Flanking sequences of rice transformation event GATV3-2931-2 and its detection method

Abstract

The present invention relates to the field of bioengineering technology, and in particular to a flanking sequence of the rice transformation event GATV3-2931-2 and a detection method thereof. The right flank sequence of the flanking sequence is shown as SEQ ID NO.1; the left flank sequence of the flanking sequence is shown as SEQ ID NO.2. The present invention provides a set of specific primers for the flanking sequence of the rice transformation event GATV3-2931-2, including nucleotide sequences shown as SEQ ID NO.11-12 and SEQ ID NO.9-10, which can be used to detect whether a biological material contains components derived from the rice transformation event GATV3-2931-2. The specific primer combination provided by the present invention can successfully detect rice transformation events and is of great significance in the biosafety detection and evaluation system of transgenic rice and its derivative lines.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, in particular to a flanking sequence of a rice transformation event GATV3-2931-2 and a detection method thereof. Background Art

[0002] To date, genetically modified crops (such as soybeans, corn, cotton, and rapeseed) have been approved for cultivation and production in some regions and can be processed into food, feed, or food additives. This raises concerns about the ecological and food safety of genetically modified products, necessitating strict regulation. Biosafety assessment of genetically modified crops is a crucial component of genetically modified product regulation. This involves identifying the specific insertion site of the transformation event within the host genome, determining whether the host genome has been inactivated or deleted based on the insertion site, and further estimating the impact of the transformation event on the host and potential safety issues.

[0003] Since the integration position of exogenous inserts in the host plant genome is random, the insertion site flanking sequences, formed by splicing the left and right end sequences of each exogenous insert with the host genome sequence, are unique. Therefore, the insertion site flanking sequences serve as unique identifiers for distinguishing 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 for exogenous inserts is mainly based on PCR technology. Established specific detection methods include inverse PCR, exogenous linker-mediated PCR, semi-random primer PCR, and whole genome resequencing technology. Among these, thermal asymmetric staggered PCR (Tail-PCR), high-efficiency thermal asymmetric PCR (hiTail-PCR), and chromosome walking (GenomeWalking) in semi-random primer PCR are currently the most commonly used methods.

[0004] Genetic Automation Technology (GAT) is a novel hybrid seed breeding technology that can successfully utilize recessive nuclear male sterile lines. The core concept of GAT is to utilize modern biotechnology to tightly link crop pollen fertility restoration genes, pollen abortion genes, herbicide sensitivity genes, and selection marker genes in a specific sequence and orientation on a GAT vector. These genes are then introduced into recessive nuclear male sterile lines through high-throughput gene transformation technology, resulting in a large number of transformation events. Summary of the Invention

[0005] To address the challenges of the prior art, the present invention provides flanking sequences of the rice transformation event GATV3-2931-2 and methods for detecting the same. Specifically, based on the genetically stable and agronomically excellent rice transformation event GATV3-2931-2, the present invention clarifies the molecular characteristics of the rice transformation event GATV3-2931-2 and facilitates biosafety assessment of GATV3-2931-2.

[0006] This study uses DNA from T0 generation GATV3-2931-2 plants as a template, isolates its T-DNA flanking sequences using hiTail-PCR, and designs detection primers based on the left and right end sequences and flanking sequences of the T-DNA. This method establishes a specific detection method for the rice transformation event GATV3-2931-2, and simultaneously verifies the specificity and sensitivity of this method. This provides a technical basis for the detection and identification of the rice transformation event GATV3-2931-2 and its derivatives. This method clarifies the molecular characteristics of the rice transformation event GATV3-2931-2 and advances the biosafety assessment of GATV3-2931-2.

[0007] The rice transformation event GATV3-2931-2 described in the present invention has been disclosed in a conference abstract (The 9th International Conference on Botany, Dec. 8-10, 2023, Characterization of GAT transformation events offered a promising option to GMS application in rice, Xiongxia Jin). The "2931-2" mentioned in the abstract of the conference paper refers to the present transformation event GATV3-2931-2.

[0008] In a first aspect, the present invention provides a flanking sequence of an exogenous insertion vector for a transgenic rice transformation event, wherein the right flanking sequence of the flanking sequence is shown as SEQ ID NO.1; the left flanking sequence of the flanking sequence is shown as SEQ ID NO.2.

[0009] Furthermore, the flanking sequences were amplified using primer pairs as shown in SEQ ID NOs. 11-12 and SEQ ID NOs. 9-10, respectively.

[0010] In a second aspect, the present invention provides a primer comprising the nucleotide sequence shown in SEQ ID NO.11-12 and / or SEQ ID NO.9-10;

[0011] The primers are used to detect the flanking sequences.

[0012] Specifically, the right flank sequence has a total of 411 bases, of which bases 1 to 313 are derived from chromosome 7 of the rice genome, and bases 314 to 411 are derived from the GATV3 vector sequence. The right flank sequence is the 3'-end border flanking sequence of the exogenous insertion vector of the rice transformation event GATV3-2931-2;

[0013] The left flank sequence consists of 574 bases, of which bases 1 to 289 are derived from chromosome 7 of the rice genome, and bases 290 to 574 are derived from the GATV3 vector sequence. The left flank sequence is the 5'-end border flanking sequence of the exogenous insertion vector of the rice transformation event GATV3-2931-2.

[0014] The above-mentioned right-wing and left-wing sequences are characteristic sequences of the rice transformation event GATV3-2931-2, which can be used to distinguish the rice transformation event GATV3-2931-2 from other transgenic / non-transgenic rice, as well as for qualitative detection and quantitative analysis of the rice transformation event GATV3-2931-2.

[0015] The present invention further provides a detection reagent or a kit, which comprises the primers.

[0016] Furthermore, it also includes water, Taq DNA polymerase, dNTPs, PCR buffer, positive control substances and negative control substances.

[0017] In a third aspect, the present invention provides the use of the flanking sequence, or the primer, or the detection reagent or kit in any of the following:

[0018] i) Detection of the insertion site of rice transformation event GATV3-2931-2;

[0019] ii) Application in the detection or identification of genetically modified rice and its derived products;

[0020] The transgenic rice is the rice transformation event GATV3-2931-2.

[0021] In a fourth aspect, the present invention provides a method for detecting transgenic rice GATV3-2931-2, comprising:

[0022] Detect whether the rice sample to be tested contains the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 at the same time.

[0023] Further, including:

[0024] Obtaining genomic DNA of the rice sample to be tested; performing PCR amplification using the primers; if the primer pair shown in SEQ ID NOs. 11-12 amplifies a specific band of 411 bp, it indicates that the rice sample to be tested includes the nucleotide sequence shown in SEQ ID NO. 1; if the primer pair shown in SEQ ID NOs. 9-10 amplifies a specific band of 574 bp, it indicates that the rice sample to be tested includes the nucleotide sequence shown in SEQ ID NO. 2.

[0025] Furthermore, if the rice sample to be tested contains the nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 at the same time, the rice sample to be tested contains components derived from the rice transgenic event GATV3-2931-2.

[0026] Furthermore, the PCR amplification procedure includes:

[0027] 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.

[0028] Preferably, the PCR amplification program includes: 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, for 30-35 cycles.

[0029] Furthermore, the T-DNA fragment of pC0309-KhvMaauMCMK5400 was inserted into the 5' end of the MYB transcription factor Os07g0438800 on chromosome 7 of the rice transgenic event GATV3-2931-2 at bases 14753623-14753795. pC0309-KhvMaauMCMK5400 has been disclosed in Chinese Patent Application No. 202010379287.9.

[0030] The present invention has the following beneficial effects:

[0031] This invention discloses for the first time the flanking sequences of the insertion site of the exogenous gene of the rice transformation event GATV3-2931-2 in the rice genome. It also identifies the sources of the different bases in the flanking sequences and the sequence of the junction site where the exogenous vector inserts into the rice genome. Based on these flanking sequences, the invention designs primers and establishes a specific detection method for the rice transformation event GATV3-2931-2, which can accurately detect the rice transformation event GATV3-2931-2. The detection method provided by the invention is suitable for the detection, monitoring, and safety management of the GATV3-2931-2 generation of the rice Genetic Intelligent Breeding Technology (GAT) transformation event and its derivatives, and is of great significance in the biosafety detection and evaluation system for this rice transformation event. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 This is the phenotypic diagram of the rice transformation event GATV3-2931-2 sprayed with 5x imazethapyr provided in Example 1 of the present invention; wherein 0d is before spraying, 14d is 14 days after spraying, WT is the wild type, CK+ is the positive control (imazethapyr-resistant plant), and 2931-2 is GATV3-2931-2.

[0034] Figure 2 This is a phenotypic diagram of the rice transformation event GATV3-2931-2 provided in Example 1 of the present invention sprayed with 3 g / L bentazon; wherein 0d is before spraying, 7d is 7 days after spraying, 14d is 14 days after spraying, WT is the wild type, CK+ is the positive control (benzyl-sensitive mutant), and 2931-2 is GATV3-2931-2.

[0035] Figure 3 This is the pollen fertility and seed fluorescence image of the rice transformation event GATV3-2931-2 provided in Example 1 of the invention; wherein ZH11 is Zhonghua 11, 2931-2 (T0) is the T0 generation of GATV3-2931-2, and 2931-2 (T1) is the T1 generation of GATV3-2931-2.

[0036] Figure 4This is a hiTail-PCRIII electrophoresis diagram of the right border flanking sequence of the rice transformation event GATV3-2931-2 provided in Example 2 of the present invention; wherein, M is a marker; ddH2O is double-distilled water; ZH11 is the non-transgenic japonica rice Zhonghua 11; 9311 is the non-transgenic indica rice 9311; P is the GATV3 vector plasmid; and 2931-2 is the rice transformation event GATV3-2931-2.

[0037] Figure 5 Schematic diagram of the integration site of the rice transformation event GATV3-2931-2 in the rice genome provided in Example 2 of the present invention; wherein, the T-DNA of the rice transformation event GATV3-2931-2 is inserted into the 5' end of the MYB transcription factor (Os07g0438800) on chromosome 7 of the rice genome at bases 14753623-14753795.

[0038] Figure 6 This is a specific qualitative PCR amplification diagram of the rice transformation event GATV3-2931-2 in Example 3 of the present invention; wherein: WT is a non-transgenic rice gene DNA template; 2931-2 is a genomic DNA template of the rice transformation event GATV3-2931-2. DETAILED DESCRIPTION

[0039] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods involved in the following examples are all conventional methods in the art. For example, reference can be made to experimental manuals in the art, or the conditions recommended by the manufacturer's instructions.

[0041] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0042] Example 1

[0043] This example provides a rice transformation event GATV3-2931-2 and its phenotypic verification, which specifically includes the following steps:

[0044] In this example, based on the genetically intelligent breeding technology GAT, pollen fertility restorer genes, pollen abortion genes, herbicide-sensitive genes, and selection marker genes were tightly linked in a specific order and orientation on the vector pC0309-KhvMaauMCMK5400 (GAT) (referred to as the GATV3 vector in this invention). The specific sequence and construction method of the vector pC0309-KhvMaauMCMK5400 were described in reference to Chinese patent application number 202010379287.9, "A genetically intelligent seed breeding system for hybrid crop breeding and its application," and the homozygous recessive male sterility gene was successfully introduced into the rice plant Zhonghua 11 (ZH11). Oscyp704b2-3 GATV3-2931-2 is one of the transformation events, which has been disclosed in the conference abstract (The 9th International Conference on Botany, Dec. 8-10, 2023, Characterization of GAT transformation events offered a promising option to GMS application in rice, Xiongxia Jin), of which 2931-2 is the current transformation event.

[0045] The present invention performs herbicide phenotype, pollen fertility, and seed fluorescence tests on the transformation event GATV3-2931-2 based on the various elements of the GAT vector. The results are as follows:

[0046] (1) According to the positive / negative herbicide screening, the transformation event GATV3-2931-2 showed a high resistance phenotype when sprayed with 5x imidazolin solution, indicating that the expression cassette of the maintainer screening element works efficiently and can be used for the purification of GAT maintainer lines, such as Figure 1 When 3 g / L bentazon solution was sprayed, the phenotype was highly sensitive, indicating that the herbicide sensitive element expression cassette was highly efficient and sensitive and could be used for impurity removal and purification of GAT sterile lines. Figure 2 shown.

[0047] (2) Using potassium iodide staining, the fertility of the pollen of the transformation event GATV3-2931-2 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, so that the maintenance line remained in a heterozygous state. Figure 3 shown.

[0048] (3) According to the results of pollen fertility identification, the self-fertilized seeds will also show a 1:1 separation when observed under a 560-595nm excitation light microscope, 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 have no fluorescence, indicating that the fluorescent protein expression can work normally and can be used for mechanical seed sorting, such as Figure 3 The above functions are present in both T1 and T2, proving that they can be stably inherited across generations.

[0049] In summary, the single-copy transformation event GATV3-2931-2, in which all elements function normally, creates an excellent initial maintainer line with genetic stability. Self-pollination of this maintainer line allows for the propagation of sterile and maintainer lines, which can be widely applied to hybrid rice, improving the efficiency of hybrid rice breeding.

[0050] Example 2

[0051] This example provides a method for isolating the flanking sequences of the rice transformation event GATV3-2931-2, which specifically comprises the following steps:

[0052] 1. Amplification of the right border flanking sequence of rice transformation event GATV3-2931-2

[0053] (1) Extraction of rice genomic DNA using TPS method

[0054] i) Grinding: Take a 3-4 cm long young leaf of the transgenic rice transformation event GATV3-2931-2 obtained in Example 1 (take a 1-2 cm long leaf of the old leaf), place it in a 2 ml centrifuge tube, add 800 μl of TPS extract, add steel balls, and grind for 120 seconds using a cell disruptor (grinding machine);

[0055] ii) After grinding, incubate in a 75°C water bath for 30 minutes;

[0056] iii) Centrifuge at 13,000 rpm for 10 min and transfer the supernatant (approximately 500 µl) to another 1.5 ml centrifuge tube;

[0057] iv) Add two volumes of pre-chilled anhydrous ethanol or an equal volume of isopropanol, mix gently, and place in a -30°C refrigerator for 2-3 hours (or in a 4°C refrigerator overnight; or in a -80°C refrigerator for 1-2 hours) until the DNA precipitates;

[0058] v) Centrifuge at 13,000 rpm for 5 minutes, discard the supernatant, and place the tube upside down on the bench to air dry.

[0059] vi) After air-drying, dissolve the aliquot in 200 µl of 1× TE solution or sterile ddH2O. Check its integrity by electrophoresis on a 1.0% agarose gel and determine the DNA concentration using a micro-UV spectrophotometer. Refrigerate at 4°C until ready for use.

[0060] (2) Isolation of T-DNA right flank sequence using hiTail-PCR

[0061] Referring to the high-efficiency thermal asymmetric PCR (hiTail-PCR) method of Liu et al. (2007), three specific primers (GATV3-RB-F1 to F3) were designed based on the right border (RB) sequence of the GATV3 (pC0309-KhvMaauMCMK5400) plasmid map. These primers were combined with the degenerate primers LAD1-1, LAD1-3, and AC1 to isolate the right flank sequence of the T-DNA. The specific primer sequences are shown in Table 1.

[0062]

[0063] The present invention utilizes hiTail-PCR technology to amplify the right flank sequence of the exogenous vector insertion site of the rice transformation event GATV3-2931-2 in three stages. The hiTail-PCR first-stage reaction uses two long random primers (LAD1-1 and LAD1-3 mixed in equal proportions) in combination with the specific primer GATV3-RB-F1, and uses the genomic DNA of the rice transformation event GATV3-2931-2 as a template for PCR amplification. ddH2O, non-transgenic rice Zhonghua 11 and 9311, and GATV3 plasmid are used as controls. The product of the first-stage PCR amplification reaction is diluted 40 times and used as a template for the second-stage Tail-PCR reaction. The primer combination for the second-stage reaction is AC1 / GATV3-RB-F2. The product of the second-stage reaction is diluted 10 times and used as a template for the third-stage reaction. The primer combination is AC1 / GATV3-RB-F3. The amplified products of the second and third-stage reactions are separated by 1.0% agarose gel electrophoresis, and the specific band 253bp is selected for sequencing (such as Figure 4 The PCR reaction system and procedure are shown in Tables 2 and 3:

[0064]

[0065]

[0066] 2. Integration site of T-DNA of rice transformation event GATV3-2931-2 in the rice genome

[0067] The present invention sequenced the specific band PCR product amplified by hiTail-PCR to obtain a 253bp right border fusion sequence, as shown in SEQ ID NO. 13. After alignment with the rice genome and the GATV3 vector sequence on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), the sequence was analyzed to be characterized as follows: positions 1 to 47 completely matched the vector right border sequence, and positions 48 to 253 were located in the rice genome sequence, completely matching the published sequence on rice chromosome chr7 (AP014963.1 (14753795 to 14754000). In other words, the 3' end of the T-DNA of the rice transformation event GATV3-2931-2 was inserted into the 5' end of the MYB transcription factor (Os07g0438800) on chromosome 7 of the rice genome at base 14753795 (as shown in FIG. Figure 5 shown).

[0068] 3. Amplification of the left border flanking sequence of rice transformation event GATV3-2931-2

[0069] According to the present invention, the insertion site information is obtained according to step 2, and a forward primer G7F1 (2931-2) is designed based on the published sequence on rice chromosome 7, the sequence of which is shown in SEQ ID NO. 9 (5'-CCGTATCCGAAAGGCATCAC-3'). A reverse primer LB-R2 is designed based on a partial sequence of the vector GATV3, the sequence of which is shown in SEQ ID NO. 10 (5'-GCAATGAATATGCTGCCATCC-3'). This pair of primers is used to amplify the left flank sequence of the rice transformation event GATV3-2931-2. The amplified product is sent for sequencing, and the obtained sequence is shown in SEQ ID NO. 2, which is 574 bp in length. Analysis of SEQ ID NO. 2 revealed that the sequence from positions 1 to 289 was located in the rice genome and completely matched the published sequence on rice chromosome chr7 (AP014963.1 (14753335 to 14753623)); positions 290 to 574 were identical to a partial sequence of the vector GATV3. This means that the 5' end of the T-DNA of the rice transformation event GATV3-2931-2 was inserted into the 5' end of the MYB transcription factor (Os07g0438800) on chromosome 7 of the rice genome at base 14753623 (e.g., Figure 5The T-DNA of rice transformation event GATV3-2931-2 was inserted into the 5' end of the MYB transcription factor (Os07g0438800) on rice chromosome 7 at bases 14753623-14753795, resulting in a 171 bp deletion (AP014963.1 (14753624 to 14753794) at the 5' end of the MYB transcription factor (Os07g0438800) on rice chromosome chr7, as shown in SEQ ID NO.14.

[0070] Example 3

[0071] This example provides a specific PCR detection method for rice transformation event GATV3-2931-2, which specifically includes the following process:

[0072] According to Example 2, the present invention obtained the right and left flanking sequences of the rice transformation event GATV3-2931-2. Specific primers were designed on rice chromosome chr7 and the exogenous vector, respectively. The specific primer sequences are shown in Table 4. PCR amplification was performed using genomic DNA from the progeny of the rice transformation event GATV3-2931-2 as a template and the wild type (WT) as a control. The specific PCR reaction system is shown in Table 5. The amplification program was: 94°C for 2 minutes; 94°C for 30 seconds; 55°C for 30 seconds; 72°C for 1 minute; 72°C for 5 minutes; 25°C for 2 minutes, for 30-35 cycles. PCR products were detected using 1.0% agarose gel (see Table 5). Figure 6 shown).

[0073]

[0074]

[0075] The results are as follows Figure 6 As shown:

[0076] (1) G7F1(2931-2) / G7R1(2931-2) combination: The rice transformation event GATV3-2931-2 template and the wild type (WT) were able to amplify the 773bp target band, which was in line with expectations and indicated that the rice transformation event GATV3-2931-2 was a heterozygous strain;

[0077] (2) RB-F2 / G7R1 (2931-2) combination: The rice transformation event GATV3-2931-2 template was able to amplify the 411bp target band of the fusion sequence of the insertion vector and the rice genome at the right border. The obtained sequence was shown in SEQ ID NO.1, while the wild type (WT) failed to amplify the target band, which was consistent with the expected result, indicating that the rice transformation event GATV3-2931-2 contained the GAT vector insert.

[0078] (3) G7F1(2931-2) / LB-R2 combination: The rice transformation event GATV3-2931-2 template was able to amplify the 574bp target band of the fusion sequence of the insertion vector and the rice genome at the left border. The obtained sequence is shown in SEQ ID NO. 2, while the wild type (WT) failed to amplify the target band, which was consistent with the expected result, indicating that the rice transformation event GATV3-2931-2 contained the GAT vector insert;

[0079] The above results indicate that the primer combinations G7F1(2931-2) / LB-R2 and RB-F2 / G7R1(2931-2) can amplify the left and right boundary sequences of the rice transformation event GATV3-2931-2 and its transformed lines (derivative lines), while no specific fusion bands can be amplified in non-transgenic or other transgenic rice varieties. These primers can be used to identify the rice transformation event GATV3-2931-2 generation and its derivative sequences.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of flanking sequences, primers, detection reagents or kits in any of the following: i) Detection of the insertion site of rice transformation event GATV3-2931-2; ii) Application in the detection or identification of genetically modified rice and its derived products; The transgenic rice is the rice transformation event GATV3-2931-2; the rice transformation event GATV3-2931-2 is a T-DNA fragment of pC0309-KhvMaauMCMK5400 inserted into the 14753623-14753795 bases of the 5' end of the MYB transcription factor Os07g0438800 on chromosome 7; The right wing sequence of the flanking sequence is shown as SEQ ID NO.1; the left wing sequence of the flanking sequence is shown as SEQ ID NO.2; The primers are used to amplify the flanking sequences; the primers include nucleotide sequences as shown in SEQ ID NO.9-12; The detection reagent or kit includes the primer.

2. The use according to claim 1, characterized in that The reagents or kit further include: water, Taq DNA polymerase, dNTPs, PCR buffer, positive control substances and negative control substances.

3. A method for detecting transgenic rice GATV3-2931-2, characterized in that: include: Detecting whether the rice sample to be tested contains the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 at the same time; The transgenic rice GATV3-2931-2 is a T-DNA fragment of pC0309-KhvMaauMCMK5400 inserted into the 14753623-14753795 bases of the 5' end of the MYB transcription factor Os07g0438800 on chromosome 7.

4. The method according to claim 3, characterized in that include: Obtaining genomic DNA of the rice sample to be tested; PCR amplification is performed using primers; the primers include nucleotide sequences as shown in SEQ ID NOs. 9-12; If the primer pair shown in SEQ ID NO.11-12 amplifies a specific band of 411 bp, it indicates that the rice sample to be tested includes the nucleotide sequence shown in SEQ ID NO.1; if the primer pair shown in SEQ ID NO.9-10 amplifies a specific band of 574 bp, it indicates that the rice sample to be tested includes the nucleotide sequence shown in SEQ ID NO.

2.

5. The method according to claim 4, characterized in that If the rice sample to be tested contains the nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 at the same time, the rice sample to be tested contains components derived from the rice transgenic event GATV3-2931-2.

6. The method according to claim 4 or 5, characterized in that The PCR amplification procedure includes: 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.

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