obtained from the screening were transferred to the MS differential medium (4.3 g / L of MS salts, vitamins MS, 300 mg / L of casein, 30 g / L of sucrose, 2 mg / L of 6 -benzyladenine, 250 mg / L of cefamycin, 5 mg / L of 4- (hydroxy (methyl) phosphinyl) -DL-homoalanine, 3 g / L of plant gel; pH 5.8), and grown for differentiation at 25 ºC. The differentiated seedlings were transferred to the rooting medium MS (2.15 g / L of MS salts, vitamins MS, 300 mg / L of casein, 30 g / L of sucrose, 250 mg / L of cephamycin, 1 mg / L of indole-3-acetic acid, 3 g / L of plant gel; pH 5.8), and grown at 25 ºC. When the seedlings reached about 10 cm in height, they were moved to the greenhouse and cultivated until fruitified. In the greenhouse, they were grown at 28 ºC for 16 hours and then at 20 ºC for 8 hours a day.

1.3 Identification and screening of transgenic events

[0133] A total of 200 unique T0 independent transgenic plants have been produced.

[0134] Since genetic transformation and insertion of genes can affect the agronomic traits of corn plants (such as dwarf and thick stem, fasciculated leaf, mosaic leaf, opposite leaf, abnormality in the female flowering stage or pollen elimination deficient fruiting), the 200 independent T0 unique transgenic plants above were moved into the greenhouse for transplantation and cultivation to identify the agronomic trait performance of the unique T0 transgenic plants in different phases (seedling to junction phase, junction for pollen release phase and filling phase for maturity phase), and a total of 136 unique transgenic T0 plants with normal agronomic traits were obtained.

[0135] By the TaqManTM analysis, the 136 transgenic corn plants above were detected for the presence of single copy genes Vip3Aa19 and pat and the absence of main vector structure sequences; and a total of 83 unique T0 transgenic plants were obtained. By analyzing the transgenic insertion sites, a total of 28 unique transgenic T0 plants were screened, in which the sequences on both sides of T-DNA were intact, T-DNA was not inserted into important genes in the corn genome and the gene insertion did not result in any new open reading frame (ORF). By assessing and comparing resistance to the main target insects (such as Agrotis ypsilon Rottemberg, Prodenia litura, Prodenia litura and Spodoptera exigua), a total of 13 unique transgenic T0 plants with good resistance to insects were screened. By assessing and comparing tolerance to the herbicide glufosinate, a total of 12 unique T0 transgenic plants with good tolerance to the herbicide glufosinate were screened. Under the condition of different generations, different geographical environments and / or different base materials, by tracking whether the agronomic traits, molecular biology, resistance to target insects and tolerance to the herbicide glufosinate of the transgenic corn plant can be acquired in a stable manner, the event GMN9501 transgenic maize was selected as an excellent event, which has a single copy of the transgene (see Example 2), good resistance to insects, tolerance to the herbicide glufosinate and performance of the agronomic trait (see Examples 5 and 6). Example 2: Detection of the DBN9501 transgenic corn event by TaqMan

[0136] Using a plant DNA extraction kit (DNeasy Plant Maxi Kit, Qiagen), genomic DNA was extracted from the leaf (about 100 mg) of the DBN9501 transgenic corn event as a sample, and the copy numbers of Vip3Aa19 and pat genes were detected by a quantitative fluorescent PCR method using Taqman probe. Meanwhile, a wild-type corn plant as a control was subjected to detection and analysis according to the methods above. The experiments were carried out in triplicate and the average value was calculated.

[0137] The specific method is as follows:

[0138] Step 1: 100 mg of leaf (after pollination) of the DBN9501 transgenic corn event was ground in a homogenate in a liquid nitrogen mortar, and each sample was prepared in triplicate;

[0139] Step 2: the genomic DNAs from the samples above were extracted using Qiagen's DNeasy Plant Maxi Kit (for detailed methods, please refer to the product instructions);

[0140] Step 3: the concentrations of genomic DNA from the samples above were measured using an ultra micro-spectrophotometer (NanoDrop 2000, Thermo Scientific);

[0141] Step 4: the concentrations of genomic DNAs from the samples above were adjusted to the same concentration value ranging from 80 to 100 ng / μL;

[0142] Step 5: the number of copies of the samples was identified by a quantitative fluorescent PCR method using the Taqman probe, in which the sample identified with a known number of copies was used as a standard and a wild-type corn plant was used as a control sample. Each sample was tested in triplicate and the average value was calculated and taken. The sequences of primers and probes for fluorescent quantitative PCR are as follows:

[0143] The following primers and probe are used to detect the Vip3Aa19 gene sequence:

[0144] Primer 1: as defined in SEQ ID No .: 16 in the SEQUENCE LISTING;

[0145] Primer 2: as defined in SEQ ID No .: 17 in the SEQUENCE LISTING;

[0146] Probe 1: as defined in SEQ ID No .: 18 in the SEQUENCE LISTING;

[0147] The following primers and probe are used to detect the pat gene sequence:

[0148] Primer 3: as defined in SEQ ID No .: 19 in the SEQUENCE LISTING;

[0149] Primer 4: as defined in SEQ ID No .: 20 in the SEQUENCE LISTING;

[0150] Probe 2: as defined in SEQ ID No .: 21 in the SEQUENCE LISTING.

[0151] The PCR reaction system comprises: JumpStart ™ Taq ReadyMix ™ (Sigma) 10 μL 50 x primer / probe mixture 1 μL Genomic DNA 3 μL Double distilled water (ddH2O) 6 μL

[0152] Where the 50 x primer / probe mixture comprises 45 μL of each primer at a concentration of 1 mM, 50 μL of the probe at a concentration of 100 μM, and 860 μL of 1 x TE buffer (Tris-HCl 10 mM, EDTA 1 mM; pH 8.0), and is stored in an amber tube at 4 ºC.

[0153] The PCR reaction condition comprises: Stage Temperature Time

1 95 ºC 5 minutes 2 95 ºC 30 seconds 3 60 ºC 1 minute 4 return to step 2 and repeat 40 times

[0154] The data were analyzed using quantitative fluorescent PCR system software in fast real time (Applied Biosystems 7900HT Fast Real-Time PCR System SDS v2.3, Applied Biosystems), which demonstrated that the resulting DBN9501 transgenic corn event is a single copy. Example 3: Analysis of the insertion sites of the DBN9501 transgenic corn event

3.1 Extraction of genomic DNA

[0155] The DNA was extracted using the conventional CTAB (cetyl trimethyl ammonium bromide) method: after 2 g of young leaf from the DBN9501 transgenic corn event was ground into liquid nitrogen, 0.5 ml of extraction buffer CTAB DNA (20 g / L CTAB, 1.4 M NaCl, 100 mM Tris-HCl, and 20 mM EDTA (edetic acid), adjusted with NaOH to pH 8.0) preheated to 65 ° C was added, then they were mixed thoroughly and extracted at 65 ºC for 90 min; 0.5 volume of phenol and 0.5 volume of chloroform were added and mixed by inversion; the mixture was centrifuged at 12,000 rpm (revolution per minute) for 10 min; the supernatant was aspirated and 2 volumes of absolute ethanol were added; the centrifuge tube was gently shaken and then allowed to rest at 4 ºC for 30 min; the centrifuge tube was centrifuged again at 12,000 rpm for 10 min such that DNA was collected to the bottom of the tube; the supernatant was removed and the pellet was washed with 1 ml of 70% ethanol (mass concentration); the mixture was centrifuged at 12,000 rpm for 5 min; the pellet was vacuum-dried or blow-dried on an extremely clean bench; and the resulting DNA pellet was dissolved in an appropriate amount of TE buffer and stored at -20 ° C.

3.2 Flanking DNA sequence analysis

[0156] The concentration of the DNA sample extracted above was measured, and adjusted to 80-100 ng / μL. Genomic DNA was digested using Kpn I (5 'end analysis) and Spe I (3' end analysis) restriction enzymes, respectively. Each enzyme digestion system was added with 26.5 μL of genomic DNA, 0.5 μL of the restriction enzymes above and 3 μL of enzyme digestion buffer (all restriction enzymes employed are enzymes and assorted buffers of these or generic buffers of NEB company (now known as NEBCutSmart) and was digested for 1 hour, after digestion, the enzyme digestion system was added with 70 μL of anhydrous ethanol, kept in an ice bath for 30 min and centrifuged at 12,000 rpm for 7 min. After discarding the supernatant, the pellet was blow-dried, then added with 8.5 μL of double distilled water, 1 μL of 10 x T4 DNA ligase buffer (NEB T4 DNA Ligase Reaction Buffer; for your specific formulation, please , visit the NEB websites or see https://www.neb.com/products/restriction-endonucleases or https://www.neb.com/products/b0202-t4-dna-ligase-reaction-buffer) and 0 , 5 μL of T4 DNA ligase, and then ligated overnight at 4 º C. The genomic DNAs at the end 5 'and 3' were isolated by PCR amplification using a series of nested primers. Specifically, the primer combination to isolate genomic DNA at the 5 'end comprises SEQ ID No .: 13 and SEQ ID No .: 26 as first primers, SEQ ID No .: 27 and SEQ ID No .: 28 as second primers , and SEQ ID No .: 13 as a sequencing primer. The primer combination to isolate genomic DNA at the 3 'end comprises SEQ ID No .: 15 and SEQ ID No .: 29 as first primers, SEQ ID No .: 30 and SEQ ID No .: 31 as second primers, and SEQ ID No .: 15 as a sequencing primer. The conditions of the PCR reaction were presented in Table 3.

[0157] The PCR amplification product was separated by electrophoresis from the amplification product resulting from the above PCR amplification on an agarose gel with a mass fraction of 2.0% and isolation of target fragment from the agarose matrix using a kit gel extraction (QIAquick Gel Extraction Kit, catalog # _28704, Qiagen Inc., Valencia, CA). Next, the purified PCR amplification product hurts sequenced (for example, using ABI PrismTM 377, PE Biosystems, Foster City, CA) and analyzed (for example, using DNASTAR sequence analysis software, DNASTAR Inc., Madison, WI ).

[0158] The 5 'and 3' flanking and joining sequences were confirmed using standard PCR procedures. The 5 'flanking and joining sequences were confirmed 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 The 3 'flanking and joining sequences were confirmed 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 systems and the amplification conditions were presented in Tables 2 and 3. It will be recognized by those skilled in the art that other primer sequences could also be used to confirm the flanking and joining sequences.

[0159] DNA sequencing of PCR amplification products provided DNA that could be used to design other DNA molecules that could be used as primers and probes for the identification of corn plants or seeds derived from the DBN9501 transgenic corn event. .

[0160] The inserted sequence of the DBN9501 transgenic corn event was found to be flanked on the left edge (5 'flanking sequence) by the genomic corn sequence shown in nucleotides 1-384 of SEQ ID No .: 5 and flanked in right edge (3 'flanking sequence) by the maize genomic sequence shown in nucleotides 7785-8559 of SEQ ID No .: 5. The 5' junction sequence was defined in SEQ ID No .: 1 and the junction sequence in 3 'was defined in SEQ ID No .: 2.

3.3. PCR assay for zygosity

[0161] The junction sequence is a relatively short polynucleotide molecule, which is a new DNA sequence and is diagnostic for the DNA of the transgenic corn event DBN9501 when detected in the detection and analysis of polynucleotide. A junction sequence in SEQ ID No .: 1 and SEQ ID No .: 2, respectively, are 11 polynucleotides on both sides of the transgenic fragment insertion site and corn genomic DNA in the DBN9501 transgenic corn event. Longer and shorter polynucleotide junction sequences can be selected from SEQ ID No .: 3 or SEQ ID No .: 4. The junction sequences (SEQ ID No .: 1 used as the 5 'junction region, and the SEQ ID No .: 2 used as a 3 'junction region) were useful in the method for detecting DNA as DNA probes or DNA primer molecules. The junction sequences in SEQ ID No .: 6 and SEQ ID No .: 7 were also new DNA sequences from the DBN9501 transgenic corn event, and could also be used to detect the presence of the DBN9501 transgenic corn event as DNA probes or DNA primer molecules. SEQ ID No .: 6 (nucleotides 385-574 of SEQ ID No .: 3) covers a DNA sequence and a transcription termination sequence tNos in construct DBN10707, and SEQ ID No .: 7 (nucleotides 252 -451 of SEQ ID No .: 4) comprises a pr35S transcription start sequence and a DNA sequence in the construct DBN10707.

[0162] In addition, an amplicon was generated by using at least one primer derived from SEQ ID No .: 3 or SEQ ID No .: 4, and said primer, when used in a PCR method, generated a diagnostic amplicon for the DBN9501 transgenic corn event.

[0163] Specifically, the PCR amplification product was generated from the 5 'end of the inserted transgenic sequence, comprising a portion of the genomic DNA flanking the 5' end of the inserted T-DNA sequence in the genome of derived plant materials of the DBN9501 transgenic corn event. The PCR amplification product comprised SEQ ID No .: 3. For PCR amplification, primer 5 (SEQ ID No .: 8), which can hybridize to a genomic DNA sequence flanking the 5 'end of the sequence transgenic insert, and primer 6 (SEQ ID No .: 9), which was on par with primer 5 and located in a transcription termination sequence tNos of the inserted T-DNA sequence, were designed.

[0164] The PCR amplification product was generated from the 3 'end of the inserted transgenic sequence, comprising a portion of the genomic DNA flanking the 3' end of the inserted T-DNA sequence in the genome of plant materials derived from the event of transgenic corn DBN9501. The PCR product comprised SEQ ID No .: 4. For PCR amplification, primer 7 (SEQ ID No .: 10), which was located in a pr35S transcription start sequence of the inserted T-DNA sequence and primer 8 (SEQ ID No .: 11), which was paired with primer 7 and can hybridize with a genomic DNA sequence flanking the 3 'end of the inserted transgenic sequence, were designed.

[0165] The DNA amplification conditions described in Table 2 and Table 3 could be used in the above PCR assays for zygosity to generate a diagnostic amplicon for the DBN9501 transgenic corn event. The detection of amplicons could be conducted using Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf Mastercycler Gradient thermocycler and the like, or by the methods and apparatus known to those skilled in the art.

Table 2: The steps and conditions of the reaction mixture in the PCR reaction for the identification of the genomic junction region / transgenic insert of the 5 'end of the DBN9501 transgenic corn event Step Reagent Quantity Note added up to a 1 Water without nuclease final volume 20 μL

The final concentration of 1 x 10 x buffer buffer; at 2 2.0 μL reaction (with MgCl2) final concentration of MgCl2: 1.5 mM dATP solution, concentration 3 dCTP, dGTP and 0.2 μL final of each dTTP at 10 mM dNTP: 200 μM Primer 5 (SEQ ID No .: 8) resuspended in 1 x buffer of A 4 TE concentration or water without 0.2 μL final: 0.1 μM nuclease with a concentration of 10 μM Primer 6 (SEQ ID No .: 9) resuspended in 1 x buffer de The concentration 5 TE or water without 0.2 μL final: 0.1 μM nuclease with a concentration of 10 μM RNase; free from 6 0.1 μL 50 ng / DNase reaction (500 μg / mL) 1.0 μL (REDTaq® DNA is recommended if you change the 7 polymerase pipette (1 1 unit / reaction before the next unit / μL) step)

Extracted DNA (Model): 200 ng leaves of DNA from samples to be genomic to be analyzed 50 ng of DNA Non-GM genomic negative control 8 No model DNA (the Negative Control solution to resuspend the DNA) 50 ng of genomic DNA from Control positive including DBN9501 Table 3: Amplification conditions of the thermal cycler Cycle No Configuration 1 94 ºC, 3 minutes 94 ºC, 30 seconds 34 64 ºC, 30 seconds 72 ºC, 1 minute 1 72 ºC, 10 minutes

[0166] The reaction solutions were mixed gently, and if there is no hot top on the thermal cycler, added with 1-2 drops of mineral oil on the top. The PCR reaction was performed on the Stratagene Robocycler (Stratagene, La Jolla, CA), Motor MJ (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA), or on the Eppendorf Mastercycler Gradient thermal cycler (Eppendorf, Hamburg, Germany) using the cycle parameters shown in Table 3. The MJ Engine or Eppendorf Mastercycler Gradient thermal cycler should be run in the calculated mode. The increase speed of the Perkin-Elmer 9700 thermal cycler should be adjusted to the maximum when it is in operation.

[0167] The results show that: when used in a PCR reaction of the genomic DNA of the transgenic corn event DBN9501, primers 5 and 6 (SEQ ID No .: 8 and 9) result in the amplification product of a 768 bp fragment , but when primers were used in PCR reactions of unprocessed corn genomic DNA and non-DBN9501 corn genomic DNA, no fragments were amplified; when used in a PCR reaction of the genomic DNA of the transgenic corn event DBN9501, primers 7 and 8 (SEQ ID No .: 10 and 11) result in the amplification product of a 1339 bp fragment, but when the primers were used in PCR reactions of unprocessed corn genomic DNA and non-DBN9501 corn genomic DNA, no fragments were amplified.

[0168] PCR assays for zygosity could also be used to identify whether the material derived from the DBN9501 transgenic corn event is homozygous or heterozygous. Primer 9 (SEQ ID No .: 12), primer 10 (SEQ ID No .: 13), primer 11 (SEQ ID No .: 14) were used in the amplification reaction to produce an amplicon diagnosis for the event of transgenic corn DBN9501. The DNA amplification conditions described in Tables 4 and 5 could be used in the above assays for zygosity to produce a diagnostic amplicon for the DBN9501 transgenic corn event.

Table 4: Reaction solution for zygosity tests. Step Reagent Quantity Note Added up to 1 Water without nuclease a final volume of 5 μL

2 x Master Mix 1x Universal (Applied 2 2.5 μL Biosystems concentration, Final Catalog No 4304437) Primer 9 (SEQ ID No .: 12), primer 10 (SEQ ID No .: 13) resuspended The concentration 3 0.05 μL in water without final nuclease: 0.25 μM to a concentration of 10 μM Primer 11 (SEQ ID No .: 14) resuspended in 1 x The concentration 4 TE buffer or water 0.01 μL final: 0.15 μM without nuclease to a concentration of 10 μM 1.0 μL REDTaq® DNA (1 5 polymerase recommended (1 change unit / reaction unit / μL reaction pipette) before the next step) extracted DNA (model): 200 ng of DNA Sample sheets to genomic 6 50 ng genomic DNA of negative Control non-transgenic corn be analyzed

No model DNA (a Negative Control solution to resuspend the DNA) 50 ng of genomic DNA of positive Control corn comprising DBN9501 Table 5: Amplification conditions of the thermocycler for zygosity assays Cycle No Configuration 1 95 ºC, 10 minutes 95 ºC, 15 10 seconds 64 ºC 1 min (- 1 ºC / cycle) 95 ºC, 15 seconds 30 54 ºC, 1 minute 1 Immersion at 10 ºC

[0169] The PCR reaction was performed on the Stratagene Robocycler (Stratagene, La Jolla, CA), Motor MJ (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA), or on the thermal cycler Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) using the cycle parameters shown in Table 5. The MJ Engine or Eppendorf Mastercycler Gradient thermal cycler should be run in the calculated mode. The increase speed of the Perkin-Elmer 9700 thermal cycler should be adjusted to the maximum when it is in operation.

[0170] In the amplification reaction, the biological sample comprising model DNA contains the diagnostic DNA for the presence of the DBN9501 transgenic corn event. Or the amplification reaction would result in two different DNA amplicons generated from the biological sample comprising the DNA derived from the corn genome, and the DNA derived from the corn genome is heterozygous with respect to the corresponding allele of the DNA inserted in the transgenic corn event. DBN9501. Two different amplicons would correspond to a first amplicon (SEQ ID No .: 12 and SEQ ID No .: 14) derived from the wild-type corn genome site and a second amplicon (SEQ ID No .: 12 and SEQ ID No .: 13) diagnosis of the presence of the event of transgenic corn DBN9501 DNA. The result that a corn DNA sample only generates a single amplicon corresponding to the second amplicon as described in relation to the heterozygous genome could be indicative of the presence of the DBN9501 transgenic corn event in that sample, and the sample was produced from seeds of corn homozygous in relation to the corresponding allele of the insert DNA in the transgenic corn plant DBN9501.

[0171] It should be noted that the primer pairs of the DBN9501 transgenic corn event are used to produce a diagnostic amplicon for the genomic DNA of the DBN9501 transgenic corn event. These primer pairs include, but are not limited to, primers 5 and 6 (SEQ ID No .: 8 and SEQ ID No .: 9), and primers 7 and 8 (SEQ ID No .: 10 and SEQ ID No .: 11), and are used in the DNA amplification method. In addition, control primers 12 and 13 (SEQ ID No .: 22 and 23) for amplification of an endogenous corn gene are included as an internal standard for reaction conditions. Analysis of the DNA extraction sample from the DBN9501 transgenic corn event should include a control of the positive tissue DNA extract from the DBN9501 transgenic corn event, a control of the negative DNA extract from a non-corn plant is the DBN9501 transgenic corn event, and a negative control that contains no model corn DNA extracts. In addition to these primer pairs, any primer pair of SEQ ID No .: 3 or a complementary sequence thereof, or SEQ ID No .: 4 or a complementary sequence thereof, can be used. When these primers are used for the DNA amplification reaction, they respectively produce amplicons that comprise SEQ ID No .: 1 or SEQ ID No .: 2 and are diagnostic for the transgenic corn plant tissue derived from the DBN9501 transgenic corn event. . DNA amplification conditions as described in Tables 2 to 5 can be used in the production of diagnostic amplicon for the DBN9501 transgenic corn event by using suitable primer pairs. The DNA extract from the corn plant or seed that produces a diagnostic amplicon for the DBN9501 transgenic corn event in a DNA amplification method, the DNA extract from the corn plant or seed that is presumed to contain the DBN9501 transgenic corn event , or a product derived from the DBN9501 transgenic corn event can be used as a model for amplification to determine the presence of the DBN9501 transgenic corn event. Example 4: Detection of the DBN9501 transgenic corn event by Southern blotting

4.1 Extraction of DNA for use in Southern blotting

[0172] Approximately 5 to 10 g of leaf tissue was crushed in liquid nitrogen using a mortar and pestle. 4 to 5 g of the crushed leaf tissue were resuspended in 20 mL of CTAB lysis buffer (100 mM Tris-HCl, pH 8.0, 20 mM EDTA, pH 8.0, 1.4 M NaCl, β-mercaptoethanol 0.2% v / v, CTAB 2% w / v), and incubated at a temperature of 65 ºC for 60 minutes. During the incubation, the sample was mixed uniformly by inversion once every 10 minutes. After incubation, an equal volume of phenol / chloroform / isoamyl alcohol (25: 24: 1) was added, and mixed gently by inversion for extraction, and then centrifuged at a rotation speed of 4,000 rpm for 20 minutes. The aqueous phase was collected and re-extracted once with an equal volume of chloroform / isoamyl alcohol (24: 1). The aqueous phase was collected and added with an equal volume of isopropanol. The mixture was mixed uniformly, allowed to stand at a temperature of -20 ºC for 1 hour to precipitate DNA, and then centrifuged at a rotation speed of 4,000 rpm for 5 minutes to obtain the DNA pellet, which was subsequently resuspended in 1 mL. of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). In order to degrade any possible RNA, the DNA was incubated at a temperature of 37 ºC for 30 minutes with 40 µL of 10 mg / mL of RNAase A, centrifuged at a rotation speed of 4,000 rpm for 5 minutes, and then centrifuged at a rotation speed of 12,000 rpm for 10 minutes to precipitate DNA in the presence of 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of anhydrous ethanol. After the supernatant was discarded, the pellet was washed with 1 ml of 70% (v / v) ethanol and dried at room temperature, and then the DNA was redissolved in 1 ml of TE buffer.

4.2 Digestion of the restriction enzyme

[0173] The concentration of genomic DNA in the sample mentioned above was measured using an ultra microvolume spectrophotometer (NanoDrop 2000, Thermo Scientific).

[0174] In the 100 μL reaction system, 5 μg of DNA was used for digestion. Genomic DNA was digested, respectively, with restriction enzymes Nco I and Nde I, using a portion of the Vip3Aa19 and pat gene sequences in T-DNA as probes. For each enzyme, the digestion products were incubated overnight at an appropriate temperature. The sample was rotated in a centrifugal vacuum concentrator (Speed Vacuum, Thermo Scientific) to reduce the volume to 20 μl.

4.3 Gel Electrophoresis

[0175] Bromophenol blue loading buffer was added to each sample in Example 4.2 and each sample was loaded onto a TAE agarose gel containing 0.7% ethidium bromide. The mixture was separated by electrophoresis using TAE electrophoresis buffer (40 mM tris-acetic acid, 2 mM EDTA, pH 8.5), and gel electrophoresis was performed overnight at a voltage of 20 V.

[0176] After electrophoresis, the gel was treated with 0.25 M HCl for 10 minutes to purify the DNA, and then treated with a denaturation solution (1.5 M NaCl, 0.5 M NaOH) and a solution neutralization (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.2) for 30 minutes, respectively. 5 × SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) were poured onto a porcelain plate, a piece of glass was covered, and then a bridge of moist filter paper, a gel, a positively charged nylon membrane (Roche, Cat. No. 11417240001), three pieces of filter paper, a paper tower, and a heavy object were placed in succession. After transferring the membrane at room temperature overnight, the nylon membrane was rinsed with deionized water twice and the DNA was immobilized on the membrane by an ultraviolet crosslinker (UVP, UV Crosslinker CL-1000).

4.4 Hybridization

[0177] A suitable DNA sequence was amplified by PCR for the preparation of the probe. The DNA probes were SEQ ID No .: 24 or SEQ ID No .: 25, or partially homologous or complementary to the above sequences. DIG labeling of the probes, Southern blotting, membrane washing, and other operations were performed using the DNA Labeling and Detection Starter Kit II kit (Roche, Cat. No 11585614910. For specific methods, please refer to your product instructions Finally, an X-ray film (Roche, Cat. No 11666916001) was used to detect the position where the probe joined.

[0178] Each Southern blotting included two control samples: (1) the DNA of the negative (untransformed) segregant, which was used to identify any endogenous corn sequences that could hybridize with the specific probe of the element; and (2) the DNA of the negative segregant, into which the plasmid DBN10707 digested by Nco I was introduced with an amount equivalent to a number of single copies based on the length of the probe, which was used as a positive control for hybridization and to show the sensitivity of the experiment.

[0179] Hybridization data provided corroborative evidence to support the TaqManTM PCR analysis, namely, the DBN9501 corn plant contained Vip3Aa19 and pat genes in a single copy. By using the Vip3Aa19 gene probe, the enzymatic digestion of Nco I and Ned I, respectively, generated unique bands of about 11 kb and 9 kb; and by using the pat gene probe, the enzymatic digestion of Nco I and Ned I, respectively, generated unique bands of about 8.5 kb and 1.3 kb. This indicated that the Vip3Aa19 and pat genes were, respectively, present in the corn plant DBN9501 in one copy. In addition, no hybridization band was obtained using the main frame probe. This indicated that no main structure sequence of the DBN10707 vector was introduced into the genome of the DBN9501 corn plant during transformation. Example 5: Detection of Event Insect Resistance

5.1 Bioassay of the DBN9501 corn plant

[0180] Two types of plants, the DBN9501 transgenic corn event and the wild type (non-transgenic, NGM) corn plant, were tested using Agrotis ypsilon Rottemberg (BCW), Spodoptera litura (TCW), Spodoptera exigua (BAW) , Sesamia inferens (PSB), Chilo sacchariphagus (SGB) and Chilo infuscatellus (MSB) using the following bioassays:

[0181] The fresh leaves (period V3-V4) of the two types of plants, the transgenic corn event DBN9501 and the wild type corn plant (non-transgenic, NGM), were prepared, respectively, rinsed with sterile water and water aspirated with gauze. Following the removal of the veins, the corn leaves were cut into long strips of about 1 cm × 3 cm. 1 to 3 pieces of the long strip sheet (the sheet number was determined based on the insect's food intake) were placed on the filter paper moistened with distilled water at the bottom of the rounded plastic Petri dishes. 5-10 larvae newly hatched by artificial feeding were placed on each plate. Then, the Petri dish was covered with lids and kept for 3 days under conditions including a temperature of 26-28 ºC, a relative humidity of 70% -80%, and a photoperiod (light / dark) of 16: 8 before obtaining a statistical result. Three indicators, rate of larvae development, mortality and leaf damage ratio, were analyzed statistically to obtain a total score of resistant trait (total score: 300 points). The total score of resistant trait = 100 × mortality + [100 × mortality + 90 × (number of newly hatched insects / larvae density) + 60 × (number of newly hatched insects - number of negative control insects / density of larvae) larvae) + 10 × (number of negative control insects / larvae density)] + 100 × (1 - leaf damage ratio), where the larvae density refers to the number of inoculated larvae, that is, 5 or 10 larvae (depending on the insect's food intake) on each plate; the larvae development rate was reflected by the formula of the total resistant trait score; and the leaf damage ratio refers to the proportion of the insect's feeding area in the total leaf area. For both the DBN9501 transgenic corn event and the wild type (non-transgenic, NGM) corn plant, five plants were tested and each sample was repeated six times. The results were shown in Tables 6 and 7.

Table 6: The result of the insect resistance bioassay of the transgenic corn event DBN9501 - Mortality (%) Insect / plant DBN9501 NGM BCW 100 17 TCW 100 8 BAW 100 12 PSB 96 5 SGB 80 1 MSB 100 3

Table 7: The result of the insect resistance bioassay of the transgenic corn event DBN9501 - Total resistance score (%).

Insects / plants DBN9501 NGM BCW 300 69 TCW 300 56 BAW 300 71 PSB 288 36 SGB 270 25 MSB 300 33

[0182] The results show that the DBN9501 transgenic corn event has good resistance to Agrotis ypsilon Rottemberg, Spodoptera litura, Spodoptera exigua, Sesamia inferens, Chilo sacchariphagus and Chilo infuscatellus, and the DBN9501 transgenic corn event shows significantly higher insect mortality tested and total score of resistant trait than the NGM.

5.2 Field effect of the DBN9501 transgenic corn event

[0183] The seeds of two types of plants, the DBN9501 transgenic corn event and wild type (non-transgenic, NGM) corn plant were designed as two treatments. Each treatment followed a randomized block design with three replicates. The site had an area of 30 m2 (5 m × 6 m), row spacing of 60 cm and plant spacing of 25 cm. The plants were cultivated conventionally and managed, avoiding spraying of insecticides throughout the growing period. The two-meter interval was adjusted between the experimental sites for inoculations of different insects to avoid spreading the insects in different locations.

(1) Agrotis ypsilon Rottemberg

[0184] Corns were only subjected to natural infestation in the area with serious natural occurrence of Agrotis ypsilon Rottemberg (the conditions for the occurrence of natural damage to insects: the larvae of Agrotis ypsilon Rottemberg that caused damage are the first generation larvae of Agrotis ypsilon Rottemberg which develop easily under suitable environmental conditions, for example, a temperature of 16-26 ºC, relative humidity of 80-90%, and soil moisture content of 15-20%, or when the first generation adults obtained with traps reach a certain amount, for example, 20 or more). In the corn seedling stage, when the materials were around the V2-V3 period, and the corn plants developed to the leaf 2-3 expansion stage, they were screened to investigate whether the plant wilting occurred in the NGM. When most of the wilted plants in the NGM were damaged near the rhizosphere by old larvae with a 4 to 6 year old instar, the damage rate (the damage rate = the number of insect-fed maize plants / the total number of insects plants × 100%) of Agrotis ypsilon Rottemberg in corn plants was investigated one by one. The result of resistance to Agrotis ypsilon Rottemberg from the DBN9501 transgenic corn event is shown in Table 8.

Table 8: The result of resistance to Agrotis ypsilon Rottemberg from the DBN9501 transgenic corn event under the condition of natural infestation Program / plant DBN9501 NGM Damage rate (%) 0 10

[0185] The results show that under the naturally occurring conditions of Agrotis ypsilon Rottemberg, when compared to NGM, the event of transgenic corn DBN9501 has a significantly reduced damage rate of Agrotis ypsilon Rottemberg, indicating that the event of transgenic corn DBN9501 has good resistance to Agrotis ypsilon Rottemberg. The field effect of the DBN9501 transgenic corn event under the naturally occurring conditions of Agrotis ypsilon Rottemberg is shown in Figure 3.

(2) Helicoverpa armigera Hubner (CBW)

[0186] Artificial inoculation was performed twice in the female flowering stage of corn. No less than 40 plants at each site were subjected to artificial inoculation. The filaments of each corn plant were subjected to inoculation of about 20 newly hatched larvae by artificial feeding. 3 days after inoculation, a second inoculation was performed with the same larval density as the first inoculation. 14-21 days after inoculation, the rate of damage to the female ears, the number of surviving larvae on each female ear and the extent of damage to the female ears were investigated one by one. The investigation was generally carried out 14 days after inoculation. If the level of damage of a negative control material (NGM) reached sensitivity or high sensitivity, it was considered valid. If the level of damage did not reach sensitivity or high sensitivity, the investigation could be delayed appropriately; however, if it has not yet reached the corresponding level after 21 days, this inoculation was considered invalid. According to the damage rate of female ears, the number of surviving larvae and the extent of damage (cm) of female ears, the average level of Helicoverpa armigera Hubner damage from female ears of corn during the ear stage in each location is calculated, and the pattern is shown in Table 9. The level of corn resistance in the ear stage to Helicoverpa armigera Hubner is determined according to the criteria in Table 10. The result of resistance to Helicoverpa armigera Hubner from the corn event transgenic DBN9501 in the female flowering stage is shown in Table 11.

Table 9: The classification pattern of the degree of damage of Helicoverpa armigera Hubner on female ears of corn Damage level of Description of symptoms female ears 0 Female ears do not suffer damage.

1 Only the filaments are damaged 2 The top of the ears is damaged by 1 cm As the damage increases by 1 cm from the top of the 3+ ears, the level of damage increases correspondingly by one level. … N Table 10: Criterion for assessing resistance to Helicoverpa armigera Hubner from female corncobs The average level of damage from the ears Female resistance level 0-1.0 High resistance (HR) 1.1-3.0 Resistance (R ) 3.1-5.0 Intermediate resistance (MR) 5.1-7.0 Sensitivity (S) 7.1 High sensitivity (HS) Table 11: The result of resistance to Helicoverpa armigera Hubner from the DBN9501 transgenic corn event in the female flowering stage Program / plant DBN9501 NGM Ear damage rate 58 100 females Number of surviving larvae 0 0.17 Ear damage extent 1.1 4.9 females Ear damage level 2.1 5.9 females RS resistance level

[0187] The results show that under the condition of artificial inoculation, the event of transgenic corn DBN9501 has a significantly lower damage rate of female ears, number of surviving larvae, extent of damage of female ears, and level of damage of female ears of the than the NGM, thus indicating that the level of resistance to Helicoverpa armigera Hubner from the DBN9501 transgenic corn event is (R). The field effect of the DBN9501 transgenic corn event inoculated with Helicoverpa armigera Hubner is shown in Figure 4.

(3) Spodoptera litura (TCW)

[0188] Corns were only subjected to natural infestation in the area with serious natural occurrence of Spodoptera litura (the conditions for the occurrence of natural damage to insects: the most serious damage occurs in July to September and the optimum temperature for the development of the pests varies from 28 to 30 ºC). 10-15 days after the first insect damage and most of the NGM was damaged by old larvae with a 4 to 6 year old instar, the damage rate of Spodoptera litura in corn plants was investigated one by one (the rate damage = the number of maize plants fed by insects / the total number of plants × 100%). The result of resistance to Spodoptera litura from the DBN9501 transgenic corn event is shown in Table 12.

Table 12: The result of resistance to Spodoptera litura of the event of transgenic corn DBN9501 under the condition of natural infestation Program / plant DBN9501 NGM Damage rate (%) 0 38

[0189] The results show that under the condition of natural occurrence of Spodoptera litura, when compared with NGM, the event of transgenic corn DBN9501 has a significantly reduced damage rate of Spodoptera litura, thus indicating that the event of transgenic corn DBN9501 has good resistance to Spodoptera litura. The field effect of the DBN9501 transgenic corn event under the naturally occurring conditions of Spodoptera litura is shown in Figure 5.

(4) Spodoptera exigua (BAW)

[0190] The experiment was carried out using substantially the same design and method as those described above for the evaluation of resistance to Spodoptera litura, except that 10-15 days after the first damage of the insects and most of the NGM was damaged by old larvae with a 4 to 5-year-old instar, the damage rate of Spodoptera exigua in corn plants was investigated one by one (the damage rate = the number of insect-fed corn plants / the total number of plants × 100% ). The result of resistance to Spodoptera exigua from the DBN9501 transgenic corn event is shown in Table 13.

Table 13: The result of resistance to Spodoptera exigua from the event of transgenic corn DBN9501 under the condition of natural infestation Program / plant DBN9501 NGM Damage rate (%) 0 56

[0191] The results show that under the condition of natural occurrence of Spodoptera exigua, when compared to NGM, the event of transgenic corn DBN9501 has a significantly reduced damage rate of Spodoptera exigua, indicating that the event of transgenic corn DBN9501 has good resistance Spodoptera exigua. The field effect of the DBN9501 transgenic corn event under the naturally occurring conditions of Spodoptera exigua is shown in Figure 6.

[0192] It should be noted in particular that, according to the disclosures as described in Chinese patent applications Nos. 201310289848.6,

201310573441.6, 201410806573.3, 201510259396.6 and 201610006375.8 and the results of the field effects against insects from the transgenic corn event

DBN9501 of the present application and their bioassays, the DBN9501 transgenic corn event of the present application allows the method and / or use of pest control, in particular, Sesamia inferens, Spodoptera litura, Chilo infuscatellus, Chilo sacchariphagus and Conogethes punctiferalis; that is, any transgenic corn plant that expresses Vip3Aa19 protein may allow the method and / or use of the control of Sesamia inferens, Spodoptera litura, Chilo infuscatellus, Chilo sacchariphagus and / or Conogethes punctiferalis.

Example 6: Detection of herbicide tolerance of the event

[0193] The herbicide Basta (in which the active ingredient is 18% aqueous solution of glufosinate-ammonium) was selected for spray application in the experiment. The randomized block design with three replicates was employed. The site had an area of 15 m2 (5 m x 3 m), row spacing of 60 cm and plant spacing of 25 cm. The plants were grown and managed conventionally and an interval of one meter was adjusted between sites. The DBN9501 transgenic corn event was subjected to the following two treatments: (1) no spray application, but spraying an equal volume of clean water when spraying the herbicide during treatment (2); and (2) spraying herbicide Basta in leaf stage V2-V3 in a dose of 800 g a.i./ha (a.i./ha means “active ingredient per hectare”). It should be noted that the herbicide glufosinate (such as Basta) is a contact type herbicide; if the herbicide is handled inappropriately in the field, for example, by accumulating too much herbicide solution locally, phytotoxicity symptoms may occur; this does not mean that the tolerance of the DBN9501 transgenic corn event has problems; and glufosinate herbicides of various concentrations and formulations are also applicable to the following conclusion if they are converted into the equivalent amount above the active ingredient glufosinate.

[0194] The investigation of phytotoxicity symptoms was carried out, respectively, at 1 week and 2 weeks after application and the yields of the sites were determined at the time of collection. The classification of phytotoxicity symptoms was shown in Table 14. Herbicide tolerance of the transformation event was assessed by using the herbicide damage rate as an indicator. In particular, the herbicide damage rate (%) = Σ (number of plants at the same damage level × level number) / (total number of plants × highest level), where the herbicide damage rate referred to damage rate of glufosinate, which was determined according to the result of the phytotoxicity investigation two weeks after treatment with glufosinate; and the level of corn tolerance to the herbicide was evaluated based on the damage rate of the herbicide (glufosinate). The corn yield of each location was obtained by weighing the total yield (by weight) of the 3-row corn kernels in the local half decade. The difference in yield between the different treatments was measured as a percentage of yield. The percentage of yield (%) = yield with spray application / yield without spray application. The results of herbicide tolerance of the DBN9501 transgenic corn event and corn yield were shown in Table 15.

Table 14: Classification standard for the degree of phytotoxicity of the herbicide glufosinate on corn Level of symptom description Phytotoxicity 1 Grows regularly without any symptoms of damage 2 Slight phytotoxicity less than 10% Intermediate phytotoxicity, which can be recovered 3 later without affecting yield Severe phytotoxicity that is difficult to recover and that 4 result in reduced yield

Severe phytotoxicity that cannot be recovered and 5 that results in significantly reduced or zero yield Table 15: Results of tolerance to the herbicide glufosinate from the event of transgenic corn DBN9501 and the yield of corn Program / plant DBN9501 Glufosinate damage rate,% (control ) 0% Glufosinate damage rate,% (800 g ai / ha) 0% Yield percentage,% (800 g ai / ha) 101.9%

[0195] The results show that, in terms of the damage rate by the herbicide glufosinate: the damage rate of the event of transgenic corn DBN9501 treated with herbicide glufosinate (800 g ai / ha) was 0. Thus, the event of transgenic corn DBN9501 has good tolerance to the herbicide glufosinate.

[0196] Regarding the yield, there is no significant difference in the yield of the DBN9501 transgenic corn event between treatment without spray application and treatment with spray application of 800 g a.i./ha of glufosinate. After being sprayed with the herbicide glufosinate, the event of transgenic corn DBN9501 has a slightly increased yield. Thus, this still shows that the DBN9501 transgenic corn event has good tolerance to the herbicide glufosinate.

Example 7

[0197] Agricultural products or commodities can be produced from the DBN9501 transgenic corn event. If sufficient expression is detected in agricultural products or commodities, they are expected to contain the nucleotide sequence that can be diagnostic for the presence of the DBN9501 transgenic corn event material in agricultural products or commodities. Agricultural products or commodities include, but are not limited to, corn oil, corn meal, corn flour, corn gluten, corn cakes, corn starch, and any other food intended for consumption as a food source by an animal, or otherwise, any other materials intended as a bulking agent or as a component in a cosmetic composition for cosmetic use, etc. Nucleic acid detection methods and / or kits based on probe or primer pairs can be developed to detect the nucleotide sequence of the DBN9501 transgenic corn event in a biological sample, such as SEQ ID No .: 1 or SEQ ID No .: 2, in order to diagnose the presence of the DBN9501 transgenic corn event, in which the probe sequence or primer sequence is selected from the sequences as shown in SEQ ID No .: 1, SEQ ID No .: 2, SEQ ID No .: 3, SEQ ID No .: 4 and SEQ ID No .: 5 or portions thereof.

[0198] In conclusion, the DBN9501 transgenic corn event of the present invention has good resistance to Lepidoptera insects and high tolerance to the herbicide glufosinate without compromising yield, and the detection methods of the present invention can accurately and quickly identify whether a biological sample contains the DNA molecule from the transgenic corn event DBN9501.

[0199] The seeds corresponding to the DBN9501 transgenic corn event were deposited according to the Budapest Treaty at the General Microbiological Center of China Microbiological Culture Collection Management Committee (CGMCC, Address: No. 3, No. 1 Precinct, Beichen West Road, Chaoyang District. Beijing, Institute of Microbiology, Academy of Sciences of China, zip code 100101) on January 23, 2019, whose classification and nomenclature are maize (Zea mays), and accession number is CGMCC No.

17099. The deposit will be deposited with the depositary for 30 years.

[0200] Finally, it should be noted that the Examples above are used only to illustrate the technical solutions of the present invention instead of limiting the present invention. Although the present invention is described in detail with reference to the Preferred Examples, those skilled in the art should understand that the technical solutions of the present invention could be modified or replaced in an equivalent manner without departing from the spirit and scope of the technical solutions of the present invention.

Claims (13)

Claims 1. Nucleic acid sequence, characterized by comprising at least 11 consecutive nucleotides in positions 1-384 of SEQ ID No .: 3 or a complementary sequence thereof, and at least 11 consecutive nucleotides in positions 385-768 of SEQ ID No .: 3 or a complementary sequence thereof; and / or at least 11 consecutive nucleotides in positions 1-564 of SEQ ID No .: 4 or a complementary sequence thereof, and at least 11 consecutive nucleotides in positions 565-1339 of SEQ ID No .: 4 or a complementary sequence thereof; preferably, the nucleic acid sequence comprises 22 to 25 consecutive nucleotides at positions 1-384 of SEQ ID No .: 3 or a complementary sequence thereof, and 22 to 25 consecutive nucleotides at positions 385-768 of SEQ ID No .: 3 or a complementary sequence thereof; and / or 22 to 25 consecutive nucleotides in positions 1-564 of SEQ ID No .: 4 or a complementary sequence thereof, and 22 to 25 consecutive nucleotides in positions 565-1339 of SEQ ID No .: 4 or a complementary sequence of this; preferably, the nucleic acid sequence comprises SEQ ID No .: 1 or a complementary sequence thereof, and / or SEQ ID No .: 2 or a complementary sequence thereof; preferably, the nucleic acid sequence comprises SEQ ID No .: 3 or a complementary sequence thereof, and / or SEQ ID No .: 4 or a complementary sequence thereof. Nucleic acid sequence according to claim 1, characterized in that the nucleic acid sequence comprises SEQ ID No .: 5 or a complementary sequence thereof. 3. Method for detecting the presence of the DNA of the transgenic corn event DBN9501 in a sample, characterized by comprising: - contact of the sample to be detected with at least two primers to amplify a target amplification product in a nucleic acid amplification reaction ; - carrying out the nucleic acid amplification reaction; e - detection of the presence of the target amplification product; wherein the target amplification product comprises the nucleic acid sequence as defined in claim 1 or 2; preferably, the target amplification product comprises SEQ ID No .: 1 or a complementary sequence thereof, SEQ ID No .: 2 or a complementary sequence thereof, SEQ ID No .: 6 or a complementary sequence thereof, and / or SEQ ID No .: 7 or a complementary sequence of this. 4. Method for detecting the presence of the DNA of the transgenic corn event DBN9501 in a sample, according to claim 3, characterized by the primers comprising a first primer and a second primer, in which the first primer is selected from the group consisting of the SEQ ID No .: 1, SEQ ID No .: 8 and SEQ ID No .: 10; and the second primer is selected from the group consisting of SEQ ID No .: 2, SEQ ID No .: 9 and SEQ ID No .: 11. 5. Method for detecting the presence of the DNA of the transgenic corn event DBN9501 in a sample, characterized by comprising: - contact of the sample to be detected with a probe, in which the probe comprises the nucleic acid sequence as defined in claim 1; preferably, the probe comprises SEQ ID No .: 1 or a complementary sequence thereof, SEQ ID No .: 2 or a complementary sequence thereof, SEQ ID No .: 6 or a complementary sequence thereof, and / or SEQ ID No .: 7 or a complementary sequence thereof; - hybridization of the sample to be detected with the probe under strict hybridization conditions; and - hybridization detection of the sample to be detected with the probe. 6. Method for detecting the presence of the DNA of the transgenic corn event DBN9501 in a sample, according to claim 5, characterized in that at least one probe is labeled with at least one fluorophore. 7. Method for detecting the presence of the DNA of the transgenic corn event DBN9501 in a sample, characterized by comprising: - contact of the sample to be detected with a nucleic acid marker molecule, in which the nucleic acid marker molecule comprises the sequence of nucleic acids as defined in claim 1; preferably, the nucleic acid marker molecule comprises at least one sequence selected from the group consisting of SEQ ID No .: 1 or a complementary sequence thereof, SEQ ID No .: 2 or a complementary sequence thereof, and / or SEQ ID Nos. : 6 to 11 or complementary sequences of these; - hybridization of the sample to be detected with the nucleic acid marker molecule under strict hybridization conditions; - detection of the hybridization of the sample to be detected with the nucleic acid marker molecule, and, also, a breeding analysis aided by marker to determine whether insect resistance and / or herbicide tolerance is genetically linked to the marker molecule. nucleic acid. 8. DNA detection kit, characterized by comprising at least one DNA molecule, in which the DNA molecule comprises the nucleic acid sequence as defined in claim 1, and the DNA molecule can act as a DNA primer or a specific probe for the event of transgenic corn DBN9501 or progeny thereof; preferably, the DNA molecule comprises SEQ ID No .: 1 or a complementary sequence thereof, SEQ ID No .: 2 or a complementary sequence thereof, SEQ ID No .: 6 or a complementary sequence thereof, and / or SEQ ID No .: 7 or a complementary sequence of this. 9. Method to protect a corn plant against insect invasion, characterized by comprising the provision of at least one transgenic corn plant cell in the target insect's diet, in which the transgenic corn plant cell comprises, in its genome , the sequence as defined in SEQ ID No .: 1, and / or SEQ ID No .: 2; and the ingestion of the transgenic corn plant cell inhibits the target insect from still feeding on the transgenic corn plant; preferably, the transgenic corn plant cell comprises, in its genome, the sequence as defined in SEQ ID No .: 3, and / or SEQ ID No .: 4; preferably, the transgenic maize plant cell successively comprises, in its genome, SEQ ID No .: 1, the nucleic acid sequence at positions 553-7491 of SEQ ID No .: 5 and SEQ ID No .: 2, or comprises the sequence as defined in SEQ ID No .: 5. 10. Method to protect a corn plant from damage caused by a herbicide or to control weeds in a corn planting field, characterized by understanding the application of an effective amount of glufosinate herbicide in a planting field of at least one plant of transgenic corn, in which the transgenic corn plant comprises, in its genome, the sequence as defined in SEQ ID No .: 1, and / or SEQ ID No .: 2, and the transgenic corn plant has tolerance to the herbicide glufosinate ; preferably, the transgenic corn plant comprises, in its genome, the sequence as defined in SEQ ID No .: 3, and / or SEQ ID No .: 4; preferably, the transgenic maize plant successively comprises, in its genome, SEQ ID No .: 1, the nucleic acid sequence at positions 553-7491 of SEQ ID No .: 5 and SEQ ID No .: 2, or comprises the sequence as defined in SEQ ID No .: 5. 11. Method to create an insect-resistant and / or tolerant to the herbicide glufosinate corn plant, characterized by comprising: - planting at least one corn seed, in which the corn seed comprises, in its genome, a sequence of acids nucleic acids encoding the insect-resistant Vip3Aa protein and / or a nucleic acid sequence encoding the PAT protein tolerant to the herbicide glufosinate, and a nucleic acid sequence from a specific region, or the corn seed comprises, in its genome, the nucleic acid sequence as defined in SEQ ID No .: 5; - corn seed growth in a corn plant; and - invasion of the corn plant with a target insect and / or spraying the corn plant with an effective amount of glufosinate herbicide, and then harvesting the plant with reduced damage to the plant compared to other plants that do not comprise the nucleic acid sequence the specific region; wherein the region-specific nucleic acid sequence is the sequence as defined in SEQ ID No .: 1, and / or SEQ ID No .: 2; preferably, the region-specific nucleic acid sequence is the sequence as defined in SEQ ID No .: 3, and / or SEQ ID No .: 4. 12. Method for producing an insect-resistant and / or tolerant to the herbicide glufosinate corn plant, characterized by comprising - introduction of a nucleic acid sequence that encodes the insect-resistant Vip3Aa protein and / or a nucleic acid sequence that encodes the PAT protein tolerant to the herbicide glufosinate, and a nucleic acid sequence of a specific region in the genome of a first maize plant, or the nucleic acid sequence as defined in SEQ ID No./16 in the genome of a first maize plant , in a second corn plant, thus producing a plurality of progeny plants; and - selection of progeny plants comprising the nucleic acid sequence of the specific region, which are also resistant to insects and / or tolerant to the herbicide glufosinate; wherein the region-specific nucleic acid sequence is the sequence as defined in SEQ ID No .: 1, and / or SEQ ID No .: 2; preferably, the region-specific nucleic acid sequence is the sequence as defined in SEQ ID No .: 3, and / or SEQ ID No .: 4; preferably, the method comprises - sexual crossing between species of the DBN9501 transgenic corn event with a corn plant that does not have the characteristic of insect resistance and / or tolerance to the herbicide glufosinate, thus producing a plurality of progeny plants; and selecting plant progenies comprising the nucleic acid sequence of the specific region; - invasion of the progeny plants with a target insect and / or treatment of the progeny plants with the herbicide glufosinate; and - selection of progeny plants that have resistance to insects and / or tolerance to the herbicide glufosinate. 13. Agricultural product or commodity derived from the DBN9501 transgenic corn event, characterized by the agricultural product or commodity being corn flake, corn flour, corn oil, corn silk, corn starch, corn gluten, corn cake, a cosmetic or a bulking agent.