Primer and method for detecting cotton MON531 transgenic sequence structure variation

Abstract

The invention relates to the technical field of biology, in particular to primers and a method for detecting cotton MON531 transgenic sequence structure variation. The invention provides a primer combination for screening cotton MON531 transgenic sequence structure variation, the primer combination comprises primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8 and P0 / P9, and the nucleotide sequences of the primer pairs are as shown in SEQ ID NO: 7-SEQ ID NO: 16. The invention also provides a method for screening cotton MON531 transgenic sequence structure variation by using the primer pair. The primer combination and the method provided by the invention can realize continuous monitoring of the structure variation of the transgenic sequence in the cotton MON531 planting process, and lay a foundation for evaluating the safety of long-term application of the cotton MON531.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to primers and methods for detecting structural variation of cotton MON531 transgenic sequences. Background Art

[0002] Genetically modified cotton is one of the main genetically modified crops planted in the world. In 2023, the global planting area reached 24.1 million hectares, accounting for 76.0% of the total cotton planting area in the world. In 2023, the planting area of ​​genetically modified cotton in my country was 2.8 million hectares, exceeding 90% of the total cotton planting area, making it the crop with the largest genetically modified planting area. Genetically modified cotton MON531 is the earliest insect-resistant cotton transformant approved for planting in my country. Since its approval in 1998, it has been continuously planted and applied in my country for more than 20 years. Continuous tracking and monitoring of the genetically modified sequence variation of cotton MON531 is one of the important contents of evaluating its long-term application safety.

[0003] Structural variation (SV) refers to long-length sequence changes in the genome, including insertion or deletion of long fragment sequences longer than 50 bp, tandem duplication, inversion, etc. There are a lot of reports on the structural variation of the genome in the study of genetic variation of plants and animals, but there are no confirmed reports in the transgenic sequences of commercially grown transgenic crops. Summary of the invention

[0004] In order to track and monitor the variation of the cotton MON531 transgenic sequence, the inventors established a rapid and sensitive method for screening structural variation of the MON531 transgenic sequence based on the structural characteristics of the cotton MON531 transgenic insertion sequence. More than 100 transgenic cotton materials containing structural variation of the MON531 transgenic sequence were screened from more than 100,000 market cotton samples, and five of the structural variants were studied in detail, and specific detection methods for these five structural variants were established.

[0005] Based on the above research results, the present invention provides a primer combination for screening for structural variation of cotton MON531 transgenic sequence, which includes primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8 and P0 / P9, and the nucleotide sequences thereof are as follows:

[0006] P0: 5'-AAATTGTCACGGTCTACCAG-3';

[0007] P1: 5'-GAAAGAACTAAACAATTAGTACCC-3';

[0008] P2: 5'-CCTCGTGCTTTACGGTATCG-3';

[0009] P3: 5'-ATGGACAACAACCCAAACATCAACG-3';

[0010] P4: 5'-CCTCTCTATGGAACTATGGG-3';

[0011] P5: 5'-GTTAGACTCAACAGCAGTGG-3';

[0012] P6: 5'-GACGAGAAGGGTGAACTCTC-3';

[0013] P7: 5'-GATGTACTGACCTGAATGAG-3';

[0014] P8: 5'-GATCGAGAACAACACTGACG-3';

[0015] P9: 5'-AGGGAACCTTCATCGTGG-3'.

[0016] The present invention also provides a kit, which comprises the primer combination for screening the structural variation of cotton MON531 transgenic sequence.

[0017] Preferably, the kit further comprises universal reagents for polymerase chain reaction.

[0018] The present invention also provides a method for screening cotton MON531 transgenic sequence structural variation, which comprises:

[0019] (1) Extracting genomic DNA of the cotton material to be tested;

[0020] (2) using the genomic DNA as a template, and using the primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8 and P0 / P9 to perform PCR respectively to obtain amplified products;

[0021] (3) Sequencing the amplified product and comparing the determined sequence with the original MON531 transgenic sequence to screen for structurally variant MON531 transgenic sequences.

[0022] Preferably, the PCR reaction system comprises 0.5 μmol / L of a forward primer and 0.5 μmol / L of a reverse primer; the annealing temperature of the PCR is 56° C. and the extension time is 90 s.

[0023] The present invention also provides a primer combination for detecting cotton MON531 transgenic sequence structural variants, which includes primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R and R8877-F / R8877-R, and the nucleotide sequences thereof are as follows:

[0024] R1164-F: 5'-CTCATGCAGGAGCTGTCAGA-3';

[0025] R1164-R: 5'-CTATCAGTGTTTCTGGTTAT-3';

[0026] R7282-F: 5'-CAAGCGTCTCAGCGACGAGA-3';

[0027] R7282-R: 5'-TAAGAGGTATCATGTAGCCT-3';

[0028] R7569-F: 5'-CGTGAATGTCCCAGGTACTG-3';

[0029] R7569-R: 5'-CCGCCTTGGGTCTTGAAGAT-3';

[0030] R8442-F: 5'-AAATCTATCCCAACAACACC-3';

[0031] R8442-R: 5'-GTGTACACTGATAACATAGC-3';

[0032] R8877-F: 5'-ACTGAATAAGTATGTAGTACTA-3';

[0033] R8877-R: 5'-GGATCAGATTGTATATAATAC-3';

[0034] Among them, R1164-F / R1164-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:1; R7282-F / R7282-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:2; R7569-F / R7569-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:3; R8442-F / R8442-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:4; R8877-F / R8877-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:5.

[0035] The present invention also provides a kit, which comprises the primer combination for detecting the structural variant of the cotton MON531 transgenic sequence.

[0036] The present invention also provides a cotton MON531 transgenic sequence structural variant, whose nucleotide sequence is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0037] The present invention also provides a method for detecting cotton MON531 transgenic sequence structural variants, which comprises:

[0038] (1) Extracting genomic DNA of the cotton material to be tested;

[0039] (2) using the genomic DNA as a template, PCR was performed using the primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R and R8877-F / R8877-R to obtain amplified products;

[0040] (3) detecting the amplification product; if the product size is 233 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:1; if the product size is 254 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:2; if the product size is 240 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:3; if the product size is 239 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:4; if the product size is 259 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:5.

[0041] Preferably, the PCR reaction system comprises 0.5 μmol / L of a forward primer and 0.5 μmol / L of a reverse primer; the annealing temperature of the PCR is 60° C. and the extension time is 30 s.

[0042] Experiments have shown that primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8 and P0 / P9 can quickly screen out transgenic cotton materials containing MON531 transgenic sequence structural variations (Example 1), and primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R and R8877-F / R8877-R can specifically detect cotton MON531 transgenic sequence structural variants R1164, R7282, R7569, R8442 and R8877 (Example 2). Therefore, the primer combination and method of the present invention can realize the continuous monitoring of the structural variation of the transgenic sequence during the planting process of cotton MON531, laying a foundation for evaluating the safety of its long-term application. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The corresponding positions of the screening primers for structural variation of MON531 transgenic sequence on the MON531 transgenic sequence are shown; P0 is used as one end primer, and P1 to P9 are used as the other end primers respectively; P 35S Indicates promoter (open arrow), T NOS and T 7Srepresents the terminator (open square), nptII represents the marker gene nptII gene (solid arrow), aad represents the marker gene aad gene (solid arrow), cry1Ac represents the insect-resistant gene cry1Ac gene (solid arrow), cry1Ac(t) represents the truncated insect-resistant gene cry1Ac gene (solid arrow); the diagonal squares at both ends represent the cotton genome.

[0044] Figure 2 The amplification results of the screening primers for structural variation of the MON531 transgenic sequence in some cotton materials are shown; P5 to P9 in the figure represent the amplification results of primer pairs P0 / P5, P0 / P6, P0 / P7, P0 / P8, and P0 / P9, respectively; R7569, R8442, and R8877 are the cotton material numbers.

[0045] Figure 3 The structural variations of five MON531 transgenic sequence structural variants relative to the original MON531 transgenic sequence are shown; R1164, R7282, R7569, R8442 and R8877 are cotton MON531 transgenic sequence structural variants; P 35S Indicates promoter (open arrow), T NOS and T 7S represents the terminator (open box), nptII represents the marker gene nptII gene (solid arrow), aad represents the marker gene aad gene (solid arrow), cry1Ac represents the insect-resistant gene cry1Ac gene (solid arrow), cry1Ac(t) represents the truncated insect-resistant gene cry1Ac gene (solid arrow); the diagonal boxes at both ends represent the cotton genome; the dotted line represents a deletion.

[0046] Figure 4The amplification results of five MON531 transgenic sequence structural variants in cotton materials are shown; from top to bottom, there are five agarose gel electrophoresis images, which are the amplification results of primer sets R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R and R8877-F / R8877-R; each agarose gel electrophoresis image is from left to right lane M and lanes 1-24, of which 1-2 are Variant material R1164, 5-6 are variant materials R7282, 7-8 are variant materials R7569, 11-12 are variant materials R8442, 13-14 are variant materials R8877, 3-4 and 9-10 are other variant materials, 15-16 are transgenic cotton MON531, 17-20 are non-transgenic cotton, 21-24 are blank controls, and M is DL2000 (molecular weights are 2000, 1500, 750, 500, 250, and 100 bp from top to bottom).

[0047] Sequence Description

[0048] SEQ ID NO: 1 Nucleotide sequence of cotton MON531 transgenic sequence structural variant R1164;

[0049] SEQ ID NO:2 Nucleotide sequence of cotton MON531 transgenic sequence structural variant R7282;

[0050] SEQ ID NO:3 Nucleotide sequence of cotton MON531 transgenic sequence structural variant R7569;

[0051] SEQ ID NO:4 Nucleotide sequence of cotton MON531 transgenic sequence structural variant R8442;

[0052] SEQ ID NO:5 Nucleotide sequence of cotton MON531 transgenic sequence structural variant R8877;

[0053] SEQ ID NO:6 Cotton MON531 original transgenic sequence;

[0054] SEQ ID NO:7 to SEQ ID NO:16 are nucleotide sequences of primers P0 to P9 used for screening for structural variation of cotton MON531 transgenic sequences;

[0055] SEQ ID NO:17 to SEQ ID NO:26 are nucleotide sequences of primers R1164-F, R1164-R, R7282-F, R7282-R, R7569-F, R7569-R, R8442-F, R8442-R, R8877-F and R8877-R for detecting structural variants of cotton MON531 transgenic sequences. DETAILED DESCRIPTION

[0056] The following implementation plans are provided:

[0057] 1. A primer combination for screening for structural variation of cotton MON531 transgenic sequence, comprising primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8 and P0 / P9, the nucleotide sequences of which are as follows:

[0058] P0: 5'-AAATTGTCACGGTCTACCAG-3';

[0059] P1: 5'-GAAAGAACTAAACAATTAGTACCC-3';

[0060] P2: 5'-CCTCGTGCTTTACGGTATCG-3';

[0061] P3: 5'-ATGGACAACAACCCAAACATCAACG-3';

[0062] P4: 5'-CCTCTCTATGGAACTATGGG-3';

[0063] P5: 5'-GTTAGACTCAACAGCAGTGG-3';

[0064] P6: 5'-GACGAGAAGGGTGAACTCTC-3';

[0065] P7: 5'-GATGTACTGACCTGAATGAG-3';

[0066] P8: 5'-GATCGAGAACAACACTGACG-3';

[0067] P9: 5'-AGGGAACCTTCATCGTGG-3'.

[0068] 2. A kit comprising the primer combination for screening the structural variation of cotton MON531 transgenic sequence according to embodiment 1.

[0069] 3. The kit described in embodiment 2 also contains universal reagents for polymerase chain reaction.

[0070] 4. A method for screening for structural variation of cotton MON531 transgenic sequence, comprising:

[0071] (1) Extracting genomic DNA of the cotton material to be tested;

[0072] (2) using the genomic DNA as a template, and using the primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8 and P0 / P9 described in Embodiment 1 to perform PCR respectively to obtain amplified products;

[0073] (3) Sequencing the amplified product and comparing the determined sequence with the original MON531 transgenic sequence to screen for structurally variant MON531 transgenic sequences.

[0074] 5. The method described in embodiment 4, wherein the PCR reaction system comprises 0.5 μmol / L of forward primer and 0.5 μmol / L of reverse primer; the annealing temperature of the PCR is 56°C and the extension time is 90s.

[0075] 6. A primer combination for detecting structural variants of cotton MON531 transgenic sequence, characterized in that it includes primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R and R8877-F / R8877-R, and its nucleotide sequence is as follows:

[0076] R1164-F: 5'-CTCATGCAGGAGCTGTCAGA-3';

[0077] R1164-R: 5'-CTATCAGTGTTTCTGGTTAT-3';

[0078] R7282-F: 5'-CAAGCGTCTCAGCGACGAGA-3';

[0079] R7282-R: 5'-TAAGAGGTATCATGTAGCCT-3';

[0080] R7569-F: 5'-CGTGAATGTCCCAGGTACTG-3';

[0081] R7569-R: 5'-CCGCCTTGGGTCTTGAAGAT-3';

[0082] R8442-F: 5'-AAATCTATCCCAACAACACC-3';

[0083] R8442-R: 5'-GTGTACACTGATAACATAGC-3';

[0084] R8877-F: 5'-ACTGAATAAGTATGTAGTACTA-3';

[0085] R8877-R: 5'-GGATCAGATTGTATATAATAC-3';

[0086] Among them, R1164-F / R1164-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:1; R7282-F / R7282-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:2; R7569-F / R7569-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:3; R8442-F / R8442-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:4; R8877-F / R8877-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:5.

[0087] 7. A kit comprising the primer combination for detecting structural variants of cotton MON531 transgenic sequence according to embodiment 6.

[0088] 8. A cotton MON531 transgenic sequence structural variant, whose nucleotide sequence is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0089] 9. A method for detecting structural variants of cotton MON531 transgenic sequence, comprising:

[0090] (1) Extracting genomic DNA of the cotton material to be tested;

[0091] (2) Using the genomic DNA as a template, PCR was performed using the primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R, and R8877-F / R8877-R described in Embodiment 6 to obtain amplified products;

[0092] (3) detecting the amplification product; if the product size is 233 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:1; if the product size is 254 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:2; if the product size is 240 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:3; if the product size is 239 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:4; if the product size is 259 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:5.

[0093] 10. The method described in embodiment 9, wherein the PCR reaction system comprises 0.5 μmol / L of forward primer and 0.5 μmol / L of reverse primer; the annealing temperature of the PCR is 60°C and the extension time is 30s.

[0094] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0095] The reagents not specifically described in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the conditions described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001), or according to the conditions recommended by the instrument or reagent manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0096] The main reagents used in the following examples are:

[0097] Plant genomic DNA extraction kit (DP305) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0098] Go GreenMaster Mix and ddH2O were purchased from Promega Biotechnology Co., Ltd.

[0099] The molecular weight marker DL2000 Marker was purchased from Dalian Bao Biotechnology Co., Ltd.

[0100] 50×TAE electrophoresis buffer was purchased from Beijing Solebow Biotechnology Co., Ltd.

[0101] Primer synthesis and Sanger sequencing were completed by Beijing Tsingke Biotech Co., Ltd.

[0102] The main instruments used in the following examples are:

[0103] Pipette (Eppendorf)

[0104] Biological sample homogenizer (Bioprep-24, Hangzhou Aosheng Instrument Co., Ltd.)

[0105] Dry thermostat (MK200-4, Hangzhou Aosheng Instrument Co., Ltd.)

[0106] Vacuum pump (Promega, Promega (Beijing) Biotechnology Co., Ltd.)

[0107] Ultra-micro-volume UV spectrophotometer (Thermo Scientific NanoDrop2000, Thermo Fisher Scientific (China) Co., Ltd.)

[0108] High-speed centrifuge (Centrifuge 5425, Eppendorf )

[0109] PCR amplification instrument (Bio-RAD T100TM Thermal Cycler, Bio-Rad )

[0110] Nucleic acid electrophoresis instrument (DYY-6C, Beijing Liuyi Instrument Factory)

[0111] Gel imaging system (EC3-Imaging System_UVP, UVP company)

[0112] Electronic balance (PL303-IC, Mettler-Toledo Instruments (Shanghai) Co., Ltd.)

[0113] Example 1. Screening of structural variations in MON531 transgenic sequences

[0114] From 2010 to 2023, commercial cotton seeds were collected from the main cotton producing areas in my country and planted at the experimental base of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Xinxiang City, Henan Province. Cotton seeds were collected from individual plants, and a total of 120,000 cotton seed materials were obtained.

[0115] 1. Sample Pretreatment

[0116] 120,000 cottonseed materials, 4 seeds were randomly selected from each material, and 100 materials were mixed into a mixed sample and ground into powder. To identify the mixed sample containing recombinants, 4 seeds were randomly selected from each cottonseed material in the mixed sample and ground into single seeds.

[0117] 2. Cotton Genomic DNA Extraction

[0118] The genomic DNA of cotton materials was extracted using a plant genomic DNA extraction kit (TIANGEN, DP305) according to the kit's operating manual. 5 μl of the extracted DNA solution was subjected to 0.8% agarose gel electrophoresis, and the quality of the DNA was preliminarily determined based on the brightness and pattern of the bands. The concentration and purity of the extracted DNA were determined using an ultraviolet spectrophotometer.

[0119] 3. Design of primers for screening structural variation of MON531 transgenic sequence

[0120] According to the structural characteristics of the cotton MON531 transgenic sequence (SEQ ID NO: 6), the structural variation occurring in the junction region of the two inserted fragments was focused on. Primer P0 was designed at the end of the 7S terminator of the truncated inserted fragment in the 3' region of the MON531 transgenic sequence, and 9 primers P1 to P9 were designed in the 5' region of the MON531 transgenic sequence. The corresponding positions of primers P0 to P9 on the MON531 transgenic sequence are shown in Figure 1 , and its nucleotide sequence is shown in Table 1.

[0121] Table 1 Primers for screening structural variations of MON531 transgenic sequences

[0122]

[0123]

[0124] 4. PCR amplification screening of MON531 transgenic sequence structural variation

[0125] Primer P0 was used as one end primer and was combined with primers P1 to P9 at the other end to obtain primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8 and P0 / P9. The genomic DNA of cotton material was used as template and PCR was performed using these 9 primer pairs. PCR reaction system: Go GreenMaster Mix 10μL, forward primer (10μmol / L) and reverse primer (10μmol / L) 1μL each, DNA template 2μL, add ultrapure water to a total volume of 20μL. Reaction procedure: 95℃ pre-denaturation for 4min; (95℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 90s) 35 cycles; 72℃ incubation for 10min.

[0126] The 1200 mixed samples were amplified separately, and the obtained amplified products were sent for sequencing. The determined sequences were analyzed and compared with the original transgenic sequence of MON531 to identify the mixed samples with structural variations. Then, the identification primer combination of the structural variation was used to identify each cotton seed material in the mixed sample to determine the individual plant with structural variation. The electrophoresis pattern of the amplified products of some cotton seed materials is shown in Figure 2 .

[0127] The results of the identification showed that a total of 110 structural variant sequences with deletions at different positions on the intact insertion fragment at the 5' end of the original transgenic sequence of MON531 were obtained. Five representative variant materials R1164, R7282, R7569, R8442 and R8877 (the numbers indicate the starting position of the sequence deletion) were selected for further detailed analysis.

[0128] Example 2. Specific detection of MON531 variants

[0129] The five representative variant materials R1164, R7282, R7569, R8442 and R8877 screened in Example 1 were planted in the laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and individual plants were identified and seeds were collected by self-pollination.

[0130] 1. Sample Pretreatment

[0131] Grind the leaves or seeds of individual cotton plants evenly.

[0132] 2. Cotton Genomic DNA Extraction

[0133] The genomic DNA of cotton materials was extracted using a plant genomic DNA extraction kit (TIANGEN, DP305) according to the kit's operating manual. 5 μl of the extracted DNA solution was subjected to 0.8% agarose gel electrophoresis, and the quality of the DNA was preliminarily determined based on the brightness and pattern of the bands. The concentration and purity of the extracted DNA were determined using an ultraviolet spectrophotometer.

[0134] 3. Determination of transgenic sequence structure of variants

[0135] The homozygous materials of the five variant materials were obtained by single plant identification, self-pollination and single plant identification of progeny materials. With reference to the transgenic sequence of cotton MON531 (SEQ ID NO: 6), the transgenic sequences of the five variant materials were obtained by PCR amplification and sequencing of amplified fragments.

[0136] Determine the sequence structure ( Figure 3) showed that the transgenic sequence lengths of the variant materials R1164, R7282, R7569, R8442 and R8877 were 3230bp, 9253bp, 9620bp, 10516bp and 10908bp, respectively, which were 7935bp, 1912bp, 1545bp, 649bp and 257bp shorter than the original transgenic sequence of MON531.

[0137] 4. Specific detection of variant materials

[0138] Specific primers were designed at both ends of the deletion region of the five variant materials. The primer sequences, primer positions and amplified fragment sizes are shown in Table 2.

[0139] Table 2 Specific detection primers for MON531 variants

[0140]

[0141] The genomic DNA of cotton materials was used as template, and PCR was performed using primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R, and R8877-F / R8877-R, respectively. Cotton materials included variant materials R1164, R7282, R7569, R8442, and R8877, other variant materials, transgenic cotton MON531, and non-transgenic cotton. Ultrapure water was used instead of DNA template for blank control.

[0142] PCR reaction system: Go GreenMaster Mix 10μL, forward primer (10μmol / L) and reverse primer (10μmol / L) 1μL each, DNA template 2μL, add ultrapure water to a total volume of 20μL. Reaction procedure: 95℃ pre-denaturation for 4min; (95℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 30s) 35 cycles; 72℃ incubation for 10min.

[0143] Test results( Figure 4 ) showed that primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R and R8877-F / R8877-R could only amplify characteristic bands from their respective corresponding MON531 variant materials, and no amplification was found in other cotton materials. Therefore, these five primer pairs can specifically detect the MON531 transgenic sequence structural variants R1164, R7282, R7569, R8442 and R8877.

Claims

1. A primer combination for screening for structural variation of cotton MON531 transgenic sequence, characterized in that: It includes primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8 and P0 / P9, and the nucleotide sequences thereof are as follows: P0: 5'-AAATTGTCACGGTCTACCAG-3'; P1: 5'-GAAAGAACTAAACAATTAGTACCC-3'; P2: 5'-CCTCGTGCTTTACGGTATCG-3'; P3: 5'-ATGGACAACAACCCAAACATCAACG-3'; P4: 5'-CCTCTCTATGGAACTATGGG-3'; P5: 5'-GTTAGACTCAACAGCAGTGG-3'; P6: 5'-GACGAGAAGGGTGAACTCTC-3'; P7: 5'-GATGTACTGACCTGAATGAG-3'; P8: 5'-GATCGAGAACAACACTGACG-3'; P9: 5'-AGGGAACCTTCATCGTGG-3'.

2. A kit, characterized in that: The primer combination for screening the structural variation of cotton MON531 transgenic sequence as described in claim 1 is included.

3. The kit according to claim 2, characterized in that Also included are general reagents for polymerase chain reaction.

4. A method for screening for structural variation of cotton MON531 transgenic sequence, characterized in that: include: (1) Extracting genomic DNA of the cotton material to be tested; (2) using the genomic DNA as a template, and using the primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8 and P0 / P9 described in claim 1 to perform PCR respectively to obtain amplified products; (3) Sequencing the amplified product and comparing the determined sequence with the original MON531 transgenic sequence to screen for structurally variant MON531 transgenic sequences.

5. The method according to claim 4, characterized in that The PCR reaction system comprises 0.5 μmol / L of a forward primer and 0.5 μmol / L of a reverse primer; the annealing temperature of the PCR is 56° C. and the extension time is 90 s.

6. A primer combination for detecting structural variants of cotton MON531 transgenic sequence, characterized in that: The primer pairs include R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R and R8877-F / R8877-R, and the nucleotide sequences thereof are as follows: R1164-F: 5'-CTCATGCAGGAGCTGTCAGA-3'; R1164-R: 5'-CTATCAGTGTTTCTGGTTAT-3'; R7282-F: 5'-CAAGCGTCTCAGCGACGAGA-3'; R7282-R: 5'-TAAGAGGTATCATGTAGCCT-3'; R7569-F: 5'-CGTGAATGTCCCAGGTACTG-3'; R7569-R: 5'-CCGCCTTGGGTCTTGAAGAT-3'; R8442-F: 5'-AAATCTATCCCAACAACACC-3'; R8442-R: 5'-GTGTACACTGATAACATAGC-3'; R8877-F: 5'-ACTGAATAAGTATGTAGTACTA-3'; R8877-R: 5'-GGATCAGATTGTATATAATAC-3'; Among them, R1164-F / R1164-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:1; R7282-F / R7282-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:2; R7569-F / R7569-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:3; R8442-F / R8442-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:4; R8877-F / R8877-R is used to detect the structural variants of the MON531 transgenic sequence with a nucleotide sequence as shown in SEQ ID NO:

5.

7. A kit, characterized in that: A primer combination for detecting cotton MON531 transgenic sequence structural variants as described in claim 6.

8. A cotton MON531 transgenic sequence structural variant, characterized in that: The nucleotide sequence thereof is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

9. A method for detecting structural variants of cotton MON531 transgenic sequence, characterized in that: include: (1) Extracting genomic DNA of the cotton material to be tested; (2) using the genomic DNA as a template, and using the primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R, and R8877-F / R8877-R described in claim 6 to perform PCR respectively to obtain amplified products; (3) Detecting the amplification product. If the product size is 233 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:1; if the product size is 254 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:2; if the product size is 240 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:3; if the product size is 239 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:4; if the product size is 259 bp, the cotton material to be tested contains a MON531 transgenic sequence structural variant with a nucleotide sequence as shown in SEQ ID NO:

5.

10. The method according to claim 9, characterized in that The PCR reaction system comprises 0.5 μmol / L of a forward primer and 0.5 μmol / L of a reverse primer; the PCR annealing temperature is 60° C. and the extension time is 30 s.

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