Insect-resistant transgenic cotton event BrsC35 as well as detection method and application thereof

Abstract

The invention discloses an insect-resistant transgenic cotton event BrsC35 as well as a detection method and application thereof. According to the invention, an epsps gene, a Cry1Ac gene and a Vip3A gene are transferred into a genome of Zhongmiansui 24 through an agrobacterium-mediated method, so that a transgenic cotton event BrsC35 of which the resistance to lepidoptera insects such as cotton bollworms, ostrinia nubilalis and spodoptera frugiperda is remarkably improved is obtained. The transgenic cotton event BrsC35 is preserved in China General Microbiological Culture Collection Center (CGMCC) on February 17, 2025, and the preservation number is CGMCC No.32181. The transgenic cotton event BrsC35 has been preserved in China General Microbiological Culture Collection Center (CGMCC). The invention also provides a method for identifying whether a plant sample is derived from the transgenic cotton event BrsC35 or an offspring of the transgenic cotton event BrsC35, the transgenic cotton event BrsC35 can be specifically detected, and the transgenic cotton event BrsC35 can be better supervised and managed. The method has an important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant genetic engineering, and particularly relates to a transgenic cotton event BrsC35 with insect resistance and a detection method and application thereof. BACKGROUND

[0002] The enzyme encoded by the epsps gene is called 5-enolpyruvate shikimate-3-phosphate synthetase, which plays an important role in the synthesis of amino acids in plants, and once it is missing, the plant will die due to the lack of amino acids. The epsps gene is a normal gene in plants, and the 5-enolpyruvate shikimate-3-phosphate synthetase encoded by the epsps gene is also a normal enzyme existing in plants. When the concentration of glyphosate reaches a certain amount, the activity of most 5-enolpyruvate shikimate-3-phosphate synthetases in plants can be inhibited, so that the plants die due to the lack of amino acids. However, after the exogenous fragment capable of overexpressing 5-enolpyruvate shikimate-3-phosphate synthetase is introduced into plant cells, under a certain concentration of glyphosate, part of the 5-enolpyruvate shikimate-3-phosphate synthetase will not be inhibited, so that the amino acids can be normally synthesized and the plants can grow normally. Therefore, the epsps gene is often used as a selection marker gene in transgenic experiments.

[0003] In recent years, cotton pests continue to occur, especially lepidopteran pests such as corn borer, cotton bollworm, spodoptera exigua, and armyworm, which cause the decrease of cotton yield and quality, and may even lead to absolute loss in severe cases. Therefore, the prevention and control of cotton pests is an important measure to ensure high and stable yield of cotton.

[0004] Cry insecticidal protein is the most widely used Bt insecticidal protein. Cry1Ac protein is one of them, which has strong specificity for corn borer and other lepidopteran insects. Cry insecticidal protein is initially ingested by insects in the form of protoxin, and then dissolved and activated into about 60-68kDa size of activated monomer insecticidal protein under high pH conditions. The activated Cry insecticidal protein binds to alkaline phosphatase and aminopeptidase N in the target pest midgut epithelial cells, thereby enriching the activated toxin on the microvilli membrane of the midgut cells. The toxin binds to the receptor cadherin with high affinity to form a four-fold oligomer pore structure. After the oligomer inserts into the cell membrane, a transmembrane pore is formed, which leads to the swelling and lysis of the target pest gut, the imbalance of the osmotic pressure in the body, and thus the death of the target pest.

[0005] Vegetative insecticidal proteins (VIP) are a new type of insecticidal proteins produced by Bacillus thuringiensis (Bt) in the vegetative growth stage, which have strong insecticidal activity on many important lepidopteran pests, and are mainly divided into VIP1, VIP2 and VIP3, etc. Among them, VIP3 has a broad-spectrum insecticidal activity on lepidopteran insects. VIP3 is a soluble protein, which can directly act without digestion by the insect midgut, and ultimately leads to the death of insects by inducing apoptosis of the midgut cells. SUMMARY

[0006] The purpose of the present application is to identify whether a plant sample is derived from a specific transgenic cotton event or its progeny. The specific transgenic cotton event can be transgenic cotton event BrsC35. The transgenic cotton event BrsC35 is Gossypium hirsutum BrsC35 CGMCC No. 32181. The plant sample can be a plant leaf, a seed, etc.

[0007] The present application first protects a method for breeding a cotton with insect resistance.

[0008] The method for breeding a cotton with insect resistance can be method A, which can comprise the following steps: crossing a cotton derived from a transgenic cotton event or its progeny with another cotton variety without insect resistance to obtain an insect-resistant cotton with higher insect resistance than the other cotton variety;

[0009] The transgenic cotton event is a cotton with insect resistance obtained by inserting an exogenous DNA molecule into the genome of a target cotton;

[0010] The upstream flanking sequence of the exogenous DNA molecule in the cotton with insect resistance is any one DNA fragment obtained by extending upstream from the upstream insertion site;

[0011] The downstream flanking sequence of the exogenous DNA molecule in the cotton with insect resistance is any one DNA fragment obtained by extending downstream from the downstream insertion site;

[0012] The upstream insertion site is a site corresponding to the first nucleotide in the DNA molecule shown in SEQ ID No. 1 from the 1st to the 511th position from the 5' end contained in the genome of the target cotton;

[0013] The downstream insertion site is a site corresponding to the last nucleotide in the DNA molecule shown in SEQ ID No. 2 from the 14700th to the 15339th position from the 5' end contained in the genome of the target cotton;

[0014] The exogenous DNA molecule comprises a gene encoding Cry1Ac protein, a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase, and a gene encoding vegetative insecticidal protein Vip3A.

[0015] The method for breeding the insect-resistant cotton can be method B, which can comprise the following steps: inserting an exogenous DNA molecule into the genome of the cotton to obtain the insect-resistant cotton.

[0016] The upstream flanking sequence of the exogenous DNA molecule in the insect-resistant cotton is any one of the DNA fragments obtained by extending upstream of the upstream insertion site.

[0017] The downstream flanking sequence of the exogenous DNA molecule in the insect-resistant cotton is any one of the DNA fragments obtained by extending downstream of the downstream insertion site.

[0018] The upstream insertion site is a site corresponding to the first nucleotide in the DNA molecule shown in SEQ ID No. 1 from 1-511 of the 5' end in the genome of the cotton.

[0019] The downstream insertion site is a site corresponding to the last nucleotide in the DNA molecule shown in SEQ ID No. 1 from 14700-15339 of the 5' end contained in the genome of the cotton.

[0020] The exogenous DNA molecule comprises a gene encoding Cry1Ac protein, a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase, and a gene encoding vegetative insecticidal protein Vip3A.

[0021] The insect-resistant cotton obtained by method B can be transgenic cotton event BrsC35. The transgenic cotton event BrsC35 is Gossypium hirsutum BrsC35 CGMCC No. 32181.

[0022] The Cry1Ac protein, the 5-enolpyruvylshikimate-3-phosphate synthase, and the vegetative insecticidal protein Vip3A can be N-terminal and / or C-terminal protein tags to obtain a fusion protein.

[0023] The protein tag refers to a polypeptide or protein fused and expressed with the target protein by DNA in vitro recombination technology to facilitate the expression, detection, tracking, and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and / or a SUMO tag.

[0024] The Cry1Ac protein of any one of the above embodiments can be a protein having 80% or more identity to the protein encoded by the Cry1Ac gene and having the same function.

[0025] The 5-enolpyruvylshikimate-3-phosphate synthase of any one of the above embodiments can be a protein having 80% or more identity to the protein encoded by the epsps gene and having the same function.

[0026] The vegetative insecticidal protein Vip3A of any one of the above embodiments can be a protein having 80% or more identity to the protein encoded by the Vip3A gene and having the same function.

[0027] The identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI homepage. For example, the identity (%) of a pair of amino acid sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and then obtaining the value of the identity (%).

[0028] In the above protein, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0029] In the method of any one of the above embodiments, the upstream insertion site can be specifically the 512th position from the 5' end of SEQ ID No. 1.

[0030] In the method of any one of the above embodiments, the downstream insertion site can be specifically the 14699th position from the 5' end of SEQ ID No. 2.

[0031] In the method of any one of the above embodiments, the nucleotide sequence of the upstream flanking sequence is as shown in SEQ ID No. 1 from the 1st to 511th position from the 5' end.

[0032] In the method of any one of the above embodiments, the nucleotide sequence of the downstream flanking sequence is as shown in SEQ ID No. 2 from the 14700th to 15339th position from the 5' end.

[0033] In any of the above-mentioned methods, the nucleotide sequence of the foreign DNA molecule consists of 29188 nucleotides, wherein the nucleotide sequence at positions 1-14489 is as shown in SEQ ID No. 1 at positions 512-15000 from the 5' end, and the nucleotide sequence at positions 14490-29188 is as shown in SEQ ID No. 2 at positions 1-14699 from the 5' end.

[0034] In any of the above-mentioned methods of inserting a foreign DNA molecule into the genome of a cotton plant, the foreign DNA molecule can be specifically DNA molecule A, which consists of 29188 nucleotides, wherein the nucleotide sequence at positions 1-14489 is as shown in SEQ ID No. 1 at positions 512-15000 from the 5' end, and the nucleotide sequence at positions 14490-29188 is as shown in SEQ ID No. 2 at positions 1-14699 from the 5' end.

[0035] In any of the above-mentioned methods of inserting a foreign DNA molecule into the genome of a cotton plant, the foreign DNA molecule can be specifically DNA molecule A, which consists of 29188 nucleotides, wherein the nucleotide sequence at positions 1-14489 is as shown in SEQ ID No. 1 at positions 512-15000 from the 5' end, and the nucleotide sequence at positions 14490-29188 is as shown in SEQ ID No. 2 at positions 1-14699 from the 5' end.

[0036] The present application also protects the use of any of the above-mentioned foreign DNA molecules, any of the above-mentioned upstream flanking sequences, or any of the above-mentioned downstream flanking sequences, which can be D1) or D2):

[0037] D1) detecting or assisting in detecting whether a plant sample is derived from any of the above-mentioned transgenic cotton events or progeny thereof, or any of the above-mentioned insect-resistant cotton plants or progeny thereof;

[0038] D2) breeding any of the above-mentioned transgenic cotton events or any of the above-mentioned insect-resistant cotton plants.

[0039] The present application also protects a method for identifying whether a plant sample to be tested is derived from any of the above-mentioned transgenic cotton events or progeny thereof, or any of the above-mentioned insect-resistant cotton plants or progeny thereof, which can be method M1 or method M2.

[0040] The method M1 can comprise the following steps:

[0041] M11) using the genomic DNA of the plant sample to be tested as a template, performing PCR amplification with a primer composition to obtain a PCR product;

[0042] M12) then the following determination is made: if the PCR product contains a DNA fragment of the expected size, then the plant sample to be tested is derived from the transgenic cotton event or its progeny or the insect-resistant cotton or its progeny; if the PCR product does not contain the DNA fragment of the expected size, then the plant sample to be tested is not derived from the transgenic cotton event or its progeny or the insect-resistant cotton or its progeny;

[0043] The primer composition comprises the following F1) or F2) or F3):

[0044] F1) a 5' end transformant detection primer pair; the upstream primer of the 5' end transformant detection primer pair is designed according to the DNA fragment shown in SEQ ID No. 1 from the 1st to the 511th position from the 5' end, and the downstream primer is designed according to any of the above-mentioned foreign DNA molecules;

[0045] F2) a 3' end transformant detection primer pair; the upstream primer of the 3' end transformant detection primer pair is designed according to any of the above-mentioned foreign DNA molecules, and the downstream primer is designed according to the DNA fragment shown in SEQ ID No. 2 from the 14700th to the 15339th position from the 5' end;

[0046] F3) the 5' end transformant detection primer pair and the 3' end transformant detection primer pair;

[0047] The method M2 comprises the following steps: sequencing the genomic DNA of the plant sample to be tested, and determining whether the plant sample to be tested is derived from the transgenic cotton event or its progeny or the insect-resistant cotton or its progeny according to the sequencing result.

[0048] In the above method, the nucleotide sequence of the upstream primer of the 5' end transformant detection primer pair is shown in SEQ ID No. 1 from the 364th to the 389th position from the 5' end; and the nucleotide sequence of the downstream primer is shown in the reverse complement sequence of SEQ ID No. 1 from the 569th to the 594th position from the 5' end.

[0049] In the above method, the nucleotide sequence of the upstream primer of the 3' end transformant detection primer pair is shown in SEQ ID No. 2 from the 14406th to the 14431st position from the 5' end; and the nucleotide sequence of the downstream primer is shown in the reverse complement sequence of SEQ ID No. 2 from the 14879th to the 14904th position from the 5' end.

[0050] The application also protects a processed product. The processed product is prepared from any of the above-mentioned transgenic cotton events or the above-mentioned insect-resistant cotton.

[0051] The processed product is not a plant as referred to in the Chinese Patent Law; the processed product is not capable of reproduction and does not have the ability to synthesize carbohydrates, proteins and the like inorganic substances such as water, carbon dioxide and inorganic salts by means of photosynthesis to sustain life.

[0052] The present application also protects the use of any of the above-mentioned primer compositions, which can be D1) or D2):

[0053] D1) detecting or assisting in detecting whether a plant sample is derived from any of the above-mentioned transgenic cotton events or progeny thereof, or, any of the above-mentioned insect-resistant cotton or progeny thereof;

[0054] D2) breeding any of the above-mentioned transgenic cotton events or any of the above-mentioned insect-resistant cotton.

[0055] The method for breeding insect-resistant cotton can be embodied as follows: the transgenic cotton event BrsC35 or its progeny is used as the male parent to sexually cross with the female parent cotton plant, thereby generating a variety of first-generation progeny plants; the first-generation progeny plants are subjected to insect resistance identification, and the progeny plants with insect resistance are screened, thereby breeding insect-resistant cotton varieties. Further, the progeny plants with insect resistance can be backcrossed with the female parent cotton plant or other parent cotton plants, and then the progeny is screened through insect resistance identification or identification of trait-related molecular markers (such as specific DNA fragments contained in the transgenic cotton event BrsC35), thereby obtaining insect-resistant cotton plants.

[0056] Any of the above-mentioned insect-resistant cotton can specifically include plants and seeds of the transgenic cotton event BrsC35 and plant cells or regenerable parts thereof. The plant parts of the transgenic cotton event BrsC35 include but are not limited to cells, pollen, ovules, flowers, sprouts, roots, stems, inflorescences, leaves and products from the transgenic cotton event BrsC35.

[0057] The progeny of any of the above-mentioned insect-resistant cotton can be transgenic materials derived from the insect-resistant cotton as the parent, including the derived progeny obtained by mutagenesis or crossing with other cottons using the insect-resistant cotton, or the progeny obtained by further derivation of the mutagenized or crossed progeny.

[0058] Any of the above-mentioned plant samples includes not only wild-type cotton or its variants and all plant varieties that can be mated with cotton, but also cells, tissues and organs (such as seeds, leaves, fruits, flowers, stems and roots) derived from the plants and propagation materials (such as pollen, ovules, roots, root tips, hypocotyls, cotyledons, stems, leaves, flowers, anthers, seeds, meristems, protoplasts and cell tissue cultures) derived from the plants, and processed products (agricultural products or commodities) derived from cotton as the main raw material, such as cotton quilts, cottonseed oil and the like.

[0059] The epsps gene, Cry1Ac gene and Vip3A gene are introduced into Zhongmiansuo 24 by a transgenic method to express, and a transgenic cotton event BrsC35 with commercial application prospect is obtained. Experiments prove that the transgenic cotton event BrsC35 has higher resistance to cotton bollworm, corn borer and Spodoptera exigua and other lepidopteran insects than wild-type cotton. The obtained transgenic cotton event BrsC35 is transplanted into cotton with different genetic backgrounds by backcrossing and other conventional breeding methods, and insect-resistant cotton varieties with the transgenic expression characteristics of the original transformant event can be bred. The application further establishes a detection method for detecting whether a to-be-tested plant sample is derived from the transgenic cotton event BrsC35 or its offspring, which can identify the junction region of the inserted foreign DNA molecule and plant genomic DNA, and is further used for accurately and rapidly identifying the plant sample derived from the transgenic cotton event BrsC35. The application has important application value.

[0060] Depository

[0061] Classification name: Gossypium hirsutum

[0062] Latin name: Gossypium hirsutum

[0063] Biomaterials referred to: BrsC35

[0064] Preservation agency: China General Microbiological Culture Collection Center

[0065] Abbreviation of preservation agency: CGMCC

[0066] Address: No. 3, Beichen West Road, Beijing City, China

[0067] Preservation date: February 17, 2025

[0068] Preservation center grade entry number: CGMCC No. 32181 BRIEF DESCRIPTION OF DRAWINGS

[0069] Figure 1 It is a structural schematic diagram of the plant expression vector DT2300-VIP3A-EPSPS-Cry1Ac.

[0070] Figure 2 It is the insect resistance identification of the transgenic cotton event BrsC35.

[0071] Figure 3 It is the 5' end specific detection of the insertion site of the transgenic cotton event BrsC35.

[0072] Figure 4Specific PCR sensitivity detection of 5' end of insertion site of transgenic cotton event BrsC35.

[0073] Figure 5 Specific detection of 3' end of insertion site of transgenic cotton event BrsC35.

[0074] Figure 6 Specific PCR sensitivity detection of 3' end of insertion site of transgenic cotton event BrsC35. DETAILED DESCRIPTION

[0075] The application will be further described in conjunction with the specific embodiments. The examples given below are only for the purpose of illustrating the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0076] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0077] In the quantitative tests in the following examples, three repeated experiments were set up, and the results were averaged.

[0078] The operation steps not specifically described in the following examples refer to the corresponding parts of the third edition of Molecular Cloning Experiments Guide (J. Sambrook and D. W. Russell, Science Press) or refer to the instructions of the reagent kits used.

[0079] The plant genomic DNA extraction kit was purchased from Beijing Bomeide Science and Technology Development Co., Ltd., and the product catalog number was DN1501. The agarose gel purification recovery kit was purchased from Beijing Bomeide Science and Technology Development Co., Ltd., and the product catalog number was DR0101. The pCambia2300 vector was purchased from Shanghai Dibo Biological Technology Co., Ltd., and the product catalog number was S209198A.

[0080] In the following examples, the cotton variety Zhongmiansuo 24 is a short-season cotton variety selected by the Cotton Research Institute of Chinese Academy of Agricultural Sciences, and was approved by the Henan Provincial Crop Variety Approval Committee, Tianjin Municipal Crop Variety Approval Committee and the National Crop Variety Approval Committee in 1995-1997. Hereinafter, the cotton variety Zhongmiansuo 24 is referred to as Zhongmiansuo 24.

[0081] Example 1, obtaining of transgenic cotton event BrsC35 and trait identification

[0082] I. Obtaining of T0 generation transgenic cotton plant BrsC35 by Agrobacterium-mediated method

[0083] 1. Construction of plant expression vector DT2300-VIP3A-EPSPS-Cry1Ac

[0084] (1) The nucleotide sequences of cotton 35S promoter, nos terminator, glyphosate-tolerant gene epsps, insect-resistant gene Cry1Ac and Vip3A gene were queried from NCBI website. The gene sequences were optimized according to plant-biased codons, and then cotton CaMV 35S promoter, nos terminator, codon-optimized epsps gene, Cry1Ac gene and Vip3A gene were artificially synthesized.

[0085] (2) The pCambia2300 vector was digested with restriction enzyme SpeI, and the vector backbone was recovered by agarose gel purification recovery kit. The vip3A gene was amplified by homologous recombination primers, and only the SpeI restriction site at one end was retained. Then homologous recombination was performed to obtain recombinant plasmid pD23-V.

[0086] (3) The recombinant plasmid pD23-V was digested with restriction enzyme SpeI, and the vector backbone was recovered by agarose gel purification recovery kit. The epsps gene was amplified by homologous recombination primers, and only the SpeI restriction site at one end was retained. Then homologous recombination was performed to obtain recombinant plasmid pD23-VE.

[0087] (4) The recombinant plasmid pD23-VE was digested with restriction enzyme SpeI, and the vector backbone was recovered by agarose gel purification recovery kit. The Cry1Ac gene was amplified by homologous recombination primers. Then homologous recombination was performed to obtain plant expression vector DT2300-VIP3A-EPSPS-Cry1Ac.

[0088] The structure diagram of plant expression vector DT2300-VIP3A-EPSPS-Cry1Ac is shown in Figure 1 In the plant expression vector DT2300-VIP3A-EPSPS-Cry1Ac, the epsps gene is expressed driven by CaMV 35S promoter, and the expression is terminated by ploy A terminator. The Cry1Ac gene and Vip3A gene are expressed driven by CaMV 35S promoter, and the expression is terminated by nos terminator.

[0089] 2. Preparation of recombinant Agrobacterium

[0090] The plant expression vector DT2300-VIP3A-EPSPS-Cry1Ac was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain recombinant Agrobacterium.

[0091] 3. Obtaining of transgenic cotton

[0092] The immature embryo of Zhongmiansuo 24 is used as an explant, and the recombinant Agrobacterium is used for genetic transformation of cotton by Agrobacterium-mediated method, that is, the T-DNA in the plant expression vector DT2300-VIP3A-EPSPS-Cry1Ac is transferred into the Zhongmiansuo 24 genome by Agrobacterium-mediated method to obtain a T0 generation transgenic cotton plant BrsC35. For specific operation steps of Agrobacterium-mediated genetic transformation of cotton, refer to Chinese patent document CN113462719A.

[0093] II. PCR detection of T0 generation transgenic cotton plant BrsC35

[0094] 1. The primers epsps-F: 5'-CGTCCTCACCCTCCAGAAGTCC-3' and epsps-R: 5'-CGTCGCTTTCCCAGTCACCAAG-3' are designed and synthesized according to the nucleotide sequence of the epeps gene. The primers Cry1Ac-F: 5'-TGGATAGGAACCTCGATGTAACCACG-3' and Cry1Ac-R: 5'-TCGCCTACGGTACTTCTTCCAACCTG-3' are designed and synthesized according to the nucleotide sequence of the Cry1Ac gene. The primers Vip-F: 5'-AGATGATGAAGTTATCGCCCCAGGCT-3' and Vip-R: 5'-CTCATCACCCTCACTTGCAAGTCAT-3' are designed and synthesized according to the nucleotide sequence of the Vip3A gene.

[0095] 1. The primers epsps-F: 5'-CGTCCTCACCCTCCAGAAGTCC-3' and epsps-R: 5'-CGTCGCTTTCCCAGTCACCAAG-3' are designed and synthesized according to the nucleotide sequence of the epeps gene. The primers Cry1Ac-F: 5'-TGGATAGGAACCTCGATGTAACCACG-3' and Cry1Ac-R: 5'-TCGCCTACGGTACTTCTTCCAACCTG-3' are designed and synthesized according to the nucleotide sequence of the Cry1Ac gene. The primers Vip-F: 5'-AGATGATGAAGTTATCGCCCCAGGCT-3' and Vip-R: 5'-CTCATCACCCTCACTTGCAAGTCAT-3' are designed and synthesized according to the nucleotide sequence of the Vip3A gene.

[0096] 2. The plant genomic DNA extraction kit is used to extract the genomic DNA of the T0 generation transgenic cotton plant BrsC35 and the genomic DNA of Zhongmiansuo 24 obtained in step 1.

[0097] 3. The genomic DNA of the T0 generation transgenic cotton plant BrsC35, the genomic DNA of Zhongmiansuo 24 (as a blank control) or the plant expression vector DT2300-VIP3A-EPSPS-Cry1Ac (as a positive control) are used as templates, and the primer pair 1 composed of the primer epsps-F and the primer epsps-R, the primer pair 2 composed of the primer Cry1Ac-F and the primer Cry1Ac-R, or the primer pair 3 composed of the primer Vip-F and the primer Vip-R are used for PCR amplification to obtain PCR amplification products.

[0098] The PCR amplification program is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 sec, annealing at 62℃ for 30 sec, extension at 72℃ for 30 sec, 30 cycles; and extension at 72℃ for 10 min.

[0099] The results show that the PCR amplification product obtained by using primer pair 1 contains a DNA band with a size of about 359 bp, the PCR amplification product obtained by using primer pair 2 contains a DNA band with a size of about 394 bp, and the PCR amplification product obtained by using primer pair 3 contains a DNA band with a size of about 490 bp, when the genomic DNA of the T0 generation transgenic cotton plant BrsC35 or the plant expression vector DT2300-VIP3A-EPSPS-Cry1Ac template is used; and the PCR amplification product obtained by using primer pair 1 does not contain a DNA band with a size of about 359 bp, the PCR amplification product obtained by using primer pair 2 does not contain a DNA band with a size of about 394 bp, and the PCR amplification product obtained by using primer pair 3 does not contain a DNA band with a size of about 490 bp, when the genomic DNA of Zhongmiansuo 24 is used. Therefore, the T0 generation transgenic cotton plant BrsC35 contains epeps gene, cry1Ac gene and vip3A gene.

[0100] III. Insect resistance identification and glyphosate resistance analysis of the transgenic cotton event BrsC35

[0101] 1. The T0 generation transgenic cotton plant BrsC35 is selfed to obtain the T1 generation transgenic cotton. The surviving plants in the T1 generation transgenic cotton line are selfed, and the seeds, i.e. T2 generation seeds, are harvested. The T2 generation transgenic cotton plant BrsC35 is obtained by planting the T2 generation seeds. The seeds obtained from the T2 generation transgenic cotton plant BrsC35 are deposited in the China General Microbiological Culture Collection Center on February 17, 2025, and the classification name is Gossypium hirsutum, and the preservation number is CGMCC No. 32181. The seeds obtained from the T2 generation transgenic cotton plant BrsC35 are the transgenic cotton event BrsC35. The full name of the transgenic cotton event BrsC35 is Gossypium hirsutum BrsC35 CGMCC No. 32181, and the abbreviation is cotton CGMCC No. 32181.

[0102] 2. Insect resistance identification of the transgenic cotton event BrsC35

[0103] Each of the three repeats was as follows: 5 cotton plants (cotton CGMCC No. 32181 or Zhongmiansuo 24) at the 5-leaf stage were collected, and 3 leaves were collected from each plant; the leaves were placed on a culture dish lined with wet filter paper, and 1 leaf was placed in each culture dish, followed by 3 2nd instar Helicoverpa armigera, 3 Ostrinia furnacalis or 3 Spodoptera exigua. When the larvae were inoculated, the activity of the larvae was confirmed to be good; after inoculation, the culture dish was closed and placed in a room at 25-28°C for 5 days. The mortality rate of the larvae was recorded and the resistance level was identified according to the identification method of the Ministry of Agriculture and Rural Affairs No. 628-5-202.

[0104] Some of the test results are shown in Figure 2 (BrsC35 is cotton CGMCC No. 32181). The statistical results are shown in Table 1 (cotton BrsC35 is cotton CGMCC No. 32181). The results show that the leaves of Zhongmiansuo 24 inoculated with Helicoverpa armigera, Ostrinia furnacalis or Spodoptera exigua were heavily eaten, while the leaves of cotton CGMCC No. 32181 were in good condition and the larvae died. Therefore, cotton CGMCC No. 32181 has resistance to Lepidoptera Helicoverpa armigera, Ostrinia furnacalis and Spodoptera exigua.

[0105] Table 1

[0106]

[0107] Example 2, Molecular characterization of transgenic cotton event BrsC35

[0108] 1. Extract 3-5 g of genomic DNA from the tender leaves of transgenic cotton event BrsC35, then use 1.2% agarose gel electrophoresis to preliminarily detect the DNA concentration and quality, and then use Qubit to accurately quantify the DNA concentration.

[0109] DNA samples with a content of 0.5 μg or more are qualified samples and are used to build a library.

[0110] 2. PacBio platform sequencing library construction

[0111] The qualified DNA samples were broken into fragments by Covaris ultrasonic crusher; the large fragment DNA was enriched and purified by magnetic beads; the sequencing adapters were connected to both ends of the DNA fragments, and the incorrectly connected fragments were removed by exonuclease. That is, DNA Sample→Fragment DNA→Damage Repair / End Repair→Ligate adapters→Purity DNA.

[0112] 3. Data quality evaluation

[0113] SMRT sequencing technology data generally have three types, CCS (Circular Consensus Sequence) read is a polymerase read on multiple subreads sequence.

[0114] (1) The original sequencing reads are dumbbell-shaped sequences containing two end adapters, called Polymerase reads. Since the data in SMAPLE.subreads.bam is the data without adapters, the statistical results are slightly smaller than the original data.

[0115] (2) Insert Length (insert sequence length is the library length of the library), is the estimated library insert length of this polymerase read according to all subreads from the same polymerase read by calculation.

[0116] (3) The original data is broken from the adapter and the adapter sequence is filtered out to get subreads, and the subreads are filtered according to the standard Filtering subreads by minimum length = 50. Generally, this part of data is the effective input data for downstream analysis.

[0117] 4. Sequence visualization

[0118] The three generations of effective sequencing data obtained by PacBio sequencing platform are aligned to the reference genome by Mininmap2 software, and the Bam file is generated by format conversion, deduplication and other operations. By SAMtools-1.9 statistics, index is constructed, and IGV-2.5.0 is used for visualization analysis. IGV can directly see the mapping position of reads in the reference genome.

[0119] 5. Transgenic cotton event BrsC35 flanking sequence verification

[0120] According to the flanking sequence of transgenic cotton event BrsC35 obtained by genome sequencing and bioinformatics analysis, the verification primer pair was designed on the plant expression vector DT2300-VIP3A-EPSPS-Cry1Ac and its flanking sequence, respectively. Fresh tender leaves of transgenic cotton event BrsC35 were used as templates, and the above primer pairs were used for PCR amplification. The DNA fragments obtained by 1.2% agarose gel electrophoresis detection of PCR amplification products were recovered and sequenced by Sanger. The sequencing results were compared with the flanking sequence obtained by PacBio sequencing to verify.

[0121] 6、By BLAST alignment analysis on the Phytozome software (https: / / phytozome-next.jgi.doe.gov / blast-results / 1491596) website, it was found that there were two cases of alignment results of the obtained DNA fragments: the 5' end of reads1 aligned to the cotton genome A07 chromosome 91750453; the 3' end of reads2 aligned to the cotton genome A07 chromosome 91750477. Through Snapgene software analysis, the 512-800th of reads1 is the sequence on the vector; the 1-296th of reads2 is the sequence on the vector. The alignment results prove that the flanking sequence of the exogenous gene in transgenic cotton event BrsC35 at the insertion site of the cotton genome is obtained. The 5' end of reads1 and the 3' end of reads2 flanking sequence is composed of 30339 nucleotides, wherein the nucleotide sequence of 1-15000 is shown in SEQ ID No. 1, and the nucleotide sequence of 15001-30339 is shown in SEQ ID No. 2.

[0122] 7、The upstream and downstream flanking sequences of the insertion site obtained in step 6 are aligned with the published BTx623 cotton genome sequence (Sorghum_bicolor_NCBIv3:

[0123] https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BL AST_SPEC=GDH_GCF_000003195.3). The results show that the transgenic cotton event BrsC35 is a transgenic event in which the nucleotide sequence between 91750453-91750477 of the A07 chromosome of the cotton variety Zhongmiansuo24 genome (i.e. the nucleotide sequence of 23bp length of 91750454-91750476 of the A07 chromosome of the Zhongmiansuo24 genome) is replaced by DNA molecule A. DNA molecule A is composed of 29188 nucleotides, wherein the nucleotide sequence of 1-14489 is shown in SEQ ID No. 1 from the 512th to the 15000th of the 5' end, and the nucleotide sequence of 14490-29188 is shown in SEQ ID No. 2 from the 1st to the 14699th of the 5' end.

[0124] Cotton CGMCC No. 32181 contains a double-stranded DNA molecule named A, the nucleotide sequence of one strand of A from position 1 to 15000 is shown in SEQ ID No. 1, and the nucleotide sequence of one strand of A from position 15001 to 30339 is shown in SEQ ID No. 2. The exogenous DNA molecule consists of 29188 nucleotides, wherein the nucleotide sequence from position 1 to 14489 is shown in SEQ ID No. 1 from position 512 to 15000 at the 5' end, and the nucleotide sequence from position 14490 to 29188 is shown in SEQ ID No. 2 from position 1 to 14699 at the 5' end; the position 512 at the 5' end of SEQ ID No. 1 is the upstream insertion site (also referred to as the 5' end insertion site) of the exogenous DNA molecule, and the position 14699 at the 5' end of SEQ ID No. 2 is the downstream insertion site (also referred to as the 3' end insertion site) of the exogenous DNA molecule; the sequence from position 1 to 511 at the 5' end of SEQ ID No. 1 is the upstream flanking sequence (i.e. the sequence in the genome of the cotton variety Zhongmiansuo 24), and the sequence from position 14700 to 15339 at the 5' end of SEQ ID No. 2 is the downstream flanking sequence (i.e. the sequence in the genome of the cotton variety Zhongmiansuo 24).

[0125] The DNA molecule shown in SEQ ID No. 1 contains two double-stranded DNA fragments including the upstream insertion site, A 499-524 and A 364-594 , i.e. the nucleotide sequence of one strand of A 499-524 is shown in SEQ ID No. 1 from position 499 to 524 at the 5' end (5'-TAACTAAGAATCCTCAAACACTGATA-3'), and the nucleotide sequence of one strand of A 364-594 is shown in SEQ ID No. 1 from position 364 to 594 at the 5' end. The DNA molecule shown in SEQ ID No. 2 contains two double-stranded DNA fragments including the downstream insertion site, A 29687-29712 and A 29406-29904 , i.e. the nucleotide sequence of one strand of A 29687-29712 is shown in SEQ ID No. 2 from position 14687 to 14712 at the 5' end (5'-TACACCACTGACAATAGATCCATTCC-3'), and the nucleotide sequence of one strand of A 29406-29904 is shown in SEQ ID No. 2 from position 14406 to 14904 at the 5' end. A 499-524 , A 364-594 , A 29687-29712 and A 29406-29904 can be used to identify whether the cotton to be tested is derived from cotton CGMCC No. 32181 or its progeny.

[0126] The sequencing of SEQ ID No. 1 shows that from the 5' end, the 1106-1783th nucleotides are the nucleotide sequence of the CaMV 35S promoter, the 1846-2643th nucleotides are the nucleotide sequence of the non-coding strand of the NPTII gene, the 2700-2874th nucleotides are the poly A nucleotide sequence, the 2951-2976th nucleotides are the LB sequence, the 3401-4198th nucleotides are the nucleotide reverse complement sequence of the non-coding strand of the NPTII gene, the 4261-4938th nucleotides are the nucleotide reverse complement sequence of the CaMV 35S promoter, the 5549-5574th nucleotides are the RB sequence, the 5637-10567th nucleotides are the vector backbone sequence, the 10568-11901th nucleotides are the genomic sequence of the E. coli ET12567 strain, the 11929-12723th nucleotides are the nucleotide reverse complement sequence of the screening gene kan gene non-coding strand, the 13148-13173th nucleotides are the LB sequence, the 13250-13424th nucleotides are the poly A nucleotide sequence, and the 13479-13727th nucleotides are the nos terminator. From the 5' end, the 13734-15000th nucleotides of SEQ ID No. 1 and the 1-588th nucleotides of SEQ ID No. 2 are the nucleotide reverse complement sequence of the Cry1Ac gene, the 627-1304th nucleotides are the nucleotide reverse complement sequence of the CaMV 35S promoter, the 1525-1699th nucleotides are the poly A nucleotide sequence, the 1706-3076th nucleotides are the nucleotide reverse complement sequence of the epsps gene, the 3403-4080th nucleotides are the CaMV 35S promoter, the 4209-4457th nucleotides are the nos terminator, the 4464-6843th nucleotides are the nucleotide reverse complement sequence of the Vip3A gene, the 6882-7559th nucleotides are the nucleotide reverse complement sequence of the CaMV 35S promoter, the 7889-7913th nucleotides are the RB sequence, the 8574-9251th nucleotides are the nucleotide sequence of the CaMV 35S promoter, the 9290-11668th nucleotides are the nucleotide sequence of the Vip3A gene, the 11675-11923th nucleotides are the nos terminator, the 12051-12728th nucleotides are the nucleotide sequence of the CaMV 35S promoter, the 13055-14425th nucleotides are the nucleotide sequence of the epsps gene, and the 14432-14606th nucleotides are the poly A nucleotide sequence.

[0127] Example 3, Establishment of a transgenic cotton event BrsC35 specific PCR detection system

[0128] I. Establishment of a 5' end transformant specific PCR detection system

[0129] 1. According to the flanking sequence of the 5' end of the foreign gene insertion site of transgenic cotton event BrsC35, the primers 5'-QF1: 5'-CGAATTGTAAAATTTAACTCGACTCA-3' (the sequence from the 5' end of SEQ ID No. 1 at positions 364-389) and primer 5'-QR1: 5'-AGCCTGAATGGCGAATGCTAGAGCAG-3' (the reverse complement sequence from the 5' end of SEQ ID No. 1 at positions 569-594) for site-specific PCR detection of the foreign gene are designed.

[0130] The primer pair composed of primer 5'-QF1 and primer 5'-QR1 is used for PCR amplification with the genomic DNA of transgenic cotton event BrsC35 as the template, and a double-stranded DNA fragment A 364-594 . A 364-594 The nucleotide sequence of one strand of A is shown in SEQ ID No. 1 at positions 364-594 from the 5' end.

[0131] It can be seen that the primer 5'-QF1 and the primer 5'-QR1 can specifically PCR amplify the segment near the upstream insertion site in the genome of transgenic cotton event BrsC35.

[0132] 2. The genomic DNA of the test plants (sorghum P184 (GenBank accession number: PQ444209), Arabidopsis thaliana Columbia (Columbia) wild type, rice variety Nipponbare, Nicotiana benthamiana, soybean, maize inbred line Zong 31, CCRI 24 or transgenic cotton event BrsC35) is extracted by using a plant genomic DNA extraction kit, and then diluted with water to a concentration of 100 ng / μL.

[0133] 3. The genomic DNA of the test plants obtained in step 2 is used as the template, and the primer pair composed of primer 5'-QF1 and primer 5'-QR1 is used for PCR amplification, and the PCR amplification product is detected by electrophoresis on a 1% agarose gel.

[0134] The reaction system is 50 μL, which is composed of 0.5 μL Taq Polymerase (5 U / μL), 5.0 μL 10×PCR Buffer (Mg 2+ Plus), 5.0 μL dNTP (2.5 mM), 1 μL primer 5'-QF1 (concentration of 10.0 μM), 1 μL primer 5'-QR1 (concentration of 10.0 μM), 1 μL template and sterilized ddH2O.

[0135] Reaction conditions: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 sec, 60°C annealing for 30 sec, 72°C extension for 30 sec, 30 cycles; 72°C extension for 10 min.

[0136] The detection results are shown in Table 2. Figure 3 (Arabidopsis is Arabidopsis Columbia wild type, rice is rice variety Nipponbare, tobacco is Nicotiana benthamiana, maize inbred line Zong 31 is maize inbred line Zong 31, and BrsC35 is transgenic cotton event BrsC35). The results show that only when the genomic DNA of transgenic cotton event BrsC35 is used as the template, a 231 bp target DNA band can be amplified, and no corresponding target band can be amplified for other plants; the 231 bp target DNA band is recovered and sequenced, and the nucleotide sequence is shown in SEQ ID No. 1 from the 364th to 594th position at the 5' end.

[0137] II. Sensitivity detection of 5' end transformant

[0138] 1. The genomic DNA of Zhongmiansuo 24 and the genomic DNA of transgenic cotton event BrsC35 are mixed according to the mass percentage to prepare DNA solutions with the content of genomic DNA of transgenic cotton event BrsC35 being 100%, 10%, 8%, 2%, 1%, 0.5%, 0.1% and 0%, respectively.

[0139] 2. The DNA solutions with different concentrations prepared in step 1 or ddH2O (as a blank control) are used as templates, a primer pair composed of primer 5'-QF1 and primer 5'-QR1 is used for PCR amplification, and the PCR amplification product is analyzed for sensitivity by 1.0% agarose gel electrophoresis.

[0140] The detection results are shown in Table 2. Figure 4 The results show that when the concentration of the genomic DNA of transgenic cotton event BrsC35 is diluted to 0.1%, primer 5'-QF1 and primer 5'-QR1 can still amplify a 231 bp specific fragment. It can be seen that the detection sensitivity of the 5' end transformant can reach 0.1%.

[0141] III. Establishment of 3' end transformant specific PCR detection system

[0142] 1. According to the flanking sequence of the 3' end of the foreign gene insertion site of transgenic cotton event BrsC35, the primers 3'-HF1: 5'- ATACAAAGGCAGCATAATAGCTCGAG-3' (the sequence from the 5' end of SEQ ID No. 2 at positions 14406-14431) and primer 3'-HR1: 5'- TCTAACGGCAGCAGAATTGATGAAGG-3' (the reverse complement sequence from the 5' end of SEQ ID No. 2 at positions 14879-14904) for site-specific PCR detection of the foreign gene were designed.

[0143] The primer pair composed of primer 3'-HF1 and primer 3'-HR1 was used for PCR amplification with the genomic DNA of transgenic cotton event BrsC35 as the template, and a double-stranded DNA fragment A 29406-29904 was obtained. 29406-29904 The nucleotide sequence of one strand of A is shown in SEQ ID No. 2 at positions 14406-14904 from the 5' end.

[0144] It can be seen that the primer 3'-HF1 and the primer 3'-HR1 can specifically PCR amplify the segment near the downstream insertion site in the genome of transgenic cotton event BrsC35.

[0145] 2. The genomic DNA of the test plant (sorghum P184, Arabidopsis thaliana Columbia wild type, rice variety Nipponbare, Nicotiana benthamiana, soybean, maize inbred line Zong 31, CCRI 24 or transgenic cotton event BrsC35) was extracted by using a plant genomic DNA extraction kit, and then diluted with water to a concentration of 100 ng / μL.

[0146] 3. The genomic DNA of the test plant obtained in step 2 was used as the template, and the primer pair composed of primer 3'-HF1 and primer 3'-HR1 was used for PCR amplification, and the PCR amplification product was detected by electrophoresis on a 1% agarose gel.

[0147] The reaction system was 50 μL, which was composed of 0.5 μL Taq Polymerase (5 U / μL), 5.0 μL 10x PCR Buffer (Mg 2+ Plus), 5.0 μL dNTP (2.5 mM), 1 μL primer 3'-HF1 (concentration of 10.0 μM), 1 μL primer 3'-HR1 (concentration of 10.0 μM), 1 μL template and sterilized ddH2O.

[0148] The reaction conditions were as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 sec, 60 °C annealing for 30 sec, 72 °C amplification for 30 sec, 30 cycles; 72 °C extension for 10 min.

[0149] The detection results are shown in Table 2 Figure 5 (Arabidopsis is Arabidopsis Columbia wild type, rice is rice variety Nipponbare, tobacco is Nicotiana benthamiana, soybean, corn is corn inbred line Zong 31, and BrsC35 is transgenic cotton event BrsC35). The results show that only the genomic DNA of transgenic cotton event BrsC35 can amplify a target DNA band of 499 bp in size, and the other plants cannot amplify the corresponding target band; the 499 bp target DNA band is recovered and sequenced, and the nucleotide sequence is shown in SEQ ID No. 2 from the 5' end 14406-14904.

[0150] IV. Sensitivity detection of 3' end transformant

[0151] 1. The genomic DNA of Zhongmiansuo 24 and the genomic DNA of transgenic cotton event BrsC35 are mixed according to the mass percentage to prepare DNA solutions of transgenic cotton event BrsC35 with the content of genomic DNA being 100%, 10%, 8%, 2%, 1%, 0.5%, 0.1% and 0%, respectively.

[0152] 2. The DNA solutions of different concentrations prepared in step 1 or ddH2O (as a blank control) are used as templates, PCR amplification is carried out using a primer pair composed of primer 3'-HF1 and primer 3'-HR1, and the PCR amplification product is analyzed by 1.0% agarose gel electrophoresis for sensitivity.

[0153] The detection results are shown in Table 2 Figure 6 The results show that the primer 3'-HF1 and the primer 3'-HR1 can still amplify a specific fragment of 499 bp when the concentration of the genomic DNA of transgenic cotton event BrsC35 is diluted to 0.1%. Therefore, the detection sensitivity of the 3' end transformant can reach 0.1%.

[0154] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In short, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application.

Claims

1. A method for breeding insect-resistant cotton, comprising the following steps: crossing cotton or its offspring obtained from a transgenic cotton event with another cotton variety that does not have insect resistance to obtain insect-resistant cotton with higher insect resistance than the other cotton variety; The transgenic cotton event refers to the insertion of exogenous DNA molecules into the genome of target cotton, resulting in cotton with insect resistance; The exogenous DNA molecule is any DNA fragment obtained by extending upstream from the upstream insertion site in the upstream flanking sequence of the insect-resistant cotton. The exogenous DNA molecule is any DNA fragment obtained by extending downstream from the downstream insertion site in the downstream flanking sequence of the insect-resistant cotton. The upstream insertion site is the site corresponding to the first nucleotide in the DNA molecule shown in SEQ ID No. 1 from the 5' end to positions 1-511 in the target cotton genome; The downstream insertion site is the site corresponding to the last nucleotide in the DNA molecule shown in SEQ ID No. 2 from the 5' end, positions 14700-15339. The exogenous DNA molecules include genes encoding Cry1Ac protein, genes encoding 5-enolpyruvate-shikimate-3-phosphate synthase, and genes encoding the vegetative insecticidal protein Vip3A.

2. A method for cultivating insect-resistant cotton, comprising the following steps: inserting exogenous DNA molecules into the genome of the target cotton to obtain insect-resistant cotton; The upstream flanking sequence of the exogenous DNA molecule in the insect-resistant cotton is any DNA fragment obtained by extending upstream from the upstream insertion site. The exogenous DNA molecule is any DNA fragment obtained by extending downstream from the downstream insertion site in the downstream flanking sequence of the insect-resistant cotton. The upstream insertion site is the site corresponding to the first nucleotide in the DNA molecule shown in SEQ ID No. 1 from the 5' end to positions 1-511 in the target cotton genome; The downstream insertion site is the site corresponding to the last nucleotide in the DNA molecule shown in SEQ ID No. 1 from the 5' end to positions 14700-15339 in the target cotton genome; The exogenous DNA molecules include genes encoding Cry1Ac protein, genes encoding 5-enolpyruvate-shikimate-3-phosphate synthase, and genes encoding the vegetative insecticidal protein Vip3A.

3. The method according to claim 1 or 2, characterized in that: The upstream insertion site is the 512th position from the 5' end of SEQ ID No. 1; The downstream insertion site is the 14699th position from the 5' end of SEQ ID No. 2; The nucleotide sequence of the upstream flanking sequence is shown in SEQ ID No. 1 from position 1 to 511 starting from the 5' end; The nucleotide sequence of the downstream flanking sequence is shown in SEQ ID No. 2, positions 14700-15339 from the 5' end.

4. The method according to claim 1 or 2, characterized in that: The nucleotide sequence of the exogenous DNA molecule consists of 29,188 nucleotides, wherein the nucleotide sequence from position 1 to 14,489 is shown as position 512-15,000 from the 5' end in SEQ ID No. 1, and the nucleotide sequence from position 14,490 to 29,188 is shown as position 1-14,699 from the 5' end in SEQ ID No.

2.

5. The method according to claim 1, 3, or 4, characterized in that: The genetically modified cotton event is the genetically modified cotton event BrsC35; the genetically modified cotton event BrsC35 is upland cotton (Gossypium hirsutum) BrsC35CGMCCNo.32181.

6. The application of any of the exogenous DNA molecules described in claims 1 to 4, any of the upstream flanking sequences described in claims 1 to 3, or any of the downstream flanking sequences described in claims 1 to 3, is D1) or D2): D1) Detect or assist in detecting whether the plant sample is derived from the transgenic cotton event of claim 1, 3, 4 or 5 or its descendants, or the insect-resistant cotton of any of claims 1 to 5 or its descendants; D2) The event of cultivating transgenic cotton as described in claim 1, 3, 4 or 5, or the insect-resistant cotton as described in any of claims 1 to 5.

7. A method for identifying whether a plant sample to be tested originates from the transgenic cotton event of claim 1, 3, 4 or 5 or its descendants, or the insect-resistant cotton of any one of claims 1 to 5 or its descendants, is method M1 or method M2; The method M1 includes the following steps: M11) Using the genomic DNA of the plant sample to be tested as a template, PCR amplification was performed using a primer combination to obtain PCR products; After M12), the following determination is made: if the PCR product contains a DNA fragment of the expected size, then the plant sample to be tested originates from the transgenic cotton event or its offspring or the insect-resistant cotton or its offspring; if the PCR product does not contain a DNA fragment of the expected size, then the plant sample to be tested does not originate from the transgenic cotton event or its offspring or the insect-resistant cotton or its offspring. The primer composition includes the following F1), F2), or F3): F1) 5' end transformant detection primer pair; the upstream primer of the 5' end transformant detection primer pair is designed based on the DNA fragment with nucleotide sequence as shown in SEQ ID No. 1 from position 1 to 511 from the 5' end, and the downstream primer is designed based on the exogenous DNA molecule according to any one of claims 1 to 4; F2) 3' end transformant detection primer pair; the upstream primer of the 3' end transformant detection primer pair is designed according to any of the exogenous DNA molecules described in claims 1 to 4, and the downstream primer nucleotide sequence is designed as shown in the DNA fragment from position 14700 to 15339 of SEQ ID No. 2 starting from the 5' end; F3) The 5' end transformant detection primer pair and the 3' end transformant detection primer pair; The method M2 includes the following steps: sequencing the genomic DNA of the plant sample to be tested, and determining, based on the sequencing results, whether the plant sample to be tested originates from the transgenic cotton event or its offspring, or from the insect-resistant cotton or its offspring.

8. The method according to claim 7, characterized in that: The nucleotide sequence of the upstream primer of the 5' end transformant detection primer pair is shown in SEQ ID No. 1, positions 364-389 from the 5' end; the nucleotide sequence of the downstream primer is shown in SEQ ID No. 1, positions 569-594, the reverse complementary sequence from the 5' end. The nucleotide sequence of the upstream primer of the 3' end transformant detection primer pair is shown in SEQ ID No. 2, positions 14406-14431 from the 5' end; the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2, positions 14879-14904, the reverse complementary sequence from the 5' end.

9. A processed product, characterized in that: The processed product is prepared from the genetically modified cotton event of claim 1, 3, 4 or 5 or the insect-resistant cotton of any of claims 1 to 5.

10. The application of the primer composition according to claim 7, as D1) or D2): D1) Detect or assist in detecting whether the plant sample is derived from the transgenic cotton event of claim 1, 3, 4 or 5 or its descendants, or the insect-resistant cotton of any of claims 1 to 5 or its descendants; D2) The event of cultivating transgenic cotton as described in claim 1, 3, 4 or 5, or the insect-resistant cotton as described in any of claims 1 to 5.

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