Finger sequence of rice gat transformation event gatv3-328-1 and detection primer and application thereof

Abstract

This invention relates to a flanking sequence of the rice GAT transformation event GATV3-328-1, its detection primers, and applications. The flanking sequence provided by this invention includes a right flanking sequence as shown in SEQ ID NO.1 and a left flanking sequence as shown in SEQ ID NO.2. This invention provides specific primers SEQ ID NO.9-10 and SEQ ID NO.11-12 for detecting this flanking sequence. PCR amplification of rice DNA samples using these specific primers can specifically indicate whether a T-DNA fragment of pC0309-KhvMaauMCMK5400 has been inserted at the 19881585-19881622 bases of the 6th exon of the zinc finger protein Os09g0511500 on rice chromosome 9. This invention successfully enables the detection and safe management of transgenic rice and its derived lines.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to the safety assessment and detection of transgenic rice, and more specifically, to the flanking sequence of an exogenous inserted fragment in a rice genetic intelligent breeding technology (GAT) transformation event, its specific primers, and their applications. Background Technology

[0002] In recent years, genetically modified (GM) crops such as soybeans, corn, cotton, and rapeseed have been approved for cultivation and production in many countries. These GM crops can be processed into food, feed, or food additives. However, the ecological and food safety of GM products remains highly controversial, necessitating strict regulation. Conducting biosafety assessments of GM crops is a crucial step in the regulation of GM products. This includes identifying the specific insertion site of the transformation event in the host genome, determining whether the insertion is inactivated or deleted based on the insertion site, and further speculating on the impact of the transformation event on the host and potential safety issues.

[0003] Because the integration location of exogenous inserts into the host plant genome is random, the flanking sequences of the insertion site formed by splicing the left and right end sequences of each exogenous insert with the host genome sequence are unique. Therefore, the flanking sequences of the insertion site are unique identifiers that distinguish different transformation events and are important technical data for establishing specific detection methods for transgenic plant lines. Specific detection of transformation events is highly specific and can accurately identify different transgenic crop lines. Currently, the isolation of flanking sequences of exogenous inserts 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.

[0004] Genetic Automation Technology (GAT) is a novel hybrid seed breeding technology that can successfully utilize recessive nuclear male sterile lines. The core idea of ​​GAT is to use modern biotechnology to construct, in a specific order and direction, tightly linked pollen fertility restoration genes, pollen abortion genes, herbicide sensitivity genes, and selection marker genes onto a GAT vector. This vector is then introduced into recessive nuclear male sterile lines using high-throughput gene transformation technology, resulting in a large number of transformation events.

[0005] This invention is based on the genetically stable and agronomically superior rice GAT transformation event GATV3-328-1. It clarifies the molecular characteristics of the rice GAT transformation event GATV3-328-1 and advances the biosafety evaluation of GATV3-328-1. Using DNA from the T0 generation of GATV3-328-1 plants as a template, this invention uses hiTail-PCR to isolate its T-DNA flanking sequences. Based on the left and right end sequences and flanking sequences of the T-DNA, detection primers are designed to establish a method for specifically detecting the rice GAT transformation event GATV3-328-1. The specificity and sensitivity of this method are also tested, providing a technical basis for the detection and identification of the rice GAT transformation event GATV3-328-1 and its derivatives. Summary of the Invention

[0006] The purpose of this invention is to provide the insertion site and flanking sequence of the rice GAT transformation event GATV3-328-1, and to provide the corresponding detection primers.

[0007] The rice GAT transformation event GATV3-328-1 has been published in the conference abstract (The 8th International Conference on Botany, Dec. 4-6, 2021, Progress of genetic automation technology breeding system based on recessive genic male Sterility, Xiongxia Jin). The "328-1" mentioned in the abstract of the conference paper refers to this transformation event GATV3-328-1.

[0008] Specifically, the purpose of this invention is to provide a flanking sequence of the exogenous vector insert fragment of the rice genetic intelligent breeding technology (GAT) transformation event GATV3-328-1, and to provide a DNA sequence, such as a PCR amplification primer sequence, for specific detection of the flanking sequence.

[0009] In a first aspect, the present invention provides flanking sequences of the exogenous insertion vector for the rice GAT transformation event GATV3-328-1, wherein the right flanking sequence is shown in SEQ ID NO.1; and the left flanking sequence is shown in SEQ ID NO.2. The flanking sequences provided by the present invention can be amplified using primer pairs shown in SEQ ID NO.11-12 and SEQ ID NO.9-10, respectively.

[0010] Specifically, the right wing sequence is composed of bases 1 to 277 from chromosome 9 of the rice genome and bases 278 to 391 from the GATV3 vector sequence. The right wing sequence is the 3' end boundary flanking sequence of the exogenous insertion vector of the rice GAT transformation event GATV3-328-1.

[0011] The left wing sequence consists of bases 1 to 292 derived from chromosome 9 of the rice genome and bases 293 to 1275 derived from the GATV3 vector sequence. The left wing sequence is the 5' end boundary flanking sequence of the exogenous insertion vector of the rice GAT transformation event GATV3-328-1.

[0012] The right and left wing sequences mentioned above are characteristic sequences of the rice GAT transformation event GATV3-328-1. They can be used to distinguish the rice GAT transformation event GATV3-328-1 from other transgenic / non-transgenic rice, as well as for the qualitative detection and quantitative analysis of the rice GAT transformation event GATV3-328-1.

[0013] Secondly, the present invention provides primers for detecting the above-mentioned flanking sequences, the nucleotide sequences of which are shown in SEQ ID NO.11-12 and SEQ ID NO.9-10.

[0014] Based on the understanding of those skilled in the art, this invention seeks protection for the use of the aforementioned flanking sequences or primers in the detection or identification of transgenic rice and its derivatives. Specifically, it seeks to determine whether a T-DNA fragment of pC0309-KhvMaauMCMK5400 is inserted at bases 19881585-19881622 of the 6th exon of the zinc finger protein (Os09g0511500) on chromosome 9 of the transgenic rice.

[0015] Thirdly, the present invention provides a PCR detection reagent or kit, the reagent or kit comprising detection primers as shown in SEQ ID NO. 11-12 and SEQ ID NO. 9-10.

[0016] The reagents or kits provided by this invention also include water, Taq DNA polymerase, dNTPs, PCR buffer, positive control and negative control.

[0017] Fourthly, the present invention provides a method for detecting transgenic rice GATV3-328-1, wherein the method detects whether the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are present simultaneously in the DNA of a rice sample.

[0018] Using the primers described above (primers shown in SEQ ID NO. 11-12 and SEQ ID NO. 9-10) or reagents or kits containing the primers described above, PCR amplification was performed using the DNA of the sample to be tested as a template.

[0019] In the method provided by this invention, based on the PCR amplification product, it is determined whether a T-DNA fragment of pC0309-KhvMaauMCMK5400 is inserted at the 19881585-19881622 bases of the 6th exon of the zinc finger protein (Os09g0511500) on chromosome 9 of the sample to be tested; pC0309-KhvMaauMCMK5400 has been disclosed in Chinese Patent Application No. 202010379287.9.

[0020] If the primer pair shown in SEQ ID NO.11-12 amplifies a target fragment of 391 bp, and the primer pair shown in SEQ ID NO.9-10 amplifies a target fragment of 1275 bp, it indicates that the sample contains components derived from the rice GAT transformation event GATV3-328-1.

[0021] In the method provided by this invention, the PCR amplification program is as follows: 93-95℃ for 1-2.5 min; 93-95℃ for 20-40 s; 50-60℃ for 20-40 s; 70-73℃ for 1-1.5 min; 70-73℃ for 5-6 min; 23-27℃ for 1.5-2.5 min, for 30-35 cycles; preferably: 94℃ for 2 min; 94℃ for 30 s; 55℃ for 30 s; 72℃ for 1 min; 72℃ for 5 min; 25℃ for 2 min, for 30-35 cycles.

[0022] The beneficial effects of this invention are:

[0023] (1) This invention discloses for the first time the flanking sequence of the insertion site of the exogenous gene GATV3-328-1 in the rice genome in the transformation event of the rice genetic intelligent breeding technology (GAT);

[0024] (2) This invention is the first to confirm the source of different bases in the flanking sequence of the insertion site of the exogenous gene GATV3-328-1 in the rice genome of the rice genetic intelligent breeding technology (GAT) transformation event, and to determine the conjugation site sequence of the exogenous vector inserted into the rice genome sequence.

[0025] (3) Using the flanking sequence discovered in this invention, a specific qualitative detection method for the transformation event GATV3-328-1 of rice genetic intelligent breeding technology (GAT) was established for the first time;

[0026] (4) This invention is applicable to the detection, monitoring and safety management of the transformation event GATV3-328-1 of rice genetic intelligent breeding technology (GAT) and its derivative lines. Attached Figure Description

[0027] Figure 1 This is a phenotypic diagram of rice GAT transformation event GATV3-328-1 after spraying with 5x imidazole ethionyl acid in Example 1 of the present invention; where 0d is before spraying, 14d is 14d after spraying, WT is wild type, CK+ is positive control (imidazole ethionyl acid resistant plant), and 328-1 is GATV3-328-1.

[0028] Figure 2 This is a phenotypic diagram of rice GAT transformation event GATV3-328-1 after spraying with 3g / L bentazon in Example 1 of the present invention; where 0d is before spraying, 7d is 7 days after spraying, 14d is 14 days after spraying, WT is wild type, CK+ is positive control (bendason-sensitive mutant), and 328-1 is GATV3-328-1.

[0029] Figure 3 This is an example of the pollen fertility and seed fluorescence of rice GAT transformation event GATV3-328-1 in Embodiment 1 of the invention; where ZH11 is the middle flower 11, 328-1(T0) is the T0 generation of GATV3-328-1, and 328-1(T1) is the T1 generation of GATV3-328-1.

[0030] Figure 4 This is a hiTail-PCRIII electrophoresis image of the right flanking sequence of the rice GAT transformation event GATV3-328-1 in Example 2 of the present invention; where M is the marker; ddH2O is double-distilled water; ZH11 is non-transgenic japonica rice Zhonghua 11; 9311 is non-transgenic indica rice 9311; P is the GATV3 vector plasmid; and 328-1 is the rice GAT transformation event GATV3-328-1.

[0031] Figure 5 This is a schematic diagram of the integration site of the rice GAT transformation event GATV3-328-1 in the rice genome in Embodiment 3 of the present invention; wherein, the T-DNA of the rice GAT transformation event GATV3-328-1 is inserted into the 6th exon of the zinc finger protein (Os09g0511500) on chromosome 9 of the rice genome at bases 19881585-19881622.

[0032] Figure 6This is a qualitative PCR amplification diagram of the rice GAT transformation event GATV3-328-1 in Example 5 of the present invention; wherein: WT is the non-transgenic rice gene DNA template; 328-1 is the genomic DNA template of the rice GAT transformation event GATV3-328-1. Detailed Implementation

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of protection of the present invention.

[0034] Unless otherwise specified, all experimental materials, reagents, instruments, etc. used in the examples of this invention are commercially available; unless otherwise specified, all technical means in the examples of this invention are conventional means well known to those skilled in the art.

[0035] Example 1: Obtaining the GAT transformation event GATV3-328-1 in rice

[0036] In this embodiment, based on the genetically intelligent breeding technology GAT, pollen fertility restoration genes, pollen abortion genes, herbicide sensitivity genes, and selection marker genes are constructed in a specific order and direction in a tightly linked manner on the vector pC0309-KhvMaauMCMK5400(GAT) (referred to as the GATV3 vector in this invention). The specific sequence of the vector pC0309-KhvMaauMCMK5400 and the vector construction method are described in accordance with Chinese Patent Application No. 202010379287.9, "A Genetically Intelligent Breeding System for Crops Hybrid Breeding and Seed Production and Its Application," and were successfully introduced into the rice plant Zhonghua 11 (ZH11, carrying the homozygous recessive male sterility gene Oscyp704b2-3). GATV3-328-1 is one of the transformation events, which has been published in the conference abstract (The 8th International Conference on Botany, Dec. 4-6, 2021, Progress of genetic automation technology breeding system based on recessive genic male Sterility, Xiongxia Jin), where 328-1 is this transformation event.

[0037] Based on the various elements of the GAT vector, the herbicide phenotype, pollen fertility, and seed fluorescence of the transformation event GATV3-328-1 were detected. The results were as follows: (1) According to the positive / negative herbicide screening, the transformation event GATV3-328-1 showed a highly resistant phenotype when sprayed with 5x imidazoline solution, indicating that the expression cassette of the maintainer line screening element works efficiently and can be used for the purification of GAT maintainer lines. See Figure 1 When sprayed with a 3 g / L bentazon solution, the phenotype was highly sensitive, indicating that the herbicide-sensitive element expression cassette is highly efficient and sensitive, and can be used for impurity removal and purification of GAT sterile lines. (See [link to relevant documentation]). Figure 2 (2) Using potassium iodide staining, the fertility of pollen from the transformation event GATV3-328-1 was tested. The ratio of aborted pollen to fertile pollen was 1:1, indicating that the working efficiency of the restoration gene element and the pollen abortion gene element was high, which enabled the maintainer line to maintain a heterozygous state. (See...) Figure 3 (3) According to the pollen fertility identification results, under a 560-595nm excitation microscope, the self-pollinated seeds will also show a 1:1 separation, that is, 50% of the seeds contain the GAT vector and show deep red fluorescence; 50% of the seeds do not contain the GAT vector and show no fluorescence. This indicates that the fluorescent protein expression can function normally and can be used for seed mechanical sorting. See Figure 3 Furthermore, the presence of these functions in both T1 and T2 demonstrates that they can be stably inherited across generations.

[0038] In summary, the single-copy transformation event GATV3-328-1 represents an excellent initial maintainer line where all elements function normally, exhibiting genetic stability. Self-pollination of the maintainer line enables the propagation of both sterile and maintainer lines, which can be widely applied to hybrid rice, improving the efficiency of hybrid rice breeding.

[0039] Example 2: Amplification of the right flanking sequence of the GAT transformation event GATV3-328-1 in rice

[0040] (1) Extraction of rice genomic DNA by TPS method

[0041] ① Grinding: Take a 3-4 cm long leaf of the young leaves of the transgenic rice GAT transformation event GATV3-328-1 obtained in Example 1 (take 1-2 cm of old leaves), put them into a 2 ml centrifuge tube, add 800 μl of TPS extraction solution, add steel balls, and grind for 120 seconds using a cell disruptor (grinding machine).

[0042] ②After grinding, place it in a 75℃ water bath for 30 minutes;

[0043] ③ Centrifuge at 13000 rpm for 10 min, and transfer the supernatant (about 500 μl) into another 1.5 ml centrifuge tube;

[0044] ④ Add two volumes of pre-cooled anhydrous ethanol or an equal volume of isopropanol, mix gently, and place in a -30°C freezer for 2-3 hours (or place in a 4°C freezer overnight; or place in a -80°C freezer for 1-2 hours) until DNA is precipitated.

[0045] ⑤ Centrifuge at 13000 rpm for 5 min, discard the supernatant, and invert the centrifuge tube on the table to air dry;

[0046] ⑥ After air drying, dissolve in 200 μl of 1×TE solution or sterile ddH2O; check its integrity by 1.0% agarose gel electrophoresis, and determine the DNA concentration by micro-ultraviolet spectrophotometer. Store at 4℃ for later use.

[0047] (2) Isolation of the right wing sequence of T-DNA using hiTail-PCR

[0048] Following the high-efficiency thermal asymmetric PCR (hiTail-PCR) method of Liu et al. (2007), three specific primers (GATV3-RB-F1~F3) were designed based on the right border (RB) sequence of the GATV3 (pC0309-KhvMaauMCMK5400) plasmid map. These primers were combined with degenerate primers LAD1-1, LAD1-3 and AC1 to separate the right wing sequence of T-DNA. The specific primer sequences are shown in Table 1.

[0049] Table 1. HiTail-PCR Primers

[0050]

[0051] B is (G / C / A), where B is either G, C, or A.

[0052] The right lateral flanking sequence of the exogenous vector insertion site in the rice GAT transformation event GATV3-328-1 was amplified in three stages using hiTail-PCR technology. In the first stage of hiTail-PCR, two long random primers (LAD1-1 and LAD1-3 in equal proportions) were combined with the specific primer GATV3-RB-F1. Genomic DNA from the rice GAT transformation event GATV3-328-1 was used as a template for PCR amplification. ddH2O, non-transgenic rice varieties Zhonghua 11 and 9311, and the GATV3 plasmid were used as controls. The product of the first-stage PCR amplification reaction was diluted 40-fold and used as the template for the second-stage Tail-PCR reaction, with the primer combination AC1 / GATV3-RB-F2. The product of the second-stage reaction was diluted 10-fold and used as the template for the third-stage reaction, with the primer combination AC1 / GATV3-RB-F3. The amplification products from the second and third stage reactions were separated by 1.0% agarose gel electrophoresis, and specific bands were selected for sequencing at 464 bp (e.g., Figure 4 The PCR reaction system and procedure are shown in Tables 2 and 3.

[0053] Table 2 hiTail-PCR reaction system

[0054]

[0055]

[0056] Table 3 hiTail-PCR reaction procedure

[0057]

[0058]

[0059] Example 3: Integration site of T-DNA of GATV3-328-1 in the rice genome during the GAT transformation event in rice.

[0060] Sequencing the specific band PCR product amplified by hiTail-PCR yielded a 464bp right-boundary fusion sequence, as shown in SEQ ID NO. 13. Alignment with the rice genome and the GATV3 vector sequence via the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) revealed the following characteristics: positions 1 to 43 perfectly match the right-boundary sequence of the vector; positions 44 to 464 are located in the rice genome sequence and perfectly match the published sequence on rice chromosome chr9 (AP014965.1 (19881622 to 19882042)). This indicates that the 3' end of the T-DNA from the rice GAT transformation event GATV3-328-1 is inserted at position 19881622 of the 6th exon of the zinc finger protein (Os09g0511500) on chromosome 9 of the rice genome (as shown in SEQ ID NO. 13). Figure 5 ).

[0061] Example 4: Amplification of the left flanking sequence of the GAT transformation event GATV3-328-1 in rice

[0062] Based on the insertion site information obtained in Example 3, a forward primer G9F1(328-1) was designed from the published sequence on rice chromosome 9, with the sequence shown in SEQ ID NO.9 (5′-TAACTGCTGCGTCTGAAACTAGAG-3′), and a reverse primer LB-R2 was designed from the partial sequence of the vector GATV3, with the sequence shown in SEQ ID NO.10 (5′-GCAATGAATATGCTGCCATCC-3′). The left wing sequence of the rice GAT transformation event GATV3-328-1 was amplified using this primer pair. The amplified product was sent for sequencing, and the obtained sequence is shown in SEQ ID NO.2, with a length of 1275 bp. Analysis of SEQ ID NO.2 revealed that positions 1 to 292 of the obtained sequence are located in the rice genome sequence and completely match the published sequence on rice chromosome chr9 (AP014965.1 (19881294 to 19881585)); positions 293 to 1275 are completely identical to a portion of the vector GATV3. This indicates that the 5' end of the T-DNA from the rice GAT transformation event GATV3-328-1 is inserted at position 19881585 of the sixth exon of the zinc finger protein (Os09g0511500) on rice chromosome 9 (e.g., ...). Figure 5 The T-DNA of the rice GAT transformation event GATV3-328-1 is inserted into the sixth exon of the zinc finger protein (Os09g0511500) on chromosome 9 of the rice genome at bases 19881585-19881622, which simultaneously causes a 36bp deletion (AP014965.1(19881586to 19881621)) of the sixth exon of the zinc finger protein (Os09g0511500) on chromosome 9 of the rice chr, as shown in SEQ ID NO.14.

[0063] Example 5: Specific PCR Detection Method for the GAT Transformation Event GATV3-328-1 in Rice

[0064] Based on Examples 3 and 4, the right and left lateral flanking sequences of the rice GAT transformation event GATV3-328-1 were obtained. Specific primers were designed for the rice chr9 chromosome and the exogenous vector, respectively. The specific primer sequences are shown in Table 4. Using the genomic DNA of the progeny of the rice GAT transformation event GATV3-328-1 as a template and wild-type (WT) as a control, PCR amplification was performed. The specific PCR reaction system is shown in Table 5. The amplification program was: 94℃ for 2 min; 94℃ for 30 s; 55℃ for 30 s; 72℃ for 1 min; 72℃ for 5 min; 25℃ for 2 min, for 30-35 cycles. PCR products were detected using a 1.0% agarose gel (see Table 4). Figure 6 ).

[0065] Table 4. Primers for specific detection of GAT transformation event in rice using GATV3-328-1

[0066]

[0067] Table 5 Reaction System

[0068]

[0069]

[0070] The results show that:

[0071] (1) G9F1(328-1) / G9R1(328-1) combination: The rice GAT transformation event GATV3-328-1 template and wild-type (WT) can both amplify a 605bp target band (e.g. Figure 6 This is in line with expectations and indicates that the rice GAT transformation event GATV3-328-1 is a heterozygous line;

[0072] (2) RB-F2 / G9R1(328-1) combination: The GATV3-328-1 template of the rice GAT transformation event can amplify the fusion sequence of the insertion vector and the rice genome at the right boundary, resulting in a 391bp target band, as shown in SEQ ID NO.1. However, the wild-type (WT) failed to amplify the target band (e.g., Figure 6 The results were in line with expectations, indicating that the rice GAT transformation event GATV3-328-1 contained a GAT vector insertion fragment.

[0073] (3) G9F1(328-1) / LB-R2 combination: The GATV3-328-1 template of the rice GAT transformation event can amplify the fusion sequence of the insertion vector and the rice genome at the left boundary, a target band of 1275 bp, as shown in SEQ ID NO.2. However, the wild type (WT) failed to amplify the target band (e.g., Figure 6 The results were in line with expectations, indicating that the rice GAT transformation event GATV3-328-1 contained a GAT vector insertion fragment.

[0074] The results indicate that the primer combinations G9F1(328-1) / LB-R2 and RB-F2 / G9R1(328-1) can amplify the left and right boundary sequences of the rice GAT transformation event GATV3-328-1 and its transgenic lines (derived lines), while no specific fusion bands were amplified in non-transgenic or other transgenic rice varieties. The primers can be used to identify the generation of the rice GAT transformation event GATV3-328-1 and its derived sequences.

[0075] 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> Flanking sequence of rice GAT transformation event GATV3-328-1, its detection primers and applications <130> KHP211124577.0 <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 391 <212> DNA <213> Artificial Sequence <400> 1 aaaccataac ggtacgaaat accggcatca tcaacataag atgacgatta atgacagcaa 60 cctgagttcc tgacactgaa gcctcagtta tttcccattc tttgctgaaa caaacccaaa 120 tatcggccaa ccccggaata tacacgatgg agggaagaca agtattcaca ggaatcgcgg 180 agtaggcaga ccattctgtg cgccataaat ggaaagcagc tctgtggctg atcttccact 240 aaaattatca taagatcgat tatgtacggg agccatctca aacactgata gtttaaactg 300 aaggcgggaa acgacaatct aagcttaaaa aagggtccag gacttggagt tcacctggta 360 aaaaatacgt acacgtgcct agggatctag t 391 <210> 2 <211> 1275 <212> DNA <213> Artificial Sequence <400> 2 taactgctgc gtctgaaact agaggccatg gccatggtat gacaagtcac gctgttcaac 60 aaacaattcc atcatccatg gcaagcaatc cacaacctcc tgcgaccaga agggttcgac 120 caagggcttt gtcaatcaca tctttcattg cagcctcatc gtcagctgag atcagagccc 180 cccatgactt tcctctcact gaaaccgcaa gcaccacaaa cggtaacatc cgtaatggtg 240 ttggtgcccc tagacatgcc aatcaatcat acagctggag ttcagagacc ttcaggatat 300[[ID=*]] attgtggtgt aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa 360 tgtactgcag gataatgaca gcctaggcgg aggtgcggta aagcttgccg aaaacatgca 420 gaagagcaac gacggcaatg aacccaatgc tcatgatgag gactgagttc ggggacatct 480 tgcgcccagc agcctcatcg gtgtagaact ggagcattgt gctggcaccg cctccaccag 540 tgccactgct ggtggttcta cgcctgcgca agcttgcagc agctgctgca ctccctctag 600 ccggggcatc tccattggcc accatcttgc ttatccctc tgcatgataa tatgagtttc 660 aaatgtaagg ttgcagcac taatattaca gaaaaccac agaacacag agtttcatcc 720 aaagtcgtat tgcatataca taggagtgt taaatatgt ctatcatttt ggaagatacg 780 gtttatgctg tcacacagca ttttggaagt gactatttta taagcacaga agttctctca 840 atgtggaata tgtcaaagg caaataga agcacagaag ttcttcaat gtggaatg 900 tcagaaggca gataaggta cacatcttgg aagtgtatga tagtactaca ccaataccag 960 tgaagtttta gttgtcacat tgagtgcta aaaaatat aaaaaagaaa tggttgctgt 1020 tgctcatgcc tatatacatt cataatctat caactact gctcctggat gctgcataac 1080 tataactaaa caagcttaag ttataatttac cacagaaaaaaaaaatga caactagtcc 1140 cagaattctg ctgaaaaatt tgggctgt cctgggctg gccaacacc cattgacatg 1200 atgctgccca agtgtaagaa ctgtaaaaca agtatagtgt ctgtgtatgt acagggatgg 1260 cagcatattc attgc 1275 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 actagatccc taggcacgtg 20 <210> 4 <211> 43 <212> DNA <213> Artificial Sequence <400> 4 acgatggact ccagtccggt taccaggtga actccaagtc ctg 43 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <400> 5 agattgtcgt ttcccgcctt cagtt 25 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <400> 6 acgatggact ccagagcggc cgcbnbnnng gaa 33 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <400> 7 acgatggact ccagagcggc cgcbbnbnnn ccaa 34 <210> 8 <211> 16 <212> DNA <213> Artificial Sequence <400> 8 acgatggact ccagag 16 <210> 9 <211> twenty four <212> DNA <213> Artificial Sequence <400> 9 taactgctgc gtctgaaact agag 24 <210> 10 <211> twenty one <212> DNA <213> Artificial Sequence <400> 10 gcaatgaata tgctgccatc c 21 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <400> 11 actagatccc taggcacg 18 <210> 12 <211> twenty four <212> DNA <213> Artificial Sequence <400> 12 aaaccataac ggtacgaaat accg 24 <210> 13 <211> 464 <212> DNA <213> Artificial Sequence <400> 13 agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt tgagatggct cccgtacata 60 atcgatctta tgataatttt agtggaagat cagccacaga gctgctttcc atttatggcg 120 cacagaatgg tctgcctact ccgcgattcc tgtgaatact tgtcttccct ccatcgtgta 180 tattccgggg ttggccgata tttgggtttg tttcagcaaa gaatgggaaa taactgaggc 240 ttcagtgtca ggaactcagg ttgctgtcat taatcgtcat cttatgttga tgatgccggt 300 atttcgtacc gttatggttt tttatttctg gaaggcaacc tcatgttagt ttcttcaagt 360 gaagatgctt tatgaattgt ctcagttaaa gtgacaatat tgtttggaat tctctatatg 420 tgctagtcct gaatcatact gaggaaaatg ctgctaaaca atgt 464 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <400> 14 ctggccacaa actggggaac ctcactggtg gagtcc 36

Claims

1. The application of primers for detecting flanking sequences in the detection or identification of transgenic rice and its derivatives; wherein the transgenic rice is rice GAT transformation event GATV3-328-1; The rice GAT transformation event GATV3-328-1 involved the insertion of a T-DNA fragment of pC0309-KhvMaauMCMK5400 at the 19881585-19881622 base position of the 6th exon of the zinc finger protein Os09g0511500 on chromosome 9. The right wing sequence of the flanking sequence is shown in SEQ ID NO.1; the left wing sequence of the flanking sequence is shown in SEQ ID NO.2; The nucleotide sequences of the primers are shown in SEQ ID NO.11-12 and SEQ ID NO.9-10.

2. A method for detecting the GAT transformation event GATV3-328-1 in rice, characterized in that, The test was conducted to determine whether the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 were present simultaneously in the DNA of rice samples. The rice GAT transformation event GATV3-328-1 involved the insertion of a T-DNA fragment of pC0309-KhvMaauMCMK5400 at bases 19881585-19881622 of the 6th exon of the zinc finger protein Os09g0511500 on chromosome 9.

3. The method according to claim 2, characterized in that, Using primers with nucleotide sequences as shown in SEQ ID NO.11-12 and SEQ ID NO.9-10, PCR amplification was performed using the DNA of the sample to be tested as a template.

4. The method according to claim 3, characterized in that, If the primer pair shown in SEQ ID NO.11-12 amplifies a target fragment of 391bp, and the primer pair shown in SEQ ID NO.9-10 amplifies a target fragment of 1275bp, it indicates that the sample contains components derived from the T-DNA fragment of pC0309-KhvMaauMCMK5400.

5. The method according to any one of claims 3-4, characterized in that, The PCR amplification program is as follows: 93-95℃ for 1-2.5 min; 93-95℃ for 20-40 s; 50-60℃ for 20-40 s; 70-73℃ for 1-1.5 min; 70-73℃ for 5-6 min; 23-27℃ for 1.5-2.5 min, for 30-35 cycles.

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