Application of OsATG8b gene and encoded protein thereof in regulation and control of plant virus resistance of rice

Abstract

The invention belongs to the technical field of biology, and particularly relates to an OsATG8b gene and application of an encoding protein of the OsATG8b gene to regulation and control of plant virus resistance of rice. The OsATG8b gene is derived from rice, the nucleotide sequence of the OsATG8b gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein coded by the OsATG8b gene is shown as SEQ ID NO.2. By cloning the OsATG8b gene and constructing a transgenic vector, OsATG8b gene overexpressed rice and OsATG8b gene knockout rice are obtained, the resistance of the OsATG8b gene overexpressed rice and OsATG8b gene knockout rice to watery straw dwarf virus and rice serrated leaf dwarf virus is measured, and the result shows that compared with wild type rice and OsATG8b gene knockout rice, the OsATG8b gene overexpressed rice and OsATG8b gene knockout rice have the advantages that the OsATG8b gene overexpressed rice and OsATG8b gene knockout rice has higher rice resistance to watery straw dwarf virus and rice serrated leaf dwarf virus; the OsATG8b gene overexpressed rice has higher resistance to rice straw dwarf virus and rice serrated leaf dwarf virus, which indicates that the OsATG8b gene plays an important regulation and control role in rice plant virus resistance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to OsATG8b Application of genes and their encoded proteins in regulating rice resistance to plant viruses. Background Art

[0002] There are many types of rice virus disease pathogens in my country, including rice dwarf virus (RDV), rice stripe virus (RSV), rice black-streaked dwarf virus (RBSDV), southern rice black-streaked dwarf virus (SRBSDV), rice grassy stunt virus (RGSV), rice gall dwarf virus (RGDV), rice ragged stunt virus (RRSV) and rice stripe mosaic virus (RSMV). These virus diseases have certain regional characteristics in distribution. For example, in the southern rice-growing areas, the damage caused by dwarf disease is particularly serious, while the northern rice-growing areas are generally plagued by stripe disease. Rice virus diseases are mainly spread by insects such as leafhoppers and planthoppers. When these insects move in the field, they spread the virus from one rice plant to another, causing the disease to spread rapidly. Since viral diseases are mostly systemic infections, once rice plants are infected, their growth and development will be seriously affected, causing the plants to shrink, the leaves to change color, growth to be stunted, and even die. This will not only significantly reduce the yield and quality of rice, but also cause huge losses to farmers' economic income.

[0003] Rice diseases caused by RRSV and RGSV are both transmitted by the insect vector brown planthopper, and complex infection often occurs in the field, making prevention and control extremely difficult. RRSV belongs to the genus Oryzavirus. Its complete particles are equiaxially symmetrical icosahedrons with double-layer capsids and a diameter of about 75~80nm. The surface of the virus particles has two types of protrusions, type A and type B, and the inner core diameter is about 57~65nm. The virus is stable in the pH range of 6.0~9.0 and can maintain structural integrity in a certain concentration of magnesium chloride, but it is easy to lose its infectivity at high temperatures. The genome of RRSV consists of 10 linear double-stranded RNA (dsRNA) fragments, encoding multiple structural proteins and non-structural proteins, which play a key role in virus replication and transmission. After RRSV infects rice, it will cause symptoms such as dwarfing of the plant, increased tillering, wrinkled and twisted leaves, and serrated notches. When severely infected, rice cannot head or the panicle is short, and filling stops, resulting in a serious decrease in yield. The virion morphology and genome structure of RGSV are different from those of RRSV. RGSV is a member of the genus Tenuvirus. Its virions are slender filaments, and its genome is single-stranded RNA (ssRNA), containing 6 segments, encoding a total of 12 proteins through ambiguity. After RGSV infects rice, it will cause symptoms such as dwarfing of the plant, increased tillering, short and narrow leaves with a light yellow color. In severe cases, brown spots will appear on the rice leaves, and they are prone to premature withering. With the development and maturity of modern molecular biotechnology, studying the interaction between viruses and hosts has become a hot topic. In 2015 and 2020, Zhang et al. successively analyzed the important pathogenic mechanisms of RRSV and RGSV. The study of miR319 by regulating its target genes TCP21 This affects the content of jasmonic acid, an endogenous hormone in rice, and changes the sensitivity of rice to RRSV. Gender The IV gene plays an important biological role in regulating rice plant development. It was found that an E3 ubiquitin ligase induced by the P3 protein encoded by RGSV acts on the RNA-mediated DNA methylation pathway by regulating the degradation of Pol IV, and participates in the pathogenicity of the virus.

[0004] Autophagy is a highly conserved intracellular degradation process that is ubiquitous in eukaryotes and is an important mechanism for organisms to maintain protein metabolic balance and intracellular environmental stability. In plants such as rice, autophagy encapsulates and degrades misfolded proteins, damaged organelles, and foreign pathogens in the cell through double-membrane autophagosomes, and then transports their contents to the vacuole for degradation and reuse. The autophagy process in rice cells involves multiple key components and complex steps. First, the cell senses starvation signals or stress conditions, and then this signal is transmitted to the pre-autophagosome, triggering the formation of the isolation membrane. The edges of the isolation membrane fuse to form a complete autophagic vacuole, which is a sign of the beginning of autophagy. The autophagic vacuole is then transported to the vacuole and fuses with the vacuole membrane, releasing the single-membrane autophagosome into the vacuole. In the vacuole, the autophagosome is disintegrated, and its contents are degraded under the action of hydrolases. Finally, the degradation products such as amino acids and lipids are released back to the cytoplasm for reuse. In rice, the regulation of cellular autophagy involves a variety of biological processes, such as responding to virus infection, growth and development regulation, and stress response. Taking RGDV as an example, the RGDV coat protein P2 interacts with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and ATG4B of the electric leafhopper to initiate the formation of autophagosomes that encapsulate the virus, thereby inducing the autophagic response of the cell. The mechanism of this incomplete autophagy is that GAPDH, by binding to ATG14, hinders the fusion of autophagosomes and lysosomes mediated by the ATG14-SNARE complex. The interaction between the Pns11 protein encoded by RGDV and ATG5 induces the formation of incomplete autophagosomes that encapsulate virus particles; at the same time, the interaction between Pns11 and the key lysosomal protein Lamp1 prevents the fusion and degradation of autophagosomes and lysosomes; and the autophagosomes that encapsulate virus particles can break through the midgut and salivary gland barriers to spread, promoting the proliferation of viruses in insects. This process not only demonstrates the important role of cellular autophagy in plant antiviral defense, but also reveals the complex interaction mechanism between viruses and host plants. In addition, rice cell autophagy is also regulated by a variety of signaling molecules, such as reactive oxygen species (ROS). As a byproduct of aerobic metabolism, ROS can form oxidative stress when accumulated in plants and participate in the regulation of autophagy. Appropriate amounts of ROS can act as signaling molecules to mediate plant responses to stress, while excessive ROS may activate mechanisms such as autophagy to remove damaged components and maintain cell homeostasis. Rice cell autophagy is a complex and sophisticated regulatory process involving multiple components and biological processes, which is of great significance for maintaining normal growth and development of rice and coping with various stresses.

[0005] Mutants constitute the cornerstone of functional genomics exploration, serving as a key strategy to identify genes and elucidate their functions. Since the 1970s, multiple mutant resource libraries have been constructed worldwide. With the successful completion of rice whole genome sequencing, rice mutants have become a powerful tool for exploring the association between rice morphology, physiological characteristics and genes, as well as the cloning and functional analysis of these genes. Integrating rice expression profile data, screening gene mutants involved in disease resistance and implementing functional complementarity verification of transgenic plants, provides an important way to deeply reveal the molecular genetic pathways of disease resistance genes and their mechanisms of action. At present, the popularization of high-throughput molecular biology technology is leading us into the era of data torrents, and the huge amount of information produced urgently needs the strong support of efficient bioinformatics platforms to analyze. Given the completion of the comprehensive sequencing of rice and RRSV and RGSV genomes, this has laid a solid data foundation for subsequent in-depth bioinformatics analysis. Summary of the invention

[0006] The object of the present invention is to provide OsATG8b Application of genes and their encoded proteins in regulating rice resistance to plant viruses.

[0007] The purpose of the present invention is achieved through the following technical solutions: Use of any of the following in regulating rice resistance to plant viruses: (1) OsATG8b Gene; (2) OsATG8b The protein encoded by the gene is OsATG8b protein; Among them, the OsATG8b The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the OsATG8b protein is shown in SEQ ID NO.2; Furthermore, the regulation includes overexpression OsATG8b Genes improve rice resistance to plant viruses, and knockout OsATG8b Genes reduce rice's resistance to plant viruses; Furthermore, the plant virus is rice grassy stunt virus or rice sawtooth leaf stunt virus.

[0008] A method for improving rice resistance to plant viruses, comprising: OsATG8b The gene was introduced into the recipient rice through the expression vector, and the OsATG8b A transgenic rice with high gene expression, wherein the transgenic rice has higher resistance to plant viruses than the recipient rice; wherein the OsATG8b The nucleotide sequence of the gene is shown in SEQ ID NO.1; Furthermore, the plant virus is rice grassy stunt virus or rice sawtooth leaf stunt virus.

[0009] A method for reducing rice resistance to plant viruses by using CRISPR / Cas9 gene editing to modify the receptor rice OsATG8b Gene knockout, OsATG8b A transgenic rice with a gene function deletion, wherein the transgenic rice has lower resistance to plant viruses than the recipient rice; wherein the OsATG8b The nucleotide sequence of the gene is shown in SEQ ID NO.1; Further, the sequence of the target site of the CRISPR / Cas9 gene editing method is 5-'TGGCCAAGAGCTCGTTCAAG-3'; Furthermore, the plant virus is rice grassy stunt virus or rice sawtooth leaf stunt virus.

[0010] Use of any of the following in breeding rice resistant to plant viruses: (1) OsATG8b Gene; (2) OsATG8b The protein encoded by the gene is OsATG8b protein; Among them, the OsATG8b The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the OsATG8b protein is shown in SEQ ID NO.2; Furthermore, the plant virus is rice grassy stunt virus or rice sawtooth leaf stunt virus.

[0011] The significant advantages of the present invention are: The present invention clones OsATG8b Gene, construct transgenic vector, obtain OsATG8b Gene overexpression in rice and OsATG8b Knockout rice, OsATG8b Gene overexpression in rice and OsATG8b The resistance of the knockout rice to rice grassy stunt virus and rice serrated leaf dwarf virus was measured and the results showed that compared with wild-type rice and OsATG8b For gene knockout rice, OsATG8b Gene overexpression rice is more resistant to rice grassy stunt virus and rice serrated leaf dwarf virus, indicating OsATG8b The gene plays an important regulatory function in rice resistance to plant viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 for OsATG8b Identification of gene overexpressing rice.

[0013] Figure 2 for OsATG8b Identification of gene knockout rice.

[0014] Figure 3 for OsATG8b Gene overexpression in rice OsATG8b qRT-PCR detection of RRSV virus-related genes in gene knockout rice and wild-type rice (Zhonghua 11) after RRSV infection.

[0015] Figure 4 Symptoms of different rice strains infected with RRSV.

[0016] Figure 5 for OsATG8b Gene overexpression in rice OsATG8b qRT-PCR detection of RGSV virus-related genes in gene knockout rice and wild-type rice (Zhonghua 11) after RGSV infection.

[0017] Figure 6 Symptoms of different rice strains infected with RGSV. DETAILED DESCRIPTION

[0018] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The experimental methods used in the embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used in the embodiments, unless otherwise specified, can be obtained from commercial sources.

[0019] Embodiment 1: OsATG8b Obtaining genes and their encoded proteins The present invention OsATG8b The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 2. Primers were designed according to the sequences shown in SEQ ID NO. 1 and 2, and the primer sequences were ATG8b-F: 5'-ATGGCCAAGAGCTCGTTCAAG-3', and ATG8b-R: 5'-CTAGAGCAGCCCAAAGGTGTT-3'.

[0020] According to the instructions, total RNA from Oryza sativa L. japonica cv. Zhonghua 11 (Xu Yu et al., "Cloning of the Glutelin Gt1 Gene in Zhonghua 11 Rice and Construction of the Gt1 Gene Expression Vector Guided by the Waxy Gene Promoter", Journal of Shanghai Normal University (Natural Science Edition), Vol. 39, No. 2, April 2010, p. 204) was extracted using TRIzol Reagent from Invitrogen, and reverse transcribed using the company's SuperScript II reverse transcriptase to obtain cDNA. The primer used for reverse transcription was a 16-nucleotide Oligod (T) primer.

[0021] The cDNA obtained by reverse transcription was used as a template and PCR amplification was performed with the primers ATG8b-F and ATG8b-R designed as described above to obtain a 360 bp PCR product having the nucleotide sequence shown in SEQ ID NO.1.

[0022] 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. The plasmid was the plasmid of SEQ ID NO.1. OsATG8b The gene was inserted into the pEASY-Blunt vector to obtain a recombinant vector, which was named pEASY-Blunt-ATG8b.

[0023] Embodiment 2: OsATG8b Obtaining Gene Overexpression Rice 1. Construction of expression vector Using the recombinant vector pEASY-Blunt-ATG8b as a template, PCR amplification was performed with primers ATG8b-5' SalI (5'-aggggatcctctagagtcgacATGGCCAAGAGCTCGTTCAAG-3') and ATG8b-3' PstI (5'-taaagcagggcatgcctgcagCTAGAGCAGCCCAAAGGTGTT-3') to obtain a 402 bp PCR product. The PCR product was purified and recovered, and connected with the pCambia2300-Actin-Flag plasmid vector backbone digested with SalI and PstI by seamless cloning (refer to Novizan, product catalog number: C112-01) to obtain the recombinant plasmid pCambia2300-Actin-FLAGATG8b. After sequencing, the recombinant plasmid pCambia2300-Actin-FLAGATG8b was a PCR product of SEQ ID NO.1. OsATG8b The gene was inserted into the vector obtained between the SalI and PstI restriction sites of the pCambia2300-Actin-Flag plasmid.

[0024] two, OsATG8b Obtaining Gene Overexpression Rice 1) Callus induction culture The seeds of Zhonghua 11 rice (hereinafter also referred to as wild-type rice) are shelled and first soaked in 70% ethanol for 10 minutes, and then soaked in 0.1% mercuric chloride for 30 minutes to sterilize the surface. Wash the solution on the surface of the seeds with a large amount of sterile water, and absorb the moisture on the surface of the seeds with sterile filter paper. Place the seeds on a mature embryo callus induction medium plate, seal the edge of the plate with Parafilm film, and culture in a 26°C incubator away from light. After about 15 days, carefully remove the grown callus tissue, transfer it to the mature embryo subculture medium, and continue to culture under the same conditions. Subculture needs to be performed 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.

[0025] 2) Cultivation of Agrobacterium The recombinant plasmid pCambia2300-Actin-FLAGATG8b was electroporated into Agrobacterium EHA105 to obtain the recombinant bacteria EHA105 / pCambia2300-Actin-FLAGATG8b.

[0026] Streak the recombinant EHA105 / pCambia2300-Actin-FLAGATG8b 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.

[0027] 3) Co-culture of rice callus and Agrobacterium Place the subcultured callus in a sterilized conical flask and pour in the Agrobacterium suspension used to transform rice callus 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.

[0028] 4) Screening and differentiation of resistant callus The co-cultivated callus tissue is washed with an appropriate amount of sterile water to remove the residual Agrobacterium on the surface, placed on the screening medium, and cultured in the dark at 26°C for screening. After two weeks, it is transferred to a new screening medium and continues to be screened for two weeks. Select the callus tissue that is in good condition after two rounds of screening, transfer it to the differentiation medium plate, culture it in the dark for 3 days, and then transfer it to the 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 the rooting medium in a conical flask and continue to culture for about two weeks. Select seedlings with good growth and developed root systems, wash the culture medium on the roots with tap water, and transplant them into the soil. Collect the seeds to obtain T1 generation transgenic rice seeds, and sow them to obtain T1 generation transgenic rice.

[0029] The T1 rice seeds were preliminarily screened with G418 (the pCambia2300 vector carries the G418 resistance screening gene). The germinated seeds indicated that the vector had been transferred into the rice. The germinated seeds were planted in the soil. After growing for 2 weeks, 0.1 g of leaves were taken and ground into powder with liquid nitrogen.

[0030] OsATG8b Add 200 μl of protein extraction buffer (0.25M Tris-HCl, pH 6.8, 8% SDS, 8% β-mercaptoethanol, 20% glycerol) to the leaf powder of the gene-overexpressing rice strain, 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 , those with a band at 20KDa were positive, indicating gene transfer and protein expression. Two strains #2 and #4 were selected for subsequent disease resistance analysis experiments (Zhonghua 11 in the figure is wild-type rice, used as a negative control).

[0031] Embodiment 3: OsATG8b Obtaining gene knockout rice 1. Construction of expression vector 1) According to the website http: / / skl.scau.edu.cn / home / . Select the target according to the PAM site of the terminal sequence NGG OsATG8b The 17-bp specific target sequence at the N-terminus of the coding region was as follows: 5′-TGGCCAAGAGCTCGTTCAAG-3′; 2) Design primers according to the DNA sequence of the target site, and add BsaI restriction sites at both ends of the primers. The primer sequences are UF: 5'-CTCCGTTTTACCTGTGGAATCG-3', ATG8b-gRT1: 5'-CTTGAACGAGCTCTTGGCCAgttttagagctagaaat-3', ATG8b-U3T1: 5'-TGGCCAAGAGCTCGTTCAAGCggcagccaagccagca-3', gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3', B1': 5'-TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3', B2: 5'-AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC-3'; 3) Perform three rounds of PCR amplification using three pairs of primers: UF / ATG8b-U3T1, ATG8b-gRT1 / gRNA-R, and B1' / B2 to obtain an expression cassette with a linker; 4) After the amplification, the final product is cut and purified, and the final vector pYLCRISPR / Cas9, restriction endonuclease and T4 ligase are added to perform cutting and ligation according to a certain system; the final vector pYLCRISPR / Cas9 uses an optimized Cas9 gene, which has been disclosed in [Ma et al., 2013, A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant. 2015], 5) 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-ATG8b:sgRNA.

[0032] two, OsATG8b Obtaining gene knockout rice 1) Callus culture and transformation Refer to Example 2, II, 1)-4), except that the recombinant vector pYLCRISPR / Cas9-ATG8b:sgRNA is transformed into the recipient rice.

[0033] 2) OsATG8b Identification of gene knockout rice OsATG8bThe leaf powder of the knockout rice strain 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 ATG8b-CF (5'-TTGGAGGAAACGTAGAGGAGTTTT-3') and ATG8b-CR (5'-ACAGTTGCTACCACACACATTC-3') were used for PCR reaction. The PCR product was directly sent for sequencing, and then sequence alignment was performed. Figure 2 As shown, DNA sequencing results showed that the two positive strains were named OsATG8b KO #6 and OsATG8b KO #9, among them, OsATG8b KO #6 in OsATG8b An "A" was inserted between bases 4 and 5 in the coding region, causing the premature termination of the amino acid sequence of OsATG8b; OsATG8b KO #9 is deleted directly from ATG, resulting in the loss of the protein.

[0034] Example 4: Overexpression OsATG8b Can improve rice resistance to RRSV and RGSV, knock out OsATG8b Can reduce rice resistance to RRSV and RGSV 1) Detection of RRSV S8 and RGSV vRNA2 expression by quantitative real-time PCR (qRT-PCR) to identify RRSV and RGSV infection T1 transgenic rice and wild-type rice Zhonghua 11 were inoculated with brown planthoppers (the pathogens were rice ragged stunt virus and rice grassy stunt virus, respectively) carrying RRSV and RGSV (see Zhang et al., 2016. Suppression of jasmonicacid-mediated defense by viral-inducible microRNA319 facilitates virus infection in rice. Molecular Plant. 9:1302-1314), 30 plants of each type of rice were inoculated, and the culture was carried out at 30℃ during the day and 22℃ at night with a humidity of 60%. Three brown planthoppers were inoculated on each plant. After three days of biting, the brown planthoppers were caught and the bitten rice were cultured in a sunny greenhouse (the experiment was repeated 3 times and the results were averaged).

[0035] Four weeks after infection, rice leaf powders from T1 transgenic rice and wild-type rice Zhonghua 11 were extracted and added with Trizol (Invitrogen) to extract RNA according to the instructions. Then, genomic DNA in RNA was digested with RQ1 DNase (Promega, catalog number: M610A) according to Table 1 below: Table 1 Digestion system Then take 2 μg of digested RNA for reverse transcription qRT-PCR. For specific methods, see invitrogen M-MLVReverse Transcriptase (Cat. No.: 28025-021). OsEF1α As internal reference, the internal reference primers were EF1α-F (5′-GCACGCTCTTCTTGCTTTCACTCT-3′) and EF1α-R (5′-AAAGGTCACCACCATACCAGGCTT-3′), the expression of RRSVS8 was detected, the detection primers of RRSV S8 were S8-F (5′-CACCAAACAACGCACTACCAT-3′) and S8-R (5′-AATCAGTCGGAGCAGCAAATC-3′), the expression of RGSV vRNA2 was detected, the detection primers of RGSV vRNA2 were vRNA2-F (5′-ATGTCTATTTCAATACCAGCAAGTGA-3′) and vRNA2-R (5′-GCAGACTGGTGCAAACTTCTC-3′). The results are shown in Figure 2 . Figure 3 and Figure 5 As shown, it can be seen that OsATG8b Gene overexpression rice ( OsATG8b The accumulation of RRSV S8 and RGSVvRNA2 in OE) was lower than that in wild-type rice Zhonghua11, while OsATG8b Knockout rice ( OsATG8b KO ) were higher than those in wild-type rice Zhonghua11 (the asterisks in the figure indicate significant differences). OsATG8b The gene can improve rice resistance to RRSV and RGSV, while knocking out OsATG8b The gene can increase the sensitivity of rice to RRSV and RGSV.

[0036] 2) Determine the infection rate of RRSV and RGSV by phenotype Four weeks after infection, the symptoms of 30 T1 transgenic rice plants and 30 wild-type rice Zhonghua 11 were observed (the RRSV-infected plants were dwarfed, with increased tillers, curled leaf tips, and notched leaf margins, and the RGSV-infected plants were dwarfed, with increased tillers and grass-like appearance, and narrowed leaves). The number of symptomatic plants was counted, and the virus infection rate was calculated, which was (the number of phenotypic plants / the total number of plants)*%. The results are shown in Tables 2 and 3: Table 2 Statistical results of virus infection rate of transgenic rice after RRSV infection Table 3 shows the statistical results of the virus-carrying rate of transgenic rice after RGSV infection From Table 2 and Table 3, we can see that OsATG8b The gene overexpression rice had a lower susceptibility to disease. OsATG8b The knockout rice had a relatively high susceptibility to disease. In addition, we took photos of different strains of susceptible rice, such as Figure 4 and Figure 6 Shown are healthy, OsATG8b Gene overexpression in rice OsATG8b The disease symptoms of gene knockout rice. It can be seen that OsATG8b The disease symptoms of gene-overexpressing rice were weaker, with the degree of dwarfing being weaker than that of wild type. OsATG8b The disease symptoms of gene knockout rice are relatively stronger, manifested as a stronger degree of dwarfing ( Figure 4 and Figure 6 , OsATG8b OE Representative OsATG8b Gene overexpression in rice, OsATG8b KO representative OsATG8b Compared with wild-type rice, OsATG8b Gene overexpression makes rice more disease resistant, OsATG8b Knockout rice is more susceptible to disease.

[0037] 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. Use of any of the following in regulating rice resistance to plant viruses: (1) OsATG8b Gene; (2) OsATG8b The protein encoded by the gene is OsATG8b protein; in, Said OsATG8b The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the OsATG8b protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The regulation includes overexpression OsATG8b Genes improve rice resistance to plant viruses, and knockout OsATG8b Gene reduces rice's resistance to plant viruses.

3. The use according to claim 1, characterized in that: The plant viruses are rice strawy stunt virus and rice sawtooth leaf stunt virus.

4. A method for improving the resistance of rice to plant viruses, characterized in that: Will OsATG8b The gene was introduced into the recipient rice through the expression vector, and the OsATG8b A transgenic rice with high gene expression, wherein the transgenic rice has higher resistance to plant viruses than the recipient rice; wherein the OsATG8b The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that: The plant viruses are rice strawy stunt virus and rice sawtooth leaf stunt virus.

6. A method for reducing resistance of rice to plant viruses, characterized in that: The CRISPR / Cas9 gene editing method was used to modify the recipient rice OsATG8b Gene knockout, OsATG8b A transgenic rice with a gene function deletion, wherein the transgenic rice has lower resistance to plant viruses than the recipient rice; wherein the OsATG8b The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. The method according to claim 6, characterized in that: The sequence of the target site of the CRISPR / Cas9 gene editing method is 5-'TGGCCAAGAGCTCGTTCAAG-3'.

8. The method according to claim 6, characterized in that: The plant viruses are rice strawy stunt virus and rice sawtooth leaf stunt virus.

9. Use of any of the following in breeding rice resistant to plant viruses: (1) OsATG8b Gene; (2) OsATG8b The protein encoded by the gene is OsATG8b protein; in, Said OsATG8b The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the OsATG8b protein is shown in SEQ ID NO.

2.

10. The use according to claim 9, characterized in that: The plant viruses are rice strawy stunt virus and rice sawtooth leaf stunt virus.

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