Application of OsAGO12 protein and coding gene thereof in regulating and controlling resistance of plants to rice tumor dwarf virus

Abstract

The invention relates to the technical field of biology, in particular to application of expression of OsAGO12 protein and a coding gene thereof in regulation and control of resistance of plants to rice tumor dwarf viruses. The amino acid sequence of the OsAGO12 protein is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene OsAGO12 of the OsAGO12 protein is as shown in SEQ ID NO. 1. According to the invention, overexpression and CRISPR / Cas9 technologies are respectively adopted to carry out overexpression or knockout mutation on the OsAGO12 gene in rice, and the result shows that the overexpression of the OsAGO12 gene in rice can significantly enhance the resistance of rice to rice tumor dwarf virus. The invention provides a new gene resource for improved breeding of rice.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of OsAGO12 protein and expression of its encoding gene in regulating plant resistance to rice gall dwarf virus. Background Art

[0002] There is a lack of effective control measures for the disaster of rice virus diseases. The study of virus-host interaction provides basic knowledge for understanding the outbreak mechanism of crop virus diseases. As one of the most important food crops in the world, rice feeds nearly half of the world's population. Pests and diseases cause global rice yield losses of up to 24.6% to 40.9%. In particular, rice virus diseases transmitted by vector insects threaten the safety of most grasses and some dicotyledonous plants with their "intermittent, migratory and explosive" characteristics. At present, facing the disaster of rice virus diseases, we lack effective prevention and control measures. Once the disease is prevalent, the losses caused by it are difficult to recover. Moreover, the genetic resources used for rice virus-resistant breeding are very limited. Therefore, it is extremely important for us to explore rice antiviral genes and analyze the mechanism of action of these antiviral genes.

[0003] Rice gall dwarf virus (RGDV) is a species of the genus Phytoreovirus in the family Reoviridae. In China, it is mainly transmitted by the insect vector, the electric leafhopper. Rice gall dwarf disease shows a fluctuating increase and decrease. The disease causes severe gall dwarfing in rice plants, with leaves becoming shorter, darker, and stiffer, and tumor-like protrusions of different sizes appearing on the back of the leaves and leaf sheaths, which are its typical symptoms. The genome of RGDV contains 12 double-stranded RNAs, encoding 6 structural proteins (P1, P2, P3, P5, P6, P8) and 6 non-structural proteins (Pns4, Pns9, Pns10, Pns11, Pns12). At present, the understanding of the pathogenic mechanism and host interaction process of RGDV is still limited, which restricts the development of genetic resources for breeding resistance to rice gall dwarf virus. However, research based on model organisms such as Arabidopsis thaliana has revealed a variety of antiviral mechanisms in plants, which provides a theoretical basis and research direction for the discovery of crop antiviral genes.

[0004] RNA silencing is a conservative way for plants to resist viruses: in the long-term interaction between viruses and plants, plants do not sit idly by, but evolve various complex defense mechanisms to resist virus infection, including antiviral immunity mediated by RNA silencing, R genes, recessive resistance, plant hormone signals, autophagy, and WUS (WUSCHEL). RNA silencing, as a gene expression regulation mechanism that is ubiquitous in eukaryotes, is of great significance for resisting pathogens, especially viruses. The key proteins involved in this mechanism include Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR), which play a role in the initiation, execution, and signal amplification stages of the RNA silencing process, respectively. Downstream of RNA silencing, small RNA (sRNA) binds to AGO proteins to form RNA-induced silencing complexes (RISCs), which then act on target genes according to the principle of sequence complementarity. RNA silencing can inhibit the transcription of homologous DNA through transcriptional gene silencing (TGS) caused by RNA-directed DNA methylation (RdDM), and can also achieve posttranscriptional gene silencing (PTGS) by cutting and degrading or inhibiting the translation of target RNA. The production of a large number of viral-derived small RNAs (vsiRNAs) in the host caused by viral infection can be loaded into the AGO protein to form RISC targeting the viral genome, thereby effectively inhibiting the replication and spread of the virus.

[0005] AGO proteins play an important role in many areas of life, especially in immune responses, where they are a key element in defending against viral invasion. In the antiviral defense mechanism of plants, AGO proteins demonstrate their core function in resisting viruses by participating in RNA silencing. As a core member and main executor in the RNA silencing pathway, AGO proteins frequently become targets of pathogens, especially viruses that require host systems to replicate and spread. Many plant viruses produce RNA silencing suppressors (VSRs), which use different mechanisms to interfere with the host's RNA silencing process, among which the RISC complex centered on AGO is often the main target of VSR attacks. For example, some virus-encoded silencing suppressors bind to AGO proteins through their GW / WG (glycine-tryptophan) motifs and further prevent AGO proteins from functioning. For example, the p38 protein encoded by Turnip crinklevirus (TCV) has been shown to inhibit RNA silencing pathways through multiple pathways. p38 can not only bind to dsRNA, but also to AGO1 and AGO12 proteins, and has a destructive effect on DCL4 enzyme, which leads to the reduction of siRNA and inhibits the RNA silencing process; the silencing suppressor P1 encoded by sweetpotato mild mottle virus (SPMMV) contains three GW / WG repeats, which may inhibit the formation of RISC through strong interaction with Arabidopsis AGO1. Some virus-encoded silencing suppressors will weaken the antiviral immunity mediated by AGO protein by degrading AGO protein, such as the silencing suppressor p25 encoded by Potato virus X (PVX), which can interact with Arabidopsis AGO1, 2, 3, 4 and then degrade these AGO proteins through the 26S proteasome pathway; the coat protein encoded by Tomato ringspot virus (ToRSV) interacts with tomato AGO1 through the GW / WG motif to inhibit its translational repression activity and promotes the degradation of AGO1 through autophagy. In addition, during the RISC assembly process, the P0 protein encoded by Beet western yellows polerovirus (BWYV) binds to the components of RISC to further prevent sRNA from being loaded into AGO1, and AGO1 that is not assembled into RISC is easily degraded. In response, plants have evolved a variety of AGO proteins, and the functions of some AGO proteins are redundant; the diversity and redundancy of AGO proteins play an important role in maintaining the function of the RNA silencing pathway and inhibiting viral invasion. In addition, plant AGO proteins have also evolved a variety of ways to deal with the interference of VSR. For example, rice AGO1 can bind to miR168 to target and cut its own mRNA, forming a negative feedback regulation mode.When the function of AGO1 is disturbed by VSR, the inhibition of AGO1 by miR168 will be released, thereby increasing the expression of AGO1 and enhancing the inhibitory effect on viruses. This ingenious negative feedback regulatory pathway can stabilize AGO1's regulation of plant growth and development and resistance to viral infection. Rice AGO18 can regulate the inhibition of AGO1 by miR168 through a "molecular lock" mode, further increasing the complexity of the pathway to cope with environmental changes and interference from pathogenic microorganisms. In the RNA silencing system of rice, the genome encodes 8 DCL proteins, 19 AGO proteins and 5 RDR proteins. Although these genes play a role in rice's viral defense, detailed information on how they work specifically needs further study.

[0006] Thanks to the rapid iteration of gene editing technology, scientists can directly locate and modify specific genes in the crop genome, thereby improving the crop's resistance to diseases. Compared with traditional breeding methods, gene editing is more efficient and can introduce and improve disease-resistant traits in a shorter period of time. In addition, gene editing avoids the introduction of exogenous DNA, which helps with public acceptance and regulatory approval. Using gene editing technology, we can test and evaluate the effects of potential antiviral genes, thereby accelerating the antiviral breeding process. Summary of the invention

[0007] The object of the present invention is to provide an application of OsAGO12 protein and the expression of its encoding gene in regulating plant resistance to rice gall dwarf virus.

[0008] To achieve the above object, the present invention adopts the following technical solutions: The amino acid sequence of OsAGO12 protein is shown in SEQ ID NO.2.

[0009] The nucleotide sequence of the gene encoding the above-mentioned OsAGO12 protein is shown in SEQ ID NO.1.

[0010] Use of the above OsAGO12 protein or encoding gene in regulating plant resistance to rice gall dwarf virus; Among them, overexpression of OsAGO12 protein or increasing the expression of the gene encoding OsAGO12 protein can improve the resistance of plants to rice gall dwarf virus; Among them, the loss of OsAGO12 protein function or down-regulation of the expression of the OsAGO12 protein encoding gene can reduce the plant's resistance to rice gall dwarf virus; Wherein, the plant is rice.

[0011] A method for improving plant resistance to rice gall dwarf virus, comprising the steps of overexpressing the above-mentioned OsAGO12 protein encoding gene in plants to obtain transgenic plants; Wherein, the plant is rice.

[0012] Furthermore, the above-mentioned method for improving plant resistance to rice gall dwarf virus specifically includes: constructing a recombinant plant expression vector containing the OsAGO12 protein encoding gene, transforming the constructed recombinant plant expression vector into a recipient plant, and cultivating and screening to obtain transgenic plants with improved resistance to rice gall dwarf virus.

[0013] A method for reducing plant resistance to rice gall dwarf virus, comprising the steps of causing the function of the OsAGO12 protein encoding gene to be lost or down-regulated to obtain a transgenic plant; Wherein, the plant is rice.

[0014] Furthermore, the above-mentioned method for reducing plant resistance to rice gall dwarf virus specifically includes: designing a CRISPR / Cas9-based sgRNA sequence with the above-mentioned OsAGO12 protein encoding gene as a target, connecting a DNA fragment containing the sgRNA sequence to a CRISPR / Cas vector, transforming a recipient plant, and thereby obtaining a transgenic plant with a functional deficiency of the OsAGO12 protein encoding gene; the transgenic plant has reduced resistance to rice gall dwarf virus compared with the recipient plant.

[0015] A method for cultivating transgenic plants, comprising: introducing the above-mentioned OsAGO12 protein encoding gene into a recipient plant to obtain a transgenic plant; the transgenic plant has improved resistance to rice gall dwarf virus compared with the recipient plant; Wherein, the plant is rice.

[0016] The significant advantages of the present invention are: The present invention uses overexpression and CRISPR / Cas9 technology to overexpress or knock out the OsAGO12 gene in rice, and the results show that overexpression of the OsAGO12 gene in rice can significantly enhance the resistance of rice to rice gall dwarf virus. The present invention provides a new gene resource for rice breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Western identification of OsAGO12 overexpressing rice.

[0018] Figure 2 Identification of OsAGO12 loss-of-function mutants in rice.

[0019] Figure 3 qRT-PCR detection of RGDV virus-related genes in OsAGO12 overexpressing rice, OsAGO12 loss-of-function mutant rice and wild-type rice (Zhonghua11) after RGDV infection.

[0020] Figure 4 Symptom diagram of different rice strains infected with RGDV. DETAILED DESCRIPTION

[0021] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0024] The amino acid sequence of the OsAGO12 protein described in the following examples is shown in SEQ ID NO.2, and the nucleotide sequence of the coding gene OsAGO12 is shown in SEQ ID NO.1.

[0025] Example 1: Obtaining OsAGO12 protein and its encoding gene Primers were designed according to the sequence shown in SEQ ID NO.2, and the primer sequences were as follows: AGO12-F: 5'-ATGTCTTCGCGCGGCGG-3', AGO12-R: 5'-TCAGCAGTAGAACATGAACCGCT-3'.

[0026] According to the instructions, total RNA from Oryza sativa L. japonica cv. Zhonghua 11 rice (Oryza sativa L. japonica cv. Zhonghua 11, Xu Yu et al. "Cloning of the rice glutenin Gt1 gene and construction of the wax gene promoter-guided Gt1 gene expression vector of Zhonghua 11", Journal of Shanghai Normal University (Natural Science Edition), Vol. 39, No. 2, April 2010, p. 204) was extracted with TRIzol Reagent from Invitrogen, and reverse transcription was performed using the company's SuperScript II reverse transcriptase to obtain cDNA. The primer used for reverse transcription was a 16-nucleotide Oligod (T) primer.

[0027] The cDNA obtained by reverse transcription was used as a template and the above primers AGO12-F and AGO12-R were used to perform PCR (Polymerase Chain Reaction) reaction to obtain a PCR product of 3177 bp having a nucleotide sequence as shown in SEQ ID NO.1.

[0028] After the PCR product was recovered, it was connected with the vector pEASY-Blunt Zero Cloning Kit (Quanshijin Company, product catalog number: CB501-01) and transformed into Escherichia coli strain DH5α to obtain transformants. The plasmid extracted from the transformant was sent for sequencing, and the plasmid with correct sequencing was named pEASY-Blunt-AGO12.

[0029] Example 2: Obtaining OsAGO12 overexpressing rice 1. Construction of overexpression vector Using pEASY-Blunt-AGO12 plasmid as template, PCR amplification was performed with primers AGO12-5'SalI and AGO12-3'PstI to obtain a PCR product containing a 3177bp target gene fragment. The obtained PCR product was purified and recovered, and connected with the pCambia2300-Actin-Flag plasmid vector backbone cut with SalI and PstI by seamless cloning (refer to Novizan, product catalog number: C112-01) to obtain the recombinant plasmid pCambia2300-Actin-FLAGAGO12. After sequencing, the recombinant plasmid pCambia2300-Actin-FLAGAGO12 was obtained by inserting the OsAGO12 gene between the SalI and PstI restriction sites of the pCambia2300-Actin-Flag plasmid, i.e., the overexpression vector.

[0030] AGO12-5'SalI:5'-aggggatcctctagagtcgacATGTCTTCGCGCGGCGG-3', AGO12-3'PstI:5'-taaagcagggcatgcctgcagTCAGCAGTAGAACATGAACCGCT-3'.

[0031] 2. Obtaining transgenic rice overexpressing OsAGO12 1) Callus induction culture The seeds of Zhonghua 11 rice (hereinafter also referred to as wild-type rice) are shelled, first soaked in 70% ethanol for 10 minutes, and then soaked in 0.1% mercuric chloride for 30 minutes; surface sterilization is performed. The solution on the surface of the seeds is washed off with a large amount of sterile water, and the moisture on the surface of the seeds is absorbed with sterile filter paper. The seeds are placed on a mature embryo callus induction medium plate, the edge of the plate is sealed with Parafilm film, and cultured in a 26°C incubator away from light. After about 15 days, the grown callus tissue is carefully removed and transferred to the mature embryo subculture medium, and the culture is continued under the same conditions. Subculture is required every two weeks. When used for transformation, it is necessary to select granular callus tissue that has been subcultured for about 5 days and is light yellow.

[0032] 2) Cultivation of Agrobacterium The overexpression vector pCambia2300-Actin-FLAGAGO12 was electroporated into Agrobacterium EHA105 to obtain the recombinant bacteria EHA105 / pCambia2300-Actin-FLAGAGO12.

[0033] Streak the recombinant strain EHA105 / pCambia2300-Actin-FLAGAGO12 on an LB plate containing antibiotics (50 mg / L Kanamycin, 50 mg / L Rifampicin) and culture at 28°C for 2 days. Pick a single colony and inoculate it into liquid LB medium and culture it at 28°C with shaking until the OD 600 The concentration of acetosyringone was about 0.5, and acetosyringone was added to a final concentration of 100 mM to obtain an Agrobacterium suspension for transforming rice callus.

[0034] 3) Co-culture of rice callus and Agrobacterium Place the subcultured callus in a sterilized conical flask and pour in the Agrobacterium suspension to submerge the callus. Leave at room temperature for 20 minutes and gently shake from time to time to allow the callus to fully contact the bacterial solution. Gently remove the callus with sterile tweezers, place it on a sterile filter paper to absorb the excess bacterial solution, and transfer it to a co-culture medium plate covered with a layer of sterile filter paper. Culture in the dark at 28°C for 3 days to obtain co-cultured callus.

[0035] 4) Screening and differentiation of resistant callus The co-cultivated callus was washed with an appropriate amount of sterile water to remove the residual Agrobacterium on the surface, placed on a screening medium, cultured in the dark at 26°C for screening, and transferred to a new screening medium after two weeks for further screening for two weeks. Select callus in good condition after two rounds of screening, transfer it to a differentiation medium plate, culture it in the dark for 3 days, and then transfer it to a light incubator (15hr / day) for light culture. Differentiated seedlings can be seen after one month. When the differentiated seedlings grow to about 2cm, transfer them to a rooting medium in a conical flask and continue to culture for about two weeks. Select seedlings with good growth and well-developed root systems, wash the culture medium from the roots with tap water, and transplant them into the soil. Collect seeds to obtain T1 transgenic rice seeds, and sow to obtain T1 transgenic rice.

[0036] The rice seeds of the T1 generation were preliminarily screened by G418 (the pCambia2300 vector carries the G418 resistance screening gene), and the germinated seeds indicated that the vector was transferred into the rice. The germinated seeds were planted in the soil, and after growing for 2 weeks, 0.1 g of leaves were taken and ground into powder with liquid nitrogen.

[0037] Add 200 μl of protein extraction buffer (0.25 M Tris-HCl, pH 6.8, 8% SDS, 8% β-mercaptoethanol, 20% glycerol) to the leaf powder of the transgenic rice line overexpressing OsAGO12, incubate on ice for 10 min, boil at 100°C for 10 min, centrifuge at 4°C and 12,000 rpm for 10 min, take the supernatant, perform SDS-PAGE, and use Western detection after transfer. SDS-PAGE and Western Blot were performed according to known methods and product instructions. The antibody used was anti-FLAG-HRP (sigma), and the antibody anti-Actin was used to detect the endogenous Actin protein in rice as an internal reference. Figure 1 , the one with a band at 120KDa was positive, indicating that the target gene was transferred and the protein was expressed. Two overexpression lines #4 and #19 were selected for subsequent disease resistance analysis experiments (Zhonghua 11 in the figure is wild-type rice, used as a negative control).

[0038] Example 3: Obtaining OsAGO12 loss-of-function mutant rice 1. Construction of expression vector 1) According to the website http: / / skl.scau.edu.cn / . Based on the PAM site with the terminal sequence of NGG, a 20 bp specific target sequence targeting the N-terminus of the OsAGO12 coding region was selected as follows: 5'-GCGGCGCGTCGACCCGTAGG-3'; 2) Design primers according to the target DNA sequence and add BsaI restriction sites at both ends of the primers. The primer sequence is: UF: 5'-CTCCGTTTTACCTGTGGAATCG-3', AGO12-gRT1: 5'-GCGGCGCGTCGACCCGTAGGgttttagagctagaaat-3', gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3', AGO12-OsU3T1: 5'-CCTACGGGTCGACGCGCCGCgccacggatcatctgc-3'; B1': 5'-TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3', BL: 5'-AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3'; 3) Referring to the CRISPR / Cas9 gene editing vector construction scheme in the reference (A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant. 2015), the intermediate vector was used as a template, and three pairs of primers, UF / AGO12-OsU3T1, AGO12-gRT1 / gRNA-R, and B1' / BL, were used for three rounds of PCR amplification to obtain an expression cassette with a linker, wherein the intermediate vector contained sgRNA (5'-GCGGCGCGTCGACCCGTAGG-3') specifically targeting the exon of OsAGO12, which can guide Cas9 to cut the target gene OsAGO12, thereby causing the gene to mutate and cause its functional loss); after the amplification, the final product was excised and purified, and the final vector pYLCRISPR / Cas9, restriction endonuclease and T4 ligase were added, and the cutting and ligation were carried out according to a certain system; 4) Transform the Escherichia coli strain DH5α, apply kanamycin-resistant culture medium to obtain transformants, extract the plasmid of the transformants and send them for sequencing. The positive transformants are the final recombinant vector, named pYLCRISPR / Cas9-AGO12:sgRNA.

[0039] 2. Obtaining OsAGO12 CRISPR / Cas9 Rice 1) Callus culture and transformation It is basically the same as Example 2, except that the transfer vector is pYLCRISPR / Cas9-AGO12:sgRNA.

[0040] 2) Identification of OsAGO12 loss-of-function mutants in rice The leaf powder of OsAGO12 loss-of-function mutant rice strains was used to extract genomic DNA. The specific method was referred to the High-Efficiency Plant Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Cat. No.: DP350). Afterwards, 0.5 g of genomic DNA was used as a template and primers AGO12-CF and AGO12-CR were used for PCR reaction. The PCR products were directly sent for sequencing and then sequence alignment was performed. Figure 2As shown, comparison of DNA sequencing results showed that two positive loss-of-function mutant strains were named ago12#1 and ago12#2, among which ago12#1 inserted an "A" between the 332nd and 333rd bases in the OsAGO12 coding region, and ago12#2 deleted 5bp between the 327th and 333rd bases in the OsAGO12 coding region, both of which caused the premature termination of the OsAGO12 amino acid sequence, thereby resulting in the loss of OsAGO12 protein.

[0041] AGO12-CF: 5'-TCGTCGGCGAGCGGCAAG-3', AGO12-CR: 5'-TTGTCCCGCGACTTGCACCAG-3'.

[0042] Example 4: Overexpression of OsAGO12 can improve the resistance of rice to RGDV, and loss of OsAGO12 function can reduce the resistance of rice to RGDV 1) The expression level of RGDVPns11 was identified by fluorescent quantitative PCR (qRT-PCR) to identify the infection of RGDV. The leaves of T1 generation OsAGO12 overexpressing rice, OsAGO12 functional loss mutant rice and wild-type rice Zhonghua 11 were inoculated with electric leafhoppers carrying RGDV (the pathogen was Rice gall dwarf virus). 30 plants of each type of rice were inoculated and cultured at 30°C during the day, 22°C at night, and 60% humidity. Three electric leafhoppers were inoculated on each plant. After three days of biting, the electric leafhoppers were caught and the bitten rice were cultured in a sunny greenhouse (natural light, temperature. Two strains of transgenic rice used in the experiment were taken to confirm each other).

[0043] Four weeks after infection, the infected rice leaf powder was extracted, Trizol (Invitrogen) was added, and RNA was extracted according to the method described in the manual. Afterwards, the genomic DNA in the RNA was digested with RQ1 DNase (Promega, catalog number: M610A) according to Table 1 below: Then 2 μg of the digested RNA was taken for reverse transcription qRT-PCR. For specific methods, see invitrogen M-MLV Reverse Transcriptase (catalog number: 28025-021). OsEF1α was used as an internal reference, and the primers for the internal reference were EF1α-F and EF1α-R. The expression level of RGDV Pns11 was detected, and the primers for RGDV Pns11 were Pns11-F and Pns11-R.

[0044] Table 1 Digestion system EF1α-F: 5'-GCACGCTCTTTCTTGCTTTCACTCT-3' EF1α-R: 5'-AAAGGTCACCACCATACCAGGCTT-3', Pns11-F: 5'-GGTGGTATTGATTGACGCATGT-3', Pns11-R: 5'-TCGGTCTCATCGTTCCCATCTT-3'.

[0045] The results are as follows Figure 3 As shown in the figure, it can be seen that the accumulation of RGDV Pns11 in the T1 generation of OsAGO12 overexpression rice (AGO12 OE) is lower than that in the wild-type rice Zhonghua11, while the accumulation of RGDV Pns11 in the OsAGO12 functional loss mutant rice is higher than that in the wild-type rice Zhonghua11 (the asterisk in the figure indicates a significant difference). Therefore, OsAGO12 overexpression can improve rice resistance to RGDV, while OsAGO12 functional loss can increase rice sensitivity to RGDV.

[0046] 2) Determine the incidence rate after RGDV inoculation by phenotype Four weeks after infection, the symptoms of 30 T1 generation OsAGO12 overexpressing rice plants (AGO12OE#4 and AGO12OE#19), 30 OsAGO12 functional loss mutant rice plants (ago12#1 and ago12#2) and 30 wild-type rice plants Zhonghua11 were observed (those infected with RGDV showed dwarfing, increased tillering, curled leaf tips and notched leaf margins), the number of symptomatic plants was counted, and the virus infection rate was calculated as (number of phenotype plants / total number of plants) ×%. The results are shown in Table 2: Table 2 shows the statistical results of virus infection rate of transgenic rice after virus infection As can be seen from Table 2, the OsAGO12 overexpression rice had a lower susceptibility rate, while the OsAGO12 loss-of-function mutant rice had a relatively high susceptibility rate. In addition, we took photos of the diseased rice of different strains, such as Figure 4The figure shows the disease symptoms of OsAGO12 overexpression and OsAGO12 loss-of-function mutant rice 4 weeks after infection. It can be seen that the disease symptoms of OsAGO12 overexpression rice are weaker, with a weaker degree of dwarfing than the wild type; while the disease symptoms of OsAGO12 loss-of-function mutant rice are relatively stronger, with a stronger degree of dwarfing (AGO12OE represents OsAGO12 overexpression rice, ago12 represents OsAGO12 loss-of-function mutant rice). Compared with wild-type rice, OsAGO12 overexpression rice is more resistant to disease, while OsAGO12 loss-of-function mutant rice is more susceptible to disease.

[0047] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. OsAGO12 protein, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding the OsAGO12 protein according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

1.

3. Use of the OsAGO12 protein according to claim 1 or the encoding gene according to claim 2 in regulating plant resistance to rice gall dwarf virus.

4. The use according to claim 3, characterized in that: By overexpressing OsAGO12 protein or increasing the expression level of the gene encoding OsAGO12 protein, the resistance of plants to rice gall dwarf virus can be improved.

5. The use according to claim 3, characterized in that: The resistance of plants to rice gall dwarf virus can be reduced by making the OsAGO12 protein function ineffective or down-regulating the expression level of the OsAGO12 protein encoding gene.

6. The use according to claim 3, characterized in that: The plant is rice.

7. A method for improving plant resistance to rice gall dwarf virus, characterized in that: The method comprises the step of overexpressing the coding gene according to claim 2 in a plant to obtain a transgenic plant.

8. The method according to claim 7, characterized in that: The plant is rice.

9. A method for cultivating transgenic plants, characterized in that: The encoding gene of claim 2 is introduced into a recipient plant to obtain a transgenic plant; the transgenic plant has improved resistance to rice gall dwarf virus compared with the recipient plant.

10. The method according to claim 9, characterized in that: The plant is rice.

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