OsDRM2 gene and application thereof in resisting rice stripe virus disease

Abstract

The invention relates to the technical field of plant disease prevention and control and transgenosis, and mainly relates to a rice OsDRM2 gene and application thereof in resisting rice stripe virus diseases.

Description

Technical Field

[0001] This invention relates to the fields of plant disease control and transgenic technology, and mainly to the rice OsDRM2 gene and its application in resistance to rice stripe virus disease. Background Technology

[0002] Rice stripe virus (RSV) is an important rice viral pathogen in East Asia, belonging to the genus Tenuivirus. This virus is primarily transmitted persistently and oviposited by the rice planthopper (Laodelphax striatellus), and can persist in insect-borne populations for extended periods, posing significant challenges to control. Infected rice plants exhibit yellowing, striping, and necrosis of leaves, as well as stunted growth. In severe cases, it can lead to heading delays or crop failure, posing a major threat to rice yield and food security.

[0003] The RSV genome consists of four negative-sense single-stranded RNA segments (RNA1-RNA4), encoding seven viral proteins that play crucial roles in viral replication, intercellular movement, vector transmission, and host interaction. With the development of molecular biology techniques, the molecular mechanisms of virus-host interaction have been gradually revealed, providing a foundation for antiviral genetic modification.

[0004] Currently, breeding resistant rice varieties remains the main strategy for controlling viral diseases. Discovering and utilizing key genes with broad-spectrum antiviral properties is crucial for elucidating the interaction mechanisms between rice and viruses, improving varietal resistance, and achieving sustainable control. However, existing resistance resources are limited, and the resistance mechanisms are not yet fully understood, necessitating the development of new antiviral genetic resources and molecular breeding techniques.

[0005] To resist various biotic stresses, plants have evolved sophisticated defense mechanisms centered on epigenetic modifications. Among these, DNA methylation, an important and highly conserved epigenetic regulatory mechanism in eukaryotes, plays a crucial role in plant disease resistance. This process is catalyzed by DNA methyltransferases, which use S-adenosylmethionine as a methyl donor to add a methyl group to the fifth carbon atom of cytosine, particularly enriching it in transposons and repetitive sequence regions. This allows for the regulation of gene expression, maintenance of genome stability, and coordination of the plant's adaptive response to pathogen invasion without altering the DNA sequence.

[0006] In plant responses to viral infection, domain rearranged methyltransferase 2 (DRM2) plays a crucial de novo methyltransferase role as a key enzyme in the RNA-mediated DNA methylation (RdDM) pathway. After the RdDM pathway is initiated, DNA-dependent RNA polymerase IV (Pol IV) and RNA-dependent RNA polymerase 2 (RDR2) sequentially synthesize double-stranded RNA, which is then processed by Dicer-like protein (DCL3) to generate siRNA. This siRNA is then loaded by Argonaute 4 protein (AGO4) to form an effector complex, ultimately guiding DRM2 to homologous DNA regions for precise methylation modification. Studies have shown that OsDRM2 in plants can interact with the P2 protein encoded by rice stripe virus, inhibiting the expression of certain disease resistance-related genes through methylation, thereby promoting rice susceptibility. However, the specific pathway remains unclear, and no further research has been conducted. Summary of the Invention

[0007] Based on the deficiencies of the existing technology, the technical problem to be solved by the present invention is how to more effectively prevent and control rice diseases caused by RSV virus. In order to solve the above technical problem, the present invention relates to a rice OsDRM2 gene and its application in resistance to rice virus diseases.

[0008] In a first aspect, the present invention relates to a rice transcription factor OsDRM2, the nucleotides of which are shown in SEQ ID NO:1:

[0009] ATGCAGGCAATAGGCGGTGACGCTTCAGTGGGTAACTGCTCTGCTTCGGCCTGTGCCCCCCAGACTCTTGAAGTCGACGAGGAGGAGGATGATACTAATTGGGATGAATATGATACTGCTGGCAATTGTGACAGGACTCCTCACTCTGATGGTTCTGGTGACGAGGATTTCTTTCAAGAAATGTCAGAGAAGGATGAAAAAATGAAGTCCTTAGTCAACATGGGTTTTCCTGAAGATGAAGCAAAGATGGCTATCGATAGATGTCTCGATGCGCCTGTAGCAGTGTTGGTTGATTCAATCTATGCATCACAGGAAGCAGGAAATGGTTACTCTGCAAACTTATCTGACTATGAGGATACAGAGTTCAGTTCCTTTGGAGGAAGAAAGAAAACAAGATTTGTGGATGGAAGCAAGAAGAGAAAGCGGTATGGAAGTGGGCCATCTGGGAATCAAGTGCCGTTTGATGGCAGCCACGAAGAGCCCATGCCTCTTCCAAATCCAATGGTGGGATTCAGCTTGCCCAATGAGAGGTTAAGGTCAGTCCACAGAAATCTTCCTGACCAAGCTCTTGGGCCACCATTCTTTTACTATGAAAACGTGGCCCTAGCTCCAAAAGGTGTATGGACAACCATCTCAAGGTTTTTATATGACATTCAGCCTGAGTTTGTGGACTCCAAGTACTTTTGTGCTGCTGCCAGGAAGAGGGGTTATATTCATAACCTGCCGATTGAGAACAGGTCACCTGTTCTCCCAATGCCTCCCAAAACAATATCTGAAGCCTTTCCTAATACCAAGAGGTGGTGGCCTTCCTGGGATCCAAGGAGGCAGTTCAACTGCTTGCAAACTTGTATGGCAAGCGCAAAGCTTACAGAGCGAATTCGTTGTGCTTTGGGTAGATTCAGTGATGTACCAACTCCACAAGTTCAGAAGTATGTCTTGGACGAATGTAGGAAATGGAACCTAGTTTGGGTCGGAAAGAATAAGGTCGCTCCTCTAGAGCCTGATGAGATGGAGTTCCTGTTAGGGTACCCTAGAAACCACACTAGGGGAGTTAGCAGGACGGAGAGGTACAGGGCTCTCGGGAATTCATTCCAAGTTGATACAGTAGCATACCATCTCTCTGTGCTGAGGGACCTGTTTCCCAATGGAATGAATGTCTTATCCCTGTTTTCTGGTATTGGAGGAGCAGAGGTAGCTCTCCACAGACTGGGGATACATATGAAAACAGTGATCTCTGTTGAGAAATCAGAAGTGAACAGAACGATTCTGAAGAGCTGGTGGGATCAGACACAAACTGGAACGCTGATTGAAATCGCCGACGTGCGGCATCTTACTACTGAAAGAATTGAAACATTCATCAGAAGGTTTGGCGGTTTTGACCTGGTGATTGGTGGCAGCCCGTGCAACAACCTTGCTGGCAGCAACCGCCATCACCGAGATGGTCTGGAGGGCGAGCACTCCGCGCTCTTCTACGATTACATTAGAATATTAGAACATGTAAAGGCTACCATGTCAGCAGTCTAG

[0010] In some embodiments, the amino acid sequence of the OsDRM2 gene is as shown in SEQ ID NO: 2:

[0011] MQAIGGDASVGNCSASACAPQTLEVDEEEDDTNWDEYDTAGNCDRTPHSDGSGDEDFFQEMSEKDEKMKSLVNMGFPEDEAKMAIDRCLDAPVAVLVDSIYASQEAGNGYSANLSDYEDTEFSSFGG RKKTRFVDGSKKRKRYGSGPSGNQVPFDGSHEEPMPLPNPMVGFSLPNERLRSVHRNLPDQALGPPFFYYENVALAPKGVWTTISRFLYDIQPEFVDSKYFCAAARKRGYIHNLPIENRSPVLPMPP KTISEAFPNTKRWWPSWDPRRQFNCLQTCMASAKLTERIRCALGRFSDVPTPQVQKYVLDECRKWNLVWVGKNKVAPLEPDEMEFLLGYPRNHTRGVSRTERYRALGNSFQVDTVAYHLSVLRDLFP NGMNVLSLFSGIGGAEVALHRLGIHMKTVISVEKSEVNRTILKSWWDQTQTGTLIEIADVRHLTTERIETFIRRFGGFDLVIGGSPCNNLAGSNRHHRDGLEGEHSALFYDYIRILEHVKATMSAV*;

[0012] On the other hand, the present invention relates to the application of the rice transcription factor OsDRM2 in the breeding of gramineous crops resistant to Tenuivirus genus;

[0013] In some embodiments, the Tenuivirus genus includes Ricestripe virus (RSV) and Maize stripe virus (MSpV), with Ricestripe virus (RSV) being preferred.

[0014] In some embodiments, the preferred gramineous food crop is rice, maize, wheat, oats, and barley; more preferably rice, and most preferably Nipponbare (Oryza);

[0015] On the other hand, the present invention relates to a method for preparing a transgenic plant resistant to rice stripe virus, the steps of which include:

[0016] 1) Construction of rice OsDRM2 gene silencing vector;

[0017] 2) Rice genetic transformation, Agrobacterium-mediated transformation and callus induction culture;

[0018] 3) Positive identification of transgenic plants:

[0019] In some embodiments, gene silencing of the rice gene OsDRM2 is achieved via an artificial miRNA RNAi system; the target sequence of the RNAi system, SEQ ID NO.3, is as follows:

[0020] AGGGGTTATATTCATAACCTG(SEQ ID NO:3)

[0021] In some embodiments, the construction steps of the rice OsDRM2 gene silencing vector further include: predicting the target sequence using software based on the OsDRM2 sequence shown in SEQ ID NO.1; designing positive and negative arms based on the selected target fragment; and tandemly cloning them in both directions. Their complementarity forms the basis for the formation of the double-stranded RNA stem. A non-palindromic circular structure is artificially synthesized, and the sequences of the positive and negative arms are amplified by PCR to ultimately form a 200bp hairpin loop. The pCAMBIA1300 vector and the hairpin sequence are then double-digested with BsaI and Eco31I restriction enzymes at 37°C, ligated with 2 μL of 10×DNALigase Buffer and 350 U of T4 ligase, transformed into E. coli DH5α, and positive clones are selected and sent to a biotechnology company for sequencing to confirm the construction of the silencing vector pCAMBIA1300-DRM2-miRNA.

[0022] The sequence of the positive arm, negative arm, and ring structure is as follows:

[0023] Arm of Justice:

[0024] tgtF(+):CAGTGGTCTCAGCAGTAGGTTATGAATATAACGCCTCAGGAGATTCAGTTTGAAGCTGGACTTCACTTTT

[0025] SEQ ID NO:4

[0026] Anti-arm:

[0027] tgtR(-):CAGTGGTCTCAGGAATAGGTTATGAAAATATCGCCTAGAGAGGCAAAAGTGAAGTCCAGCTTCA

[0028] SEQ ID NO:5

[0029] Ring structure:

[0030] M-SEQ:CAGGAGATTCAGTTTGAAGCTGGACTTCACTTTTGCCTCCT

[0031] SEQ ID NO:6

[0032] In some embodiments, the steps of rice genetic transformation, Agrobacterium transformation, and callus induction culture further include:

[0033] Plasmid transformation: Add 1 μL of plasmid to 50 μL of EHA105 Agrobacterium competent cells, mix thoroughly, and then transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix thoroughly, and then transfer to a 1.5 mL centrifuge tube. Incubate at 30°C and 180 rpm for 30 min on a shaker. Inoculate 50 μL of the activated Agrobacterium culture onto LB solid medium and incubate in the dark for 48 h.

[0034] Synthesize the corresponding detection primers;

[0035] As shown in Table 1 below, prepare the PCR amplification system, mix thoroughly after preparation, and use a PCR instrument for amplification. The amplification program should be set according to the primer information and other relevant settings.

[0036] Table 1: PCR amplification system

[0037] Components quantity Forward primer (10 μM) 1μL Negative primer (10 μM) 1μL 2μTaq PCR Mix 10μL ddH2O 7μL template 1μL total 20μL

[0038] Gel electrophoresis detection: Prepare a 1% agarose gel, spot the sample, and complete the electrophoresis process;

[0039] If the PCR amplification results show clear and correctly sized bands in both the positive control and the sample, and no bands are observed in the negative control, then the sample can proceed to the next step.

[0040] Induction: Select rice grains without mold spots and with normal sprouts, disinfect with 75% alcohol for 1 min, rinse with sterile water for 1 min each time; disinfect with 15% sodium hypochlorite for 20 min, rinse with sterile water 3 times for 1 min each time; inoculate the disinfected rice grains into the induction medium and culture at 26℃ under light for 20 days.

[0041] Agrobacterium infection: Agrobacterium was picked up and placed in the infection solution to prepare OD. 600 Use 0.2% Agrobacterium resuspension to pick up callus and place it in an Erlenmeyer flask. Add Agrobacterium resuspension and infect for 10-15 minutes. Discard the bacterial solution and inoculate the callus onto co-culture medium. Co-culture at 20°C for 48-72 hours.

[0042] Callus screening: Inoculate the callus from the Agrobacterium infection step onto the screening medium and culture in the dark for 20-30 days; inoculate the positive callus onto the secondary screening medium and culture in the dark for 7-10 days.

[0043] Differentiation and rooting: Inoculate positive callus into differentiation medium and culture at 25-27℃ under light for 15-20 days. After the shoots of 2-5cm have differentiated, inoculate them into rooting medium and culture at 30℃ under light for 7-10 days.

[0044] In some implementations, the positive seedling identification step of the transgenic plant includes:

[0045] Total RNA was extracted from transgenic plants that had undergone hygromycin screening and quantitatively reverse transcribed into cDNA, using the rice OsUBQ5 gene as an internal control; the OsDRM2 quantitative primers are shown in SEQ ID NO:7-10:

[0046] qRT-OsDRM2-F:GACATGTCGGAGCTCATCTG (SEQ ID NO:7);

[0047] qRT-OsDRM2-R: CAATCTGGTTGGCTGATCTG (SEQ ID NO:8);

[0048] OsUBQ5-F:ACCACTTCGACCGCCACTACT(SEQ ID NO:9);

[0049] OsUBQ5-R: ACGCCTAAGCCTGCTGGTT (SEQ ID NO: 10);

[0050] Based on the above gene expression level detection results, it is determined whether the rice plant to be tested is resistant rice. If the relative expression level of the OsDRM2 gene in the rice plant to be tested is significantly lower than that in the control, then it is resistant rice.

[0051] This invention utilizes plant transgenic technology to transform rice with an OsDRM2 silencing vector, followed by screening to obtain transgenic lines with low expression levels. Experiments show that after RSV infection of rice, the OsDRM2-silencing transgene exhibits significantly increased resistance to the virus compared to the wild-type control. OsDRM2 plays a negative regulatory role in rice's antiviral process.

[0052] The present invention mainly achieves the above-mentioned objectives by adopting the following solutions:

[0053] This invention targets the rice DNA methyltransferase OsDRM2. An OsDRM2 silencing expression vector, pCAMBIA1300-DRM2-miRNA, was constructed and transformed into rice Nipponbare (NIP) embryos using Agrobacterium EHA105-mediated genetic transformation. The embryos were then placed in a symbiotic culture medium to induce callus formation. After obtaining transgenic rice seedlings, the expression level of the OsDRM2 gene in rice was detected using quantitative real-time PCR (qRT-PCR). After several generations of screening and identification, homozygous transgenic rice with stable inheritance in the T3 generation was obtained. Resistance to RSV was assessed by artificial inoculation with OsDRM2-silencing transgenic rice. The results showed that the symptoms of RSV infection in OsDRM2-silencing transgenic rice were milder than those in wild-type rice, and the viral load was significantly lower in OsDRM2-silencing transgenic rice. This indicates that OsDRM2-silencing transgenic rice significantly enhances rice resistance to RSV. These findings not only demonstrate that OsDRM2 affects RSV infection efficiency but also lay the foundation for research on rice-virus interactions.

[0054] Compared with the prior art, the beneficial effects of the present invention are:

[0055] This invention utilizes the RNAi silencing system to silence the OsDRM2 gene in wild-type NIP rice, obtaining two homozygous OsDRM2 silencing mutant rice lines, named RNAi-DRM2#2 and RNAi-DRM2#7, respectively. Further qRT-PCR detection confirmed the successful construction of the OsDRM2 silencing mutant plants. Artificial inoculation of these rice materials with rice stripe virus (RSV) showed that, compared with wild-type Nipponbare NIP rice, the OsDRM2 silencing mutant rice lines exhibited stronger disease resistance. This invention provides a new direction for breeding transgenic rice with RSV resistance and adds valuable genetic resources to the rice germplasm resource bank, laying the foundation for further research on disease resistance theory and the breeding of resistant varieties. Attached Figure Description

[0056] Figure 1 Experimental results on the relative expression level of OsDRM2 in OsDRM2-silenced transgenic rice.

[0057] Figure 2 Comparison of disease symptoms between OsDRM2-silenced transgenic and wild-type control groups after RSV infection.

[0058] Figure 3 Results of experiments on the viral load of RSV-infected OsDRM2-silenced transgenes and control wild-type. Detailed Implementation

[0059] The rice variety used in this series of experiments is: Nipponbare (Oryza, Sativa L. spp. japonica).

[0060] The relevant culture medium components are as follows:

[0061] Induction medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1g / L, 2mg / L 2,4-D, pH=5.8.

[0062] Subculture medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: 15HBZ0601) 24.1 g / L, 2,4-D, 50 mg / L hygromycin, 300 mg / L cephalosporin, pH=5.8.

[0063] Co-culture medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2 mg / L 2,4-D, 200 μmol / L acetylsylgenone, pH=5.8.

[0064] Rooting medium: 1 / 2MS (manufacturer: Haibo Biotechnology Co., Ltd., product number: HB8469-6) 39.45g / L, 0.5mg / L NAA, 50mg / L hygromycin, pH=5.8.

[0065] Example 1: Construction of rice OsDRM2 gene knockout vector

[0066] Based on the OsDRM2 sequence shown in SEQ ID NO.1, the target sequence (AGGGGTTATATTCATAACCTG) was predicted using WMD3 Web MicroRNA Designer software. According to the selected target fragment, sense and antisense arms were designed and cloned in tandem in both directions. A non-palindromic circular structure was artificially synthesized. The sequences connecting the sense and antisense arms were amplified by PCR, ultimately forming a 200 bp hairpin loop. The pCAMBIA1300 vector and hairpin sequence were then double-digested with BsaI and Eco31I restriction enzymes at 37°C. Ligation was performed with 2 μL of 10× DNA ligase buffer and 350 U of T4 ligase. The mixture was transformed into *E. coli* DH5α, and positive clones were selected and sent to a biotechnology company for sequencing, confirming the construction of the silencing vector pCAMBIA1300-DRM2-miRNA. This vector was then transformed into *Agrobacterium EAH105*.

[0067] The sequence of the positive arm, negative arm, and ring structure is as follows:

[0068] Arm of Justice:

[0069] tgtF(+):CAGTGGTCTCAGCAGTAGGTTATGAATATAACGCCTCAGGAGATTCAGTTTGAAGCTGGACTTCACTTTT

[0070] Anti-arm:

[0071] tgtR(-):CAGTGGTCTCAGGAATAGGTTATGAAAATATCGCCTAGAGAGGCAAAAGTGAAGTCCAGCTTCA

[0072] Ring structure:

[0073] M-SEQ:CAGGAGATTCAGTTTGAAGCTGGACTTCACTTTTGCCTCCT Example 2: Genetic transformation of rice

[0074] Agrobacterium preparation:

[0075] Plasmid transformation: Add 1 μL of plasmid to 50 μL of EHA105 Agrobacterium competent cells, mix thoroughly, and then transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix thoroughly, and then transfer to a 1.5 mL centrifuge tube. Incubate at 30°C and 180 rpm for 30 min on a shaker. Inoculate 50 μL of the activated Agrobacterium culture onto LB solid medium and incubate in the dark for 48 h.

[0076] Agrobacterium detection:

[0077] As shown in the table below, prepare the PCR amplification system, mix thoroughly after preparation, and perform amplification using a PCR instrument. The amplification program should be set according to the primer information, etc. For gel electrophoresis detection, prepare a 1% agarose gel (weigh 1.5g of agarose powder and dissolve it in 150mL of 1×TAE buffer, microwave for about 3 minutes until the liquid becomes transparent. Add EB to the gel casting plate, pour the dissolved agarose liquid into the plate, mix well, insert a comb, and let stand for 40 minutes until the gel turns milky white). Load the sample and complete the electrophoresis process. Check the PCR amplification results. If the electrophoretic bands of the positive control and the sample are clear and of the correct size, and the negative control shows no bands, the sample can proceed to the next step.

[0078] Table 2: PCR reaction system

[0079] Components quantity forward primer 1ul negative primer 1ul 2uTaq PCR Mix 10ul ddH2O 7ul template 1ul total 20ul

[0080] Callus induction: Select rice grains without mold spots and with normal bud openings, disinfect with 75% alcohol for 1 min, rinse with sterile water for 1 min each time; disinfect with 15% sodium hypochlorite for 20 min, rinse with sterile water 3 times for 1 min each time; inoculate the disinfected rice grains into the induction medium and culture at 26℃ under light for 20 days.

[0081] Agrobacterium infection: Agrobacterium was picked into the infection solution to prepare an Agrobacterium resuspension with OD600=0.2. Callus was picked into an Erlenmeyer flask, the Agrobacterium resuspension was added, and after infection for 10-15 min, the bacterial solution was discarded. The callus was then inoculated into a co-culture medium and co-cultured at 20℃ for 48-72 h.

[0082] Callus screening: Inoculate the callus tissue during the Agrobacterium infection process into the screening medium and culture in the dark for 20-30 days; inoculate the positive callus tissue into the secondary screening medium. During the callus tissue picking process, be sure to pick single-clonal callus tissue and culture in the dark for 7-10 days.

[0083] Differentiation and rooting: Inoculate positive callus onto differentiation medium and culture at 25-27℃ under light for 15-20 days. After the shoots differentiate into 2-5cm buds, inoculate them onto rooting medium and culture at 30℃ under light for 7-10 days.

[0084] Positive seedling detection: Rice genomic DNA was extracted using the CTAB method and then detected by PCR.

[0085] Example 3: Positive identification of transgenic plants

[0086] Total RNA was extracted from the transgenic plants to be tested and reverse transcribed into cDNA, with the rice OsUBQ5 gene used as an internal control. Quantitative primers are shown in SEQ ID NO:7-10. The relative expression levels of the OsDRM2 gene-silenced transgenic lines RNAi-DRM2#2 and RNAi-DRM2#7 were significantly lower than the control. Figure 1 The identification process involved the following primers:

[0087] qRT-OsDRM2-F:GACATGTCGGAGCTCATCTG (SEQ ID NO:7);

[0088] qRT-OsDRM2-R: CAATCTGGTTGGCTGATCTG (SEQ ID NO:8);

[0089] OsUBQ5-F:ACCACTTCGACCGCCACTACT(SEQ ID NO:9);

[0090] OsUBQ5-R: ACGCCTAAGCCTGCTGGTT (SEQ ID NO: 10);

[0091] Example 4: Artificial inoculation with RSV and antiviral detection

[0092] OsDRM2 silent transgenic rice seeds and control rice seeds (Nipponbare) were soaked and cultured in a 37℃ incubator for 2-3 days. After the seeds sprouted white, they were sown in 1L glass beakers with 30-35 seedlings per beaker, with 3 biological replicates. The beakers were placed in a 25℃ artificial climate chamber and cultured under 16h light and 8h dark conditions. 1-2 instar virus-free planthoppers were transferred to RSV-infected rice seedlings to acquire the virus. After 3-5 days, the planthoppers were transferred to healthy rice seedlings using a vacuum pump to complete the cycle (10-12 days).

[0093] The virus-carrying rate of the insects was detected, and the number of insects inoculated per seedling was calculated based on the virus-carrying rate. Virus-carrying / non-virus-carrying planthoppers were inoculated onto rice seedlings at the three-to-four-leaf stage (approximately 15 days old). After 3 days, all insects were removed, and the seedlings were placed in a 30℃ greenhouse for growth. After 30 days, the rice disease symptoms were observed, and the incidence rate was statistically analyzed to determine the disease situation. Two silent transgenic lines of OsDRM2 (RNAi-DRM2#2 and RNAi-DRM2#7) showed significant resistance to RSV, such as... Figure 2 As shown. qRT-PCR was used to further detect the virus content, and the results showed that the virus content in the infected transgenic rice lines was significantly lower than that in the wild type, such as... Figure 3 As shown.

[0094] In summary, the experimental results show that, compared with wild-type NIP rice, the two OsDRM2 silencing mutant rice lines, RNAi-DRM2#2 and RNAi-DRM2#7, can enhance the resistance of rice to RSV. Therefore, this invention successfully obtained RSV-resistant gene-silencing mutant rice through the RNAi system, adding valuable genetic resources to the rice germplasm resource bank and having significant implications for agricultural development and national food security.

Claims

1. A transcription factor OsDRM2 derived from rice, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:1 in the sequence listing, and the amino acid sequence encoded by the gene is shown in SEQ ID NO:2 in the sequence listing.

2. The use of the OsDRM2 gene or the protein encoded by it according to claim 1 in the breeding of Tenuivirus-resistant Gramineae food crops.

3. The use as described in claim 2, wherein the fibrillvirus genus includes rice stripe virus (RSV) and maize stripe virus (MSpV), preferably rice stripe virus (RSV).

4. The use as described in claim 2, wherein the gramineous food crop includes rice, corn, wheat, oats or barley, preferably rice, and most preferably Nipponbare variety.

5. A gene silencing vector that confers resistance to rice stripe virus (RSV) on rice, characterized in that: The vector contains an artificial miRNA hairpin expression cassette capable of downregulating the expression of rice transcription factor OsDRM2. The hairpin expression cassette includes complementary sense and antisense arms and a non-palindromic loop structure connecting the two. The target sequence of the hairpin is the OsDRM2 target sequence as shown in SEQ ID NO:

3. The hairpin expression cassette is operatively connected between a plant-expressible promoter and a terminator; and the vector is a binary or dinomial vector that can be replicated or integrated into plant cells.

6. The carrier according to claim 5, wherein the carrier is constructed based on the pCAMBIA1300 skeleton, and the positive arm, negative arm, and ring structure are respectively: Arm of Justice: CAGTGGTCTCAGCAGTAGGTTATGAATATAACGCCTCAGGAGATTCAGTTT GAAGCTGGACTTCACTTTT; Anti-arm: CAGTGGTCTCAGGAATAGGTTATGAAAATATCGCCTAGAGAGGCAAAAGTG AAGTCCAGCTTCA; Non-palindromic cycle: CAGGAGATTCAGTTTGAAGCTGGACTTCACTTTTGCCTCTCT.

7. A method for preparing transgenic rice resistant to rice stripe virus, comprising the following steps: 1) Construction of the rice OsDRM2 gene silencing vector according to any one of claims 5-6; 2) Rice genetic transformation, Agrobacterium-mediated transformation and callus induction culture; 3) Positive identification of transgenic plants.

8. The method of claim 7, wherein the positive identification step of the transgenic plant includes: Total RNA was extracted from transgenic plants that had been screened for hygromycin and quantitatively reverse transcribed into cDNA, with the OsUBQ5 gene of rice as an internal control. The OsDRM2 quantitative primers are shown in SEQ ID NO:7-10: qRT-OsDRM2-F:GACATGTCGGAGCTCATCTG (SEQ ID NO:7); qRT-OsDRM2-R: CAATCTGGTTGGCTGATCTG (SEQ ID NO:8); OsUBQ5-F:ACCACTTCGACCGCCACTACT(SEQ ID NO:9); OsUBQ5-R: ACGCCTAAGCCTGCTGGTT (SEQ ID NO: 10); Based on the above gene expression level detection results, it is determined whether the rice plant to be tested is resistant rice. If the relative expression level of the OsDRM2 gene in the rice plant to be tested is significantly lower than that in the control, then it is resistant rice.

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