Rice transformation event gatv3-1314-3 and methods of use thereof

Abstract

The present application relates to the technical field of bioengineering, and in particular to a rice transformation event GATV3-1314-3 and an application method thereof. The flanking sequences provided by the present application include a right flanking sequence as shown in SEQ ID NO. 1 and a left flanking sequence as shown in SEQ ID NO. 2. The present application provides specific primers for detecting the flanking sequences, namely SEQ ID NO. 9-10 and SEQ ID NO. 11-12. The specific primers are used for PCR amplification on a DNA sample of a rice to be detected, and can specifically indicate whether a T-DNA fragment of pC0309-KhvMaauMCMK5400 is inserted at the 8826884-8826912 base of the non-coding region of the 3rd chromosome of the rice. The present application successfully realizes detection and safety management on transgenic rice and its derivative lines.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, in particular to a rice transformation event GATV3-1314-3 and an application method thereof. BACKGROUND

[0002] Transgenic technology is a technology of transferring genes with known functional traits such as high yield, stress resistance, disease and pest resistance, and improved nutritional quality into target organisms through modern scientific and technological means, so that the recipient organisms can have new functional characteristics on the basis of the original genetic characteristics, and new varieties and products can be produced.

[0003] Genetically modified crops (GMC) are crops cultivated by using tissue culture technology and gene recombination technology. Genetically modified crops usually have increased yield, improved quality, improved drought resistance, cold resistance, pest resistance and other characteristics, and have better quality.

[0004] In recent years, genetically modified crops such as soybeans, corns, cottons and rapeseeds have been approved for planting and production in many countries. Genetically modified crops can be processed into food, feed or food additives. Due to the current controversy over the ecological safety and edible safety of genetically modified products, strict supervision of genetically modified products is required. Biological safety evaluation of genetically modified crops is an important link in the supervision of genetically modified products, including identification of the specific insertion site of the transformation event in the host genome, judgment of the inactivation or deletion of the host genome according to the insertion site, further speculation of the influence of the transformation event on the host and possible safety problems.

[0005] Since the integration position of the exogenous insertion fragment in the host plant genome is random, the insertion site flanking sequence spliced by the left and right end sequences of each exogenous insertion fragment and the host genome sequence is unique. Therefore, the insertion site flanking sequence is the only unique identifier to distinguish different transformation events, and is an important technical material for establishing a specific detection method for genetically modified plant lines. Specific detection of transformation events has high specificity and can accurately identify different genetically modified crop lines. At present, the flanking sequence of the exogenous insertion fragment is mainly based on PCR technology, and the specific detection methods established include reverse PCR, exogenous adapter-mediated PCR, semi-random primer PCR, whole genome resequencing technology, etc., among which the hot asymmetric staggered PCR (Tail-PCR) in semi-random primer PCR, high-efficiency hot asymmetric PCR (hiTail-PCR) or chromosome walking method (Genome Walking) are the commonly used methods at present.

[0006] Genetic Automation Technology (GAT) is a new type of hybrid seed production technology, which can successfully use recessive cytoplasmic male sterile lines. The core idea of GAT is to use modern biotechnology to construct a GAT vector by tightly linking crop pollen fertility restoration genes, pollen abortion genes, herbicide-sensitive genes, and screening marker genes in a specific order and direction, and then introducing them into a recessive cytoplasmic male sterile line through high-throughput gene transformation technology to obtain a large number of transformation events.

[0007] The present application is based on the obtained rice GAT transformation event GATV3-1314-3 with genetic stability and excellent agronomic traits. The molecular characteristics of the rice GAT transformation event GATV3-1314-3 are determined, and the biological safety evaluation of GATV3-1314-3 is promoted. The present application uses the DNA of the T0 generation plant of GATV3-1314-3 as a template, and uses hiTail-PCR to isolate the T-DNA flanking sequence. According to the T-DNA left and right end sequences and the left and right flanking sequences, detection primers are designed to establish a method for specifically detecting the rice GAT transformation event GATV3-1314-3. The specificity and sensitivity of the method are tested, providing a technical basis for the detection and identification of the rice GAT transformation event GATV3-1314-3 and its derivative products. SUMMARY

[0008] The purpose of the present application is to provide the insertion site and flanking sequence of the rice GAT transformation event GATV3-1314-3, and to provide corresponding detection primers, which can be used for safety assessment and detection of transgenic rice.

[0009] The rice GAT transformation event GATV3-1314-3 has been disclosed in a conference abstract (The 9th International Conference on Botany, Dec. 8-10, 2023, Characteriztation of GAT transformation events offered a promising option to GMS application in rice, Xiongxia Jin). The "1314-3" mentioned in the abstract of the conference paper refers to the present transformation event GATV3-1314-3.

[0010] Specifically, the purpose of the present application is to provide the flanking sequence of the exogenous vector insertion fragment of the rice Genetic Automation Technology (GAT) transformation event GATV3-1314-3, and to provide DNA sequences for specific detection of the flanking sequence, such as PCR amplification primer sequences.

[0011] In a first aspect, the present application provides flanking sequences of a foreign inserted vector of rice GAT transformation event GATV3-1314-3, wherein a right flanking sequence of the flanking sequences is shown as SEQ ID NO. 1; a left flanking sequence of the flanking sequences is shown as SEQ ID NO. 2; and the flanking sequences can be amplified by using a primer pair shown as SEQ ID NO. 11-12 and a primer pair shown as SEQ ID NO. 9-10, respectively.

[0012] Specifically, the right flanking sequence is composed of the first to 435th bases derived from the chromosome 3 of the rice genome and the 436th to 547th bases derived from the GATV3 vector sequence, and the right flanking sequence is the 3' end boundary flanking sequence of the foreign inserted vector of the rice GAT transformation event GATV3-1314-3.

[0013] The left flanking sequence is composed of the first to 180th bases derived from the chromosome 3 of the rice genome and the 181th to 1152th bases derived from the GATV3 vector sequence, and the left flanking sequence is the 5' end boundary flanking sequence of the foreign inserted vector of the rice GAT transformation event GATV3-1314-3.

[0014] The above-mentioned right and left flanking sequences are characteristic sequences of the rice GAT transformation event GATV3-1314-3, and can be used for distinguishing the rice GAT transformation event GATV3-1314-3 from other transgenic / non-transgenic rice, as well as for qualitative detection and quantitative analysis of the rice GAT transformation event GATV3-1314-3.

[0015] In a second aspect, the present application provides primers for detecting the above-mentioned flanking sequences, wherein the nucleotide sequences of the primers are shown as SEQ ID NO. 11-12 and SEQ ID NO. 9-10.

[0016] According to the understanding of those skilled in the art, the present application claims the use of the above-mentioned flanking sequences or the above-mentioned primers in detecting or identifying transgenic rice and its derived products. Specifically, whether a T-DNA fragment of pC0309-KhvMaauMCMK5400 is inserted at the 8826884-8826912th base of the non-coding region of the chromosome 3 of the transgenic rice.

[0017] In a third aspect, the present application provides a PCR detection reagent or kit, wherein the detection reagent or kit comprises the detection primers shown as SEQ ID NO. 11-12 and SEQ ID NO. 9-10.

[0018] The reagent or kit also comprises water, Taq DNA polymerase, dNTPs, PCR buffer, positive control and negative control.

[0019] In a fourth aspect, the present application provides a method for detecting transgenic rice GATV3-1314-3, which detects whether the sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 exist simultaneously in the DNA of a rice sample.

[0020] The primers (shown in SEQ ID NO. 11-12 and shown in SEQ ID NO. 9-10) or the reagent or kit containing the primers are used to perform PCR amplification on the DNA of the sample to be tested as a template.

[0021] In the method provided by the present application, according to the PCR amplification product, it is judged whether the T-DNA fragment of pC0309-KhvMaauMCMK5400 is inserted at the 8826884-8826912 base of the non-coding region of chromosome 3 of the sample to be tested; pC0309-KhvMaauMCMK5400 has been disclosed in Chinese patent with application number 202010379287.9.

[0022] If the primers shown in SEQ ID NO. 11-12 amplify a 547bp target fragment, and the primers shown in SEQ ID NO. 9-10 amplify a 1152bp target fragment, it indicates that the sample contains components derived from rice GAT transformation event GATV3-1314-3.

[0023] In the method provided by the present application, the PCR amplification procedure is: 93-95℃ 1-2.5min; 93-95℃ 20-40s; 50-60℃ 20-40s; 70-73℃ 1-1.5min; 70-73℃ 5-6min; 23-27℃ 1.5-2.5min, 30-35 cycles; preferably: 94℃ 2min; 94℃ 30s; 55℃ 30s; 72℃ 1min; 72℃ 5min; 25℃ 2min, 30-35 cycles.

[0024] The present application has the following beneficial effects:

[0025] (1) The present application first discloses the flanking sequence of the insertion site of the foreign gene of rice genetic intelligent breeding technology (GAT) transformation event GATV3-1314-3 in the rice genome;

[0026] (2) The application firstly confirms the source of different bases in the flanking sequence of the insertion site of the foreign gene in the rice genome of the rice genetic intelligent breeding technology (GAT) transformation event GATV3-1314-3, and determines the junction site sequence of the foreign vector inserted into the rice genome sequence;

[0027] (3) The specific qualitative detection method of the rice genetic intelligent breeding technology (GAT) transformation event GATV3-1314-3 is firstly established by using the flanking sequence found by the application.

[0028] (4) The application is suitable for detecting, monitoring and safety management of the rice genetic intelligent breeding technology (GAT) transformation event GATV3-1314-3 generation and its derived lines. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0030] Figure 1 is the phenotype diagram of spraying 5x imidazopyrazinyl acetanilide of the rice GAT transformation event GATV3-1314-3 in the embodiment 1 of the application; wherein 0d is before spraying, 14d is 14d after spraying, WT is wild type, CK+ is positive control (imidazopyrazinyl acetanilide resistant plant), and 1314-3 is GATV3-1314-3.

[0031] Figure 2 is the phenotype diagram of spraying 3g / L bentaluron of the rice GAT transformation event GATV3-1314-3 in the embodiment 1 of the application; wherein 0d is before spraying, 7d is 7d after spraying, 14d is 14d after spraying, WT is wild type, CK+ is positive control (bentaluron sensitive mutant), and 1314-3 is GATV3-1314-3.

[0032] Figure 3 is the pollen fertility and seed fluorescence diagram of the rice GAT transformation event GATV3-1314-3 in the embodiment 1 of the application; wherein ZH11 is Zhonghua 11, 1314-3 (T0) is T0 generation of GATV3-1314-3, and 1314-3 (T1) is T1 generation of GATV3-1314-3.

[0033] Figure 4is a hiTail-PCR III electropherogram of the right border flanking sequence of rice GAT transformation event GATV3-1314-3 in Example 2 of the present application; wherein, M is Marker; ddH2O is double distilled water; ZH11 is non-transgenic japonica rice Zhonghua 11; 9311 is non-transgenic indica rice 9311; P is GATV3 vector plasmid; 1314-3 is rice GAT transformation event GATV3-1314-3.

[0034] Figure 5 is a schematic diagram of the integration site of rice GAT transformation event GATV3-1314-3 in the rice genome in Example 3 of the present application; wherein, the T-DNA of rice GAT transformation event GATV3-1314-3 is inserted at the 8826884-8826912 base of the non-coding region of chromosome 3 of the rice genome.

[0035] Figure 6 is a specific qualitative PCR amplification diagram of rice GAT transformation event GATV3-1314-3 in Example 5 of the present application; wherein: WT is a non-transgenic rice gene DNA template; 1314-3 is a rice GAT transformation event GATV3-1314-3 genomic DNA template. DETAILED DESCRIPTION

[0036] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0037] If not specifically indicated, the experimental materials, reagents, instruments and the like used in the examples of the present application can be commercially available; if not specifically indicated, all technical means in the examples of the present application are conventional means well known to those skilled in the art.

[0038] Example 1 Obtaining rice GAT transformation event GATV3-1314-3

[0039] In this embodiment, based on the genetic intelligent breeding technology GAT, the pollen fertility restoration gene, pollen abortion gene, herbicide sensitive gene, screening marker gene, etc. are tightly linked in a specific order and direction on the vector pC0309-KhvMaauMCMK5400 (GAT) (referred to as GATV3 vector in this application). The specific sequence of the vector pC0309-KhvMaauMCMK5400 and the vector construction method are referred to the Chinese patent "Genetic Intelligent Breeding System for Crop Hybrid Breeding and Its Application" with application number 202010379287.9, which has been successfully introduced into rice Zhonghua 11 (ZH11, carrying homozygous recessive male sterility gene Oscyp704b2-3 ). GATV3-1314-3 is one of the transformation events, which has been disclosed in the conference abstract (The 9th International Conference on Botany, Dec. 8-10, 2023, Characteriztation of GAT transformation events offered a promising option to GMS application in rice, Xiongxia Jin), in which 1314-3 is the transformation event.

[0040] According to the various elements of the GAT vector, the herbicide phenotype, pollen fertility, and seed fluorescence detection of the transformation event GATV3-1314-3 were carried out, and the results were as follows: (1) According to the positive / negative herbicide screening, the transformation event GATV3-1314-3 showed high resistance when sprayed with 5x imidazolinone solution, indicating that the selection element expression box of the maintainer line was highly efficient, which could be used for GAT maintainer line impurity purification, see Figure 1 ; when sprayed with 3g / L bendazone solution, the phenotype was highly sensitive, indicating that the herbicide sensitive element expression box was highly efficient and sensitive, which could be used for GAT sterile line impurity purification, see Figure 2 .(2) Using potassium iodide staining method, the fertility of the pollen of the transformation event GATV3-1314-3 was detected, and the ratio of aborted pollen to fertile pollen was 1:1, indicating that the work efficiency of the restoration gene element and the pollen abortion gene element was high, which made the maintainer line maintain the heterozygous state, see Figure 3 .(3) According to the pollen fertility identification results, under the 560-595nm excitation light microscope, the seeds of self-pollination would also show a 1:1 segregation, i.e. 50% of the seeds containing the GAT vector showed deep red fluorescence, and 50% of the seeds not containing the GAT vector showed no fluorescence, indicating that the fluorescence protein expression could work normally and could be used for seed mechanical sorting, see Figure 3 . At the same time, T1 and T2 have the above functions, which prove that they can be stably inherited in generations.

[0041] In summary, the single copy of the transformation event GATV3-1314-3 is a good initial maintainer line with normal function of each element, and has genetic stability. The inbred line is sterile, and can be widely used in hybrid rice to improve the breeding efficiency of hybrid rice.

[0042] Example 2 Amplification of the right border flanking sequence of the rice GAT transformation event GATV3-1314-3

[0043] (1) Extraction of rice genomic DNA by TPS method

[0044] ① Grinding sample: take the 3-4 cm long leaves of the transgenic rice GAT transformation event GATV3-1314-3 obtained in Example 1 (1-2 cm 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 with a cell crusher (sample grinder);

[0045] ② After grinding, incubate in a 75 ℃ water bath for 30 min;

[0046] ③ Centrifuge at 13,000 rpm for 10 min, and take the supernatant (about 500 μl) to another 1.5 ml centrifuge tube;

[0047] ④ Add double volume of pre-cooled anhydrous ethanol or equal volume of isopropanol, mix gently, and place in a -30 ℃ refrigerator for 2-3 hours (or in a 4 ℃ refrigerator overnight; or in a -80 ℃ refrigerator for 1-2 h) until the DNA precipitates;

[0048] ⑤ Centrifuge at 13,000 rpm for 5 min, and pour off the supernatant, and place the centrifuge tube upside down on the table to air dry;

[0049] ⑥ After air drying, add 200 μl of 1×TE solution or sterilized ddH2O to dissolve; detect the integrity by 1.0% agarose gel electrophoresis, and measure the DNA concentration by a micro UV spectrophotometer, and store in a 4 ℃ refrigerator.

[0050] (2) Isolation of the T-DNA right flanking sequence by hiTail-PCR

[0051] According to the high efficient thermal asymmetric PCR (hiTail-PCR) method of Liu et al. (2007), three specific primers (GATV3-RB-F1~F3) were designed according to the sequence of the right border (RB) of the GATV3 (pC0309-KhvMaauMCMK5400) plasmid map, combined with the degenerate primers LAD1-1, LAD1-3 and AC1 to isolate the right flanking sequence of T-DNA, and the specific primer sequences are shown in Table 1.

[0052] Table 1 hiTail-PCR primer table

[0053]

[0054] N is (A / T / G / C), A or T or G or C; V is (G / C / A), V is G or C or A.

[0055] The right flanking sequence of the exogenous vector insertion site of the rice GAT transformation event GATV3-1314-3 was amplified by three stages of hiTail-PCR. The first stage of hiTail-PCR used two long random primers (LAD1-1 and LAD1-3 mixed at an equal ratio) combined with the specific primer GATV3-RB-F1 to amplify the rice GAT transformation event GATV3-1314-3 genomic DNA as a template, and ddH2O, non-transgenic rice Zhonghua 11 and 9311 and GATV3 plasmid as controls. The first stage of PCR amplification product was diluted 40 times to be used as a template for the second stage of Tail-PCR, and the primer combination of the second stage was AC1 / GATV3-RB-F2. The second stage reaction product was diluted 10 times to be used as a template for the third stage reaction, and the primer combination was AC1 / GATV3-RB-F3. The amplification products of the second and third stages of reaction were separated by 1.0% agarose gel electrophoresis, and the specific band 436bp was sequenced (as shown in Figure 4 ). The PCR reaction system and procedure are shown in Tables 2 and 3:

[0056] Table 2 hiTail-PCR reaction system

[0057]

[0058] Table 3 hiTail-PCR reaction procedure

[0059]

[0060] Example 3 Integration site of T-DNA of rice GAT transformation event GATV3-1314-3 in the rice genome

[0061] The PCR product of the specific band amplified by hiTail-PCR was sequenced to obtain a 436 bp right border fusion sequence, the sequence of which is shown as SEQ ID NO. 13. By comparing with the rice genome on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and with the sequence of the GATV3 vector, the sequence was analyzed to have the following characteristics: the first to the 54th matched the sequence of the vector right border completely, and the 55th to the 436th matched the sequence of the rice chromosome 3 (AP014959.1 (8826503 to 8826884) completely. That is, the 3' end of the T-DNA of the rice GAT transformation event GATV3-1314-3 was inserted into the 8826884th base of the non-coding region of the rice chromosome 3 (as shown in Figure 5 ).

[0062] Example 4 Amplification of the left border flanking sequence of the rice GAT transformation event GATV3-1314-3

[0063] According to the insertion site information obtained in Example 3, a forward primer LB-F2 was designed based on the partial sequence of the vector GATV3, the sequence of which is shown as SEQ ID NO. 9 (5'-GCAATGAATATGCTGCCATCC-3'), and a reverse primer G3R1 (1314-3) was designed based on the sequence published on the rice chromosome 3, the sequence of which is shown as SEQ ID NO. 10 (5'-GCTAAGCGGCGTATCGTATT-3'), and the left flanking sequence of the rice GAT transformation event GATV3-1314-3 was amplified using the primers. The amplification product was sequenced to obtain a sequence of 1152 bp, as shown in SEQ ID NO. 2. By analyzing SEQ ID NO. 2, it was found that the obtained sequence matched the sequence of the rice chromosome 3 (AP014959.1 (8826912 to 8827091) completely from the first to the 180th, and matched the partial sequence of the vector GATV3 completely from the 181st to the 1152nd. That is, the 5' end of the T-DNA of the rice GAT transformation event GATV3-1314-3 was inserted into the 8826912th base of the rice chromosome 3 (as shown in Figure 5 ). The T-DNA of the rice GAT transformation event GATV3-1314-3 was inserted into the 8826884-8826912th base of the rice chromosome 3, which resulted in a deletion of 27 bp on the rice chromosome 3 (AP014959.1 (8826885 to 8826911), the sequence of which is shown as SEQ ID NO. 14.

[0064] Example 5 Specific PCR detection method for rice GAT transformation event GATV3-1314-3

[0065] The right and left flanking sequences of rice GAT transformation event GATV3-1314-3 were obtained according to Examples 3 and 4, and specific primers were designed on the rice chromosome 3 and the exogenous vector part, respectively. The specific primer sequences are shown in Table 4. The PCR amplification was performed using the genomic DNA of the progeny of rice GAT transformation event GATV3-1314-3 as the template and wild type (WT) as the control. The specific PCR reaction system is shown in Table 5. The amplification program was as follows: 94°C for 2 min; 94°C for 30 s; 55°C for 30 s; 72°C for 1 min; 72°C for 5 min; 25°C for 2 min, 30-35 cycles. The PCR products were detected by 1.0% agarose gel (see Figure 6 ).

[0066]

[0067] Table 5 Reaction system

[0068]

[0069] The results show that:

[0070] (1) G3F1(1314-3) / G3R1(1314-3) combination: the 642 bp target band can be amplified from both the template of rice GAT transformation event GATV3-1314-3 and wild type (WT) (as shown in Figure 6 ), which is consistent with the expectation and indicates that the rice GAT transformation event GATV3-1314-3 is a heterozygous line;

[0071] (2) G3F1(1314-3) / RB-R2 combination: the 547 bp target band of the fusion sequence of the inserted vector and the rice genome at the right border can be amplified from the template of rice GAT transformation event GATV3-1314-3, and the obtained sequence is shown as SEQ ID NO. 1, while the wild type (WT) cannot amplify the target band (as shown in Figure 6 ), which is consistent with the expected result, indicating that the rice GAT transformation event GATV3-1314-3 contains the GAT vector insertion fragment.

[0072] (3) LB-F2 / G3R1(1314-3) combination: the 1152 bp target band of the fusion sequence of the inserted vector and the rice genome at the left border can be amplified from the template of rice GAT transformation event GATV3-1314-3, and the obtained sequence is shown as SEQ ID NO. 2, while the wild type (WT) cannot amplify the target band (as shown in Figure 6), which is in accordance with the expected result, indicating that the GAT vector insertion fragment is contained in the rice GAT transformation event GATV3-1314-3;

[0073] The above results show that the primer combination of LB-F2 / G3R1 (1314-3) and G3F1 (1314-3) / RB-R2 can amplify the left and right border sequences of the rice GAT transformation event GATV3-1314-3 and its breeding lines (derivative lines), and no specific fusion bands can be amplified in non-transgenic or other transgenic rice varieties. The above primers can be used to identify the generations of the rice GAT transformation event GATV3-1314-3 and its derivative sequences.

[0074] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for detecting the transformation event GATV3-1314-3 in transgenic 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 transformation event GATV3-1314-3 involved the insertion of a T-DNA fragment pC0309-KhvMaauMCMK5400 at bases 8826884-8826912 in the non-coding region of chromosome 3.

2. The method according to claim 1, characterized in that, Using primers or reagents or kits containing said primers, PCR amplification is performed using the DNA of the sample to be tested as a template; The nucleotide sequences of the primers are shown in SEQ ID NO.11-12 and SEQ ID NO.9-10.

3. The method according to claim 2, characterized in that, Based on the PCR amplification products, determine whether the T-DNA fragment pC0309-KhvMaauMCMK5400 is inserted at the 8826884-8826912 bases of the non-coding region of chromosome 3 in the sample to be tested.

4. The method according to claim 2 or 3, 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.

5. Application of flanking sequences or primers in detecting or identifying GAT transformation events in rice product GATV3-1314-3; The method involves detecting or identifying whether the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are simultaneously present in the DNA of the rice sample; The rice transformation event GATV3-1314-3 is the insertion of a T-DNA fragment pC0309-KhvMaauMCMK5400 at the 8826884-8826912 bases of the non-coding region of chromosome 3. 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.

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