Primer sets, kits, and methods for detecting vector gatv2 transformation events

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a primer set, a kit and a method for detecting a vector GATV2 transformation event. The primer set comprises left border-specific primers and / or right border-specific primers, the left border-specific primers comprise GATV2-LB-R1 / R2 / R3, and the sequences of the left border-specific primers are shown in SEQ ID NO: 1-3 in sequence respectively; the right border-specific primers comprise GATV2-RB-F1 / F2 / F3, and the sequences of the right border-specific primers are shown in SEQ ID NO: 4-6 in sequence respectively. The primer set of the application can accurately and quickly find the left and right flanking sequences of GATV2, and no specific bands can be amplified in non-transgenic or other transgenic plants, and is suitable for detecting, monitoring and safety management of a rice genetic and artificial technology (GAT) maintainer line GATV2 and a derivative line thereof.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to methods for safety assessment and detection of transgenic plants, specifically to a primer set, reagent kit, and method for detecting transformation events of the vector GATV2. Background Technology

[0002] Rice is one of the world's most important food crops. With population growth and improved living standards, it is projected that annual rice production will need to increase by 1-2 times by 2050 to meet the demands of human development. Plant transgenic technology is one of the core technologies of modern biotechnology. It involves integrating exogenous DNA fragments into the genomic DNA of a recipient plant through biological, physical, or chemical methods, aiming to improve the recipient organism's original traits or endow it with new traits.

[0003] Unlike the "three-line method" and "two-line method" in hybrid rice breeding, intelligent male sterility lines are controlled by only one pair of recessive nuclear genes, unaffected by light, temperature, or environmental factors. Furthermore, because these genes are recessive mutations, theoretically almost any variety can be used as a restorer line to produce fertile offspring. However, for a long time, their industrial application has been limited by the inability to maintain male sterility in offspring. Therefore, Genetic Automation Technology (GAT) for rice hybrid breeding was developed. The GATV2 vector constructs expression cassettes for plant male fertility restoration genes, plant pollen abortion genes, herbicide resistance genes, herbicide sensitivity genes, and fluorescent genes onto a GAT vector and transforms them into sterile mutants to create the GATV2 maintainer line.

[0004] In recent years, genetically modified (GM) crops such as soybeans, corn, cotton, and rapeseed have been approved for cultivation and production in many countries, with some being processed into food, feed, or used as food additives. However, the ecological and food safety of GM products remains highly controversial and requires strict regulation. Identifying the specific insertion site of the transformation event in the genome, determining the insertion, inactivation, or deletion of the host genome based on the insertion site of the exogenous vector, and inferring the impact of the transformation event on the host and the resulting safety issues are crucial aspects of regulation.

[0005] In transgenic crops obtained through Agrobacterium-mediated genetic transformation, the integration location of T-DNA in the recipient plant genome is random. The flanking sequences of the T-DNA insertion site, formed by splicing the left and right ends of the T-DNA with the recipient genome sequence for each transgenic event, are unique. These flanking sequences are unique identifiers distinguishing different transformation events and are crucial technical data for establishing specific detection methods for transgenic plant lines. Currently, the isolation of exogenous T-DNA flanking sequences is mainly based on PCR technology. Specific detection methods include reverse PCR, exogenous adapter-mediated PCR, semi-random primer PCR, and whole-genome resequencing. Among these, thermal asymmetric interleaved PCR (Tail-PCR), high-performance thermal asymmetric PCR (hiTail-PCR), or chromosome walking are commonly used methods in semi-random primer PCR. Summary of the Invention

[0006] The purpose of this invention is to provide a dedicated PCR detection primer, kit, and method for identifying the transformation event insertion site of the Genetically Intelligent Breeding Technology (GAT) vector pC0308-MMMaauCK5400 (GATV2). This invention is based on the transgenic rice GATV2 provided by Hainan Bolian Rice Gene Technology Co., Ltd., and relevant information about this vector has been disclosed in CN202010379287.9, "A Genetically Intelligent Breeding System for Crop Hybrid Breeding and Seed Production and Its Application".

[0007] To clarify the molecular characteristics of the GATV2 vector inserted into the genome and facilitate its detection, and to advance its biosafety evaluation, this invention designs specific detection primers based on the left and right end sequences of the GATV2 vector's T-DNA, uses Tail-PCR to separate its T-DNA flanking sequences, and establishes a method for detecting the insertion site of GATV2 transformation events. This provides a technical basis for the detection and identification of transformation events created by GATV2 and its derivative products.

[0008] In one aspect, the present invention first designs left-boundary specific primers GATV2-LB-R1 / R2 / R3 (SEQ ID NO: 1-3) and right-boundary specific primers GATV2-RB-F1 / F2 / F3 (SEQ ID NO: 4-6) based on the left-boundary (LB) and right-boundary specific sequences of the vector GATV2 (e.g., ...). Figure 1 (As shown).

[0009] Accordingly, the present invention first provides a primer set for detecting the transformation event of the GATV2 vector, which includes a left boundary specific primer and / or a right boundary specific primer;

[0010] The left-bound specific primers include GATV2-LB-R1, GATV2-LB-R2 and GATV2-LB-R3, whose sequences are shown in SEQ ID NO:1 to 3 respectively.

[0011] The right-boundary specific primers include GATV2-RB-F1, GATV2-RB-F2 and GATV2-RB-F3, whose sequences are shown in SEQ ID NO:4 to 6 respectively.

[0012] Preferably, the primer set further includes degenerate primers. In this invention, the degenerate primers preferably include random degenerate primers AD2, AD8 and AD10, whose sequences are shown in SEQ ID NO:7 to 9 respectively.

[0013] Secondly, the present invention also provides a kit comprising the aforementioned primers.

[0014] As a preferred embodiment, the kit includes the left-bound-specific primer, the right-bound-specific primer, and the random degenerate primer.

[0015] More preferably, the kit further includes one or more of the following: hot-start Taq DNA polymerase, dNTPs, PCR reaction buffer, positive control, and negative control.

[0016] Thirdly, the present invention also provides the application of the primer set or the kit in detecting the transformation event of the GATV2 vector.

[0017] Preferably, the application specifically includes applications in any of the following aspects:

[0018] (1) Detect whether the GATV2 carrier conversion event exists in the sample to be tested;

[0019] (2) Identify the flanking sequence specific to the GATV2 transformation event of the vector;

[0020] (3) Identify the insertion site of the GATV2 transformation event in the vector.

[0021] Furthermore, the present invention also provides a method for detecting the transformation event of the vector GATV2, comprising: using the primer set or the kit described herein, and using the DNA of the sample to be tested as a template, performing PCR amplification.

[0022] Preferably, the method includes: performing Tail-PCR amplification on the left and / or right boundaries of GATV2; specifically including:

[0023] a) Using the DNA of the sample to be tested as a template, and AD2, AD8, AD10 and the GATV2-LB-R1 / GATV2-RB-F1 as primers, the first round of PCR amplification was performed;

[0024] b) After diluting the first round of PCR amplification products (e.g., diluting 50 times), use them as templates and AD2, AD8, AD10 and the GATV2-LB-R2 / GATV2-RB-F2 described above as primers to perform the second round of PCR amplification.

[0025] c) After diluting the second round of PCR amplification products (e.g., diluting 20 times), use them as templates and AD2, AD8, AD10 and the GATV2-LB-R3 / GATV2-RB-F3 described above as primers to perform the third round of PCR amplification.

[0026] Preferably, if a specific band can be amplified, it is determined that the GATV2 conversion event exists in the sample to be tested; otherwise, it is determined that the GATV2 conversion event does not exist in the sample to be tested.

[0027] Preferably, the method further includes:

[0028] The final PCR amplification products were sequenced to determine the flanking sequences specific to the GATV2 transformation event.

[0029] Preferably, the method further includes:

[0030] The final PCR amplification product was compared with the sequence of the corresponding transformed plant genome to determine the insertion site of the GATV2 vector transformation event.

[0031] The targets of the detection mentioned in this invention include, but are not limited to, genetically modified crops such as soybeans, corn, cotton, and rapeseed and their derivatives. As long as the GATV2 vector transformation event exists, it can be detected by the primer set, kit and method of this invention.

[0032] As a preferred embodiment, the carrier GATV2 conversion event described in this invention includes any of the following:

[0033] 1) The vector GATV2 is inserted into GATV2-88-4 at nucleotides 27241465 to 27241510 on chromosome 12 of the rice genome. The LB side sequence is shown in SEQ ID NO:10.

[0034] 2) The LB end of the vector GATV2 is inserted into GATV2-11-1 at nucleotide position 33460100 on chromosome 1 of the rice genome, as shown in SEQ ID NO:11.

[0035] 3) The RB end of the vector GATV2 is inserted into GATV2-53-1 at nucleotide position 27344067 on chromosome 4 of the rice genome, as shown in SEQ ID NO:12.

[0036] Based on the above technical solution, the beneficial effects of the present invention are as follows:

[0037] This invention discloses for the first time a primer set, kit, and method for detecting the transformation event of the vector GATV2. It can accurately and quickly identify the left and right flanking sequences of GATV2, while failing to amplify specific bands in non-transgenic or other transgenic plants. It is suitable for the detection, monitoring, and safety management of the rice genetic intelligent breeding technology (GAT) maintainer line GATV2 and its derivative lines. Attached Figure Description

[0038] Figure 1 The T-DNA consists of a GAT-pC0308-MMMaauCK5400-L188(GATV2) map and specific primers for the left and right boundaries distributed on the vector. The T-DNA includes a left boundary sequence (LB), a plant male fertility restoration gene expression cassette, a plant pollen abortion gene expression cassette, a herbicide resistance gene expression cassette, a herbicide sensitivity gene expression cassette, a fluorescent gene expression cassette, and a right boundary sequence (RB).

[0039] Figure 2 The results of the third round of electrophoresis for Tail-PCR at the left boundary are shown. M: D2000 DNA Marker; lanes 1-8 are the PCR products amplified by random degenerate primer AD2; lanes 9-16 are the PCR products amplified by random degenerate primer AD8; and lanes 17-24 are the PCR products amplified by random degenerate primer AD10.

[0040] Figure 3 The results of the third round of electrophoresis for Tail-PCR at the right boundary are shown. M: D2000 DNA Marker; lanes 1-8 are the PCR products amplified by random degenerate primer AD2; lanes 9-16 are the PCR products amplified by random degenerate primer AD8; and lanes 17-24 are the PCR products amplified by random degenerate primer AD10.

[0041] Figure 4 The left boundary sequencing results are for the transformation event GATV2-88-4. The bold boxed part is the LB left boundary sequence of the GATV2 gene, and the italic part is the rice genome sequence.

[0042] Figure 5 This is a schematic diagram of the integration site of the rice maintainer line GATV2-88-4 in the rice genome.

[0043] Figure 6 The left boundary sequencing results are for the transformation event GATV2-11-1. The bold boxed part is the right boundary sequence of the RB of the GATV2 gene, and the italic part is the rice genome sequence.

[0044] Figure 7 The image shows the right boundary sequencing results of the transformation event GATV2-53-1. The bold boxed portion is the RB right boundary sequence of the GATV2 gene, and the italic portion is the rice genome sequence. Detailed Implementation

[0045] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0046] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0047] Example 1: Construction of GAT vector and acquisition of GATV2 plants

[0048] The GAT vector is constructed in a plant binary expression vector by linking several of the following five gene expression cassettes through adapter sequences, as described in patent CN202010379287.9, "A Genetic Intelligent Breeding System for Crop Hybridization and Seed Production and Its Application." The five gene expression cassettes are:

[0049] (1) The plant male fertility restoration gene expression cassette is composed of a promoter, a male fertility restoration gene coding region, and a terminator connected in sequence. The function of this expression cassette is to restore the male fertility of the recessive homozygous mutant oscyp704b2 of the OsCYP704B2 gene.

[0050] (2) A plant pollen abortion gene expression cassette, which is composed of a plant pollen-specific promoter, an abortion gene coding region, and a terminator connected in sequence. The function of the expression cassette is to cause pollen containing GAT transgenes to abort, thereby maintaining the heterozygous state of GAT transformants or GAT maintainer lines and preventing GAT transgene drift.

[0051] (3) The herbicide resistance gene expression cassette consists of a promoter, a herbicide resistance gene coding region, and a terminator connected in sequence. The function of this expression cassette is to provide resistance for gene transformation screening and to distinguish between GAT maintainer lines and sterile lines.

[0052] (4) The herbicide-sensitive gene expression cassette is composed of a promoter, a herbicide-dominant sensitive element, and a terminator connected in sequence. The function of this element is to prevent GAT transgene drift and remove contamination of non-transgenic plants by GAT plants.

[0053] (5) A fluorescent gene expression cassette, consisting of a seed-specific promoter, a fluorescent gene, and a terminator connected in sequence. This element is used to distinguish GAT maintainer line seeds from GAT sterile lines or GAT hybrids.

[0054] The GAT vector was transferred into the male-sterile material of Zhonghua 11 using Agrobacterium tumefaciens-mediated genetic transformation, and T0 generation GAT transgenic plants were obtained.

[0055] Single-copy or low-copy GAT plants were selected as GAT maintainer lines. Self-pollination of the GAT maintainer lines yielded seeds of a 1:1 segregation ratio between the GAT maintainer line and the GAT sterile line (the Zhonghua 11 male-sterile material). These seeds were separated using fluorescent markers provided by a fluorescent gene expression cassette, enabling self-propagation of the GAT maintainer line. Pollination of the GAT maintainer line with the GAT sterile line resulted in seed production in the sterile line, maintaining male sterility in its offspring, thus achieving the propagation of the recessive male-sterile line. Herbicide resistance and susceptibility differences provided by herbicide resistance and susceptibility gene expression cassettes were used to screen for GAT maintainer and GAT sterile lines.

[0056] The five gene expression cassettes in the GAT vector provided by this invention work together organically, and combined with mechanized and automated processing, can successfully enable the commercial use of recessive nuclear male sterile lines in plants. The GAT vector of this invention can be applied to hybridization breeding and seed production of recessive nuclear male sterile materials in plants, thereby obtaining high-quality, high-yielding, widely adaptable, and highly resistant new plant varieties and their seeds.

[0057] Example 2: Design of specific primers for the left and right boundaries (LB / RB) of vector GATV2

[0058] Referring to Liu et al.'s (1995) TAIL PCR (specific method refers to Liu, Y.-G., Whittier, RF, 1995. Thermal Asymmetric Interlaced PCR: Automatable Amplification and Sequencing of Insert End Fragments from PI and YAC Clones for Chromosome Walking. Genomics 25, 674-681.), based on GAT-pC0308-MMMaauCK5400-L188 (GATV2), the specific sequence of pC0308-MMMaauCK5400 refers to Chinese Patent Application No. 202010379287.9, "A Genetic Intelligent Breeding System for Crop Hybrid Breeding and Seed Production and Its Application", three specific primers (GATV2-LB-R1 / R2 / R3) were designed based on the left border (LB) sequence of the plasmid map, as shown in SEQ ID NO: 1-3; based on the right border (Right) sequence... Three specific primers (GATV2-RB-F1 / F2 / F3) were designed based on the border (RB) sequence, as shown in SEQ ID NO:4~6 (specific primer sequences are shown in Tables 1~2). Figure 1 As shown.

[0059] Example 3: Amplification of the flank segments of the left and right boundaries of GATV2

[0060] 1. DNA extraction

[0061] (1) Sample collection: Leaves of different lines of the transgenic rice maintainer line GATV2 grown in the transgenic greenhouse were collected and stored at -80℃.

[0062] (2) DNA extraction: Genomic DNA was extracted from the rice leaves using the traditional CTAB method.

[0063] (3) Detection of DNA concentration and purity: The concentration and purity of the DNA sample described in step (2) were detected using NanoDrop2000.

[0064] 2. Obtaining the left and right flanking sequences

[0065] In this embodiment, the flanking sequences of the left and right boundaries of the exogenous T-DNA integration site in transgenic rice GATV2 were obtained using thermal asymmetric PCR (TAIL-PCR) invented by Academician Liu Yaoguang of South China Agricultural University.

[0066] (1) The left boundary specific primers designed based on the GATV2 sequence are shown in Table 1, the right boundary specific primers are shown in Table 2, and the random degenerate primers used are shown in Table 3.

[0067] Table 1. Specific primers for the left border

[0068]

[0069] Table 2. Specific primers for the right boundary

[0070]

[0071] Table 3 Random degenerate primers for TAIL-PCR

[0072]

[0073] Where N represents A, T, G, or C, and W represents A or T.

[0074] (2) The Tail-PCR reaction system consisted of: 1 μL 10× reaction buffer, 0.25 μL dNTPs, and 0.25 μL of 10 μmol·L⁻¹ solution. -1 Forward primer and 0.25 μL of 10 μmol·L⁻¹ concentration -1 Reverse primer, concentration 100 μmol·L -1 Add 1 μL each of random degenerate primers AD2, AD8, and AD10, 0.5 U Taq DNA polymerase, 1 μL of 10 ng / μL template DNA, and ultrapure water to bring the total volume to 10 μL.

[0075] The PCR reaction was performed in three steps: The first step used specific primer pairs GATV2-LB-R1 or GATV2-RB-F1 and random degenerate primers AD2, AD8, and AD10. The template DNA was genomic DNA from different transformation events of transgenic rice GATV2. The reaction program was as follows: preheating at 93℃ for 2 min, denaturation at 95℃ for 1 min, followed by the following cycles: denaturation at 94℃ for 30 s, annealing at 60℃ for 1 min, extension at 72℃ for 3 min, for 10 cycles; then denaturation at 94℃ for 30 s, annealing at 20℃ for 2 min, extension at 72℃ for 3 min, followed by the following cycles: denaturation at 94℃ for 20 s, annealing at 58℃ for 1 min, extension at 72℃ for 3 min, for 25 cycles; after the first cycle, a final extension at 72℃ for 5 min was performed to terminate the reaction.

[0076] The second step uses specific primer pairs GATV2-LB-R2 or GATV2-RB-F2 and random degenerate primers AD2, AD8, and AD10. The template DNA is a 50-fold dilution of the product from reaction 1. The reaction program is as follows: perform the following cycles: denaturation at 94°C for 20 s, annealing at 65°C for 1 min, extension at 72°C for 3 min, for one cycle. Then perform the following cycles: denaturation at 94°C for 20 s, annealing at 68°C for 1 min, extension at 72°C for 3 min, denaturation at 94°C for 20 s, annealing at 68°C for 1 min, extension at 72°C for 3 min, denaturation at 94°C for 20 s, annealing at 50°C for 1 min, extension at 72°C for 3 min, for 13 cycles. After the first cycle, perform a final extension at 72°C for 5 min to terminate the reaction.

[0077] The third step uses specific primer pairs GATV2-LB-R3 or GATV2-RB-F3 and random degenerate primers AD2, AD8, and AD10. The template DNA is a 20-fold dilution of the product from reaction 2. The reaction program is as follows: denaturation at 94℃ for 20 s, annealing at 68℃ for 1 min, extension at 72℃ for 3 min, denaturation at 94℃ for 20 s, annealing at 68℃ for 1 min, extension at 72℃ for 3 min, denaturation at 94℃ for 20 s, annealing at 50℃ for 1 min, extension at 72℃ for 3 min, for 6-7 cycles; after the cycles, perform an additional 5 min extension at 72℃ to terminate the reaction.

[0078] Prepare a 1.5% agarose gel and electrophoresis at a 5V / cm electric field for 30 min; recover the PCR products of reaction 2 using a commercially available DNA gel recovery kit.

[0079] Figure 2 This shows the third round of PCR reactions using primers AD2, AD8, and AD10 on the left wing. Lanes 1-8 show the PCR products amplified by random degenerate primer AD2; lanes 9-16 show the PCR products amplified by random degenerate primer AD8; and lanes 17-24 show the PCR products amplified by random degenerate primer AD10. Figure 3 The right-wing primers AD2, AD8, and AD10 are shown in the third round of PCR reaction. Lanes 1-8 show the PCR product results of random degenerate primer AD2; lanes 9-16 show the PCR product results of random degenerate primer AD8; and lanes 17-24 show the PCR product results of random degenerate primer AD10.

[0080] 3. Determination of the integration site of exogenous T-DNA in the rice genome.

[0081] Different exogenous T-DNA integration sites in transgenic rice GATV2 were compared on the rice.plantbiology.msu website (http: / / rice.plantbiology.msu.edu / analyses_search_blast.shtml).

[0082] The detection results for the conversion event GATV2-88-4 are shown below. Figure 4-6 The sequencing results of its left boundary are shown in Figure 4 The left-wing sequence is continuous on the rice genome Chr12:27241310..27241465 (SEQ ID NO:10), meaning that the T-DNA of the rice GAT maintainer line GATV2-88-4 is inserted into chromosome 12 of the rice genome at bases 27241465 to 27241510. A schematic diagram of its integration site in the rice genome is shown below. Figure 5 .

[0083] Sequencing results of the left boundary of transformation event GATV2-11-1 are shown below. Figure 6 The LB end of the vector GATV2 is inserted at nucleotide position 33460100 on chromosome 1 of the rice genome, as shown in SEQ ID NO:11.

[0084] Sequencing results of the right boundary of the transformation event GATV2-53-1 are shown below. Figure 7 The RB end of the vector GATV2 is inserted into GATV2-53-1 at nucleotide position 27344067 on chromosome 4 of the rice genome, as shown in SEQ ID NO:12.

[0085] In summary, this invention provides a kit for amplifying flanking sequences of transformation events in the GATV2 vector genome. The kit for amplifying the left flanking sequence includes: left-boundary specific primers GATV2-LB-R1 / R2 / R3, dNTPs, PCR buffer, and hot-start Taq DNA polymerase, along with corresponding amplification procedures and methods. The kit for amplifying the right flanking sequence includes: right-boundary specific primers GATV2-RB-F1 / F2 / F3, dNTPs, PCR buffer, and hot-start Taq DNA polymerase, along with corresponding amplification procedures and methods. These flanking sequence kits can accurately and rapidly identify the left and right flanking sequences of GATV2, while specific bands were not amplified in non-transgenic or other transgenic rice varieties. Therefore, these flanking sequence kits are suitable for identifying insertion sites of other transformation events generated by the GATV2 vector.

[0086] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Hainan Bolian Rice Gene Technology Co., Ltd. <120> Primer set, kit, and method for detecting GATV2 vector transformation events. <130> KHP211123761.4 <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <400> 1 attgaatcct gttgccggtc tt 22 <210> 2 <211> twenty four <212> DNA <213> Artificial Sequence <400> 2 gcatgacgtt atttatgaga tggg 24 <210> 3 <211> twenty four <212> DNA <213> Artificial Sequence <400> 3 cccgcaatta tacatttaat acgc 24 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <400> 4 ccagcttata taccttagca ggagaca 27 <210> 5 <211> 52 <212> DNA <213> Artificial Sequence <400> 5 acgatggact ccagtccggg acattccttc cgtatctttt acgcagcggt at 52 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <400> 6 tttttcaaag ttgttttcaa agttggcgta taaca 35 <210> 7 <211> 16 <212> DNA <213> Artificial Sequence <400> 7 agtgnagaan caaagg 16 <210> 8 <211> 16 <212> DNA <213> Artificial Sequence <400> 8 agwgnagwan cawagg 16 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <400> 9 wgtgnagwan canaga 16 <210> 10 <211> 209 <212> DNA <213> Artificial Sequence <400> 10 aattatcgcg cgcggtgtca tctatgttac tagatcctgc agtgtttaca ccacaattaa 60 ttatcaaatt aaaggccctg caaatctcat atgaaattcc ttcgtttcag aatgataacg 120 agacgtacag agagaaatgg aatgtccata gctgtgcgtg tgccatcgtt gactgggccg 180 gccgctcgtg cgtgctcaag tactcaacc 209 <210> 11 <211> 427 <212> DNA <213> Artificial Sequence <400> 11 aattatcgcg cgcggtgtca tctatgttac tagatcctgc agtgtttacg ccgtattcac 60 gtgttatatt ccatggtcca taccataaag gacaatacac tagcgcaaat tgctgggccg 120 ggccatgctc ctgtactaag ctaaagccta cctatgtagc taagacgaaa gtaagcccaa 180 taacttgctt ttttttttct ttttttacgg ggccaaatct gctttgaaaa cgtcaaacct 240 tatctctgca actgatgcga aaagtccctg aaaaacaacg caggcataag gtcagtcaaa 300 ctcaaatctt attcttacac aatcattctg ttttgatctg atcgtttgtg aatgaaaaat 360 catcaattta taatccaaaa aaaaaaaaat ttacatgctg tttaggtccc tttgttttct 420 cccataa 427 <210> 12 <211> 198 <212> DNA <213> Artificial Sequence <400> 12 agttgccgtt cttccgaata gcatcggtaa catgagcaaa gtctgccgcc ttacaacggc 60 tctccaggca cgggagggcg ccgccactgc cctagctgtc cgagggagcc gccgccgcat 120 tccgtctcca cgcgcccaac tgcggatgag aaaggggcga cgggataaga aacaggcgga 180 ggggaacgat gactgcat 198

Claims

1. A primer set for detecting transformation events of the transgenic rice vector GATV2, characterized in that, It includes left-boundary specific primers, right-boundary specific primers, and degenerate primers; The left-bound specific primers include GATV2-LB-R1, GATV2-LB-R2 and GATV2-LB-R3, whose sequences are shown in SEQ ID NO:1~3 respectively; The right boundary specific primers include GATV2-RB-F1, GATV2-RB-F2 and GATV2-RB-F3, whose sequences are shown in SEQ ID NO:4~6 respectively; The degenerate primers include AD2, AD8 and AD10, whose sequences are shown in SEQ ID NO:7~9 respectively.

2. A reagent kit, characterized in that, It includes the primer set as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, It also includes one or more of the following: hot-start Taq DNA polymerase, dNTPs, PCR reaction buffer, positive control, and negative control.

4. The application of the primer set of claim 1 or the kit of claim 2 or 3 in detecting the transformation event of the transgenic rice vector GATV2.

5. The application according to claim 4, characterized in that, The application specifically includes applications in any of the following aspects: (1) Detect whether the GATV2 carrier conversion event exists in the sample to be tested; (2) Identify the flanking sequence specific to the GATV2 transformation event of the vector; (3) Identify the insertion site of the GATV2 transformation event in the vector.

6. A method for detecting transformation events of the transgenic rice vector GATV2, characterized in that, It includes: Using the primer set described in claim 1 or the kit described in claim 2 or 3, PCR amplification was performed using the DNA of the sample to be tested as a template.

7. The method according to claim 6, characterized in that, It includes: Tail-PCR amplification was performed on the left and / or right boundaries of GATV2, specifically including: a) Using the DNA of the sample to be tested as a template, and AD2, AD8, AD10 and the GATV2-LB-R1 / GATV2-RB-F1 described above as primers, the first round of PCR amplification was performed; b) After diluting the product of the first round of PCR amplification, use it as a template and AD2, AD8, AD10 and the GATV2-LB-R2 / GATV2-RB-F2 as primers to perform the second round of PCR amplification; c) After diluting the product from the second round of PCR amplification, use it as a template and AD2, AD8, AD10 and the GATV2-LB-R3 / GATV2-RB-F3 as primers to perform the third round of PCR amplification.

8. The method according to claim 6 or 7, characterized in that, If a specific band can be amplified, it is determined that the GATV2 conversion event exists in the sample to be tested; otherwise, it is determined that the GATV2 conversion event does not exist in the sample to be tested.

9. The method according to claim 6 or 7, characterized in that, The method further includes: The final PCR amplification products were sequenced to determine flanking sequences specific to the GATV2 transformation event; and / or The final PCR amplification product was compared with the sequence of the corresponding transformed plant genome to determine the insertion site of the GATV2 vector transformation event.

10. The method according to claim 8, characterized in that, The method further includes: The final PCR amplification products were sequenced to determine flanking sequences specific to the GATV2 transformation event; and / or The final PCR amplification product was compared with the sequence of the corresponding transformed plant genome to determine the insertion site of the GATV2 vector transformation event.

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