Application of rice WRKY protein OsWRKY102 in resisting rice virus diseases

Abstract

The invention relates to application of targeted editing of a rice OsWRKY102 gene by using a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 (CRISPR associated protein 9) technology in resisting rice virus diseases.

Description

Technical Field

[0001] This invention relates to the fields of transgenic technology and plant disease control, specifically to the targeted editing of the rice OsWRKY102 gene using CRISPR / Cas9 technology and its application in resistance to rice viral diseases. Background Technology

[0002] Rice stripe virus (RSV), also known as rice stripe leaf blight virus, belongs to the genus Tenuivirus. It is a multispecific, negative-sense, single-stranded RNA virus transmitted by the planthopper (Laodelphax striatellus). Under natural conditions, RSV only infects grasses, but in laboratory conditions, it can infect Nicotiana benthamiana through mechanical inoculation. Infected rice typically exhibits yellowing and necrosis of newly developed leaves, followed by stunted growth, abnormal heading, and reduced fruit production, which can lead to death in severe cases. Overall, RSV is an insect-borne Tenuivirus transmitted persistently and proliferatingly by the planthopper, causing severe yield reductions in rice in Southeast Asia. This virus is a major viral disease affecting rice production.

[0003] Southern rice black-streaked dwarf virus (SRBSDV) belongs to the genus Fijivirus in the family Reoviridae. This virus is transmitted via the vector white-backed planthopper and is not transmitted through eggs, but it can be persistently transmitted. After infecting the plant host, it causes malformed growth, with main symptoms including stunted growth, increased tillering, dark green leaves, and white wart-like protrusions on the underside of leaves and stems. In areas south of the Yangtze River in my country, this virus primarily damages rice, causing rice black-streaked dwarf disease, while in areas north of the Yangtze River, it damages maize, causing maize rough dwarf disease.

[0004] Rice stripe mosaic virus (RSMV) is a new rice virus species reported by the Plant Virus Research Laboratory of South China Agricultural University in 2017. It is the first cytoplasmic rhabdovirus discovered to infect rice. In recent years, this virus has spread rapidly in rice-growing areas of South my country, and the damage has become increasingly severe year by year. However, the infection mechanism of this virus on its host rice is still unclear.

[0005] Based on the above-mentioned deficiencies of existing technologies, the inventors of this invention used the CRISPR / Cas9 gene editing system to target and edit OsWRKY102. A knockout vector of YL-Hu-OsWRKY102 was constructed, and callus tissue induced by mature NIP embryos of rice (Nipponbare) was transformed using rice callus transfection technology. After obtaining T0 generation transgenic rice seeds, propagation was continued to obtain T1 generation seeds. DNA was extracted from the young leaves of T1 generation individual plants, and the sgRNA target site was amplified using target-specific primers and sequenced to obtain stably inherited homozygous mutant transgenic plants.

[0006] Experimental results showed that transgenic OsWRKY102 mutant plants inoculated with RSV exhibited significantly lower symptoms and viral RNA expression levels compared to susceptible wild-type control NIP plants. This demonstrates that OsWRKY102 mutant transgenic rice significantly enhances rice's resistance to RSV infection. Similar experiments indicated that the OsWRKY102 mutant also possesses some resistance to RSMV and SRBSDV infections. The OsWRKY102 mutant plants have significant application value in the field of transgenic technology. This invention provides practical guidance for cultivating transgenic plants resistant to rice stripe virus blight and related diseases, and also holds important application prospects in the field of plant disease control. Summary of the Invention

[0007] This invention relates to a rice OsWRKY102 gene and its application in resistance to rice stripe virus, rice black-streaked dwarf virus, and rice stripe mosaic virus.

[0008] On the one hand, the rice OsWRKY102 gene has a nucleotide sequence as shown in SEQ ID NO:1 or has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:1, or is composed of nucleotides thereof.

[0009] On the other hand, the OsWRKY102 gene encodes a protein whose amino acid sequence is as shown in SEQ ID NO:2 or whose amino acid sequence has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid encoded by SEQ ID NO:2, or is composed of the amino acid sequence shown in SEQ ID NO:2.

[0010] In some embodiments, the nucleotide sequence (SEQ ID NO:1) of the OsWRKY102 gene is as follows:

[0011] ATGTTCCCATCACCAGGGAGGGCGGTGATGGCGCTAGGCCACCACGGCGCCGCCCGCCAACCGCCGACCACCATGGCGGCGGCGGCCTCCTCGTCGACGACCTCCGCCGCCGCCGCCCCGGCCACGGCCACCACCACCGTCGCCTTCTCCTTCCAGCATCCCACGCCCACGCCGTCTCACCACCACCACCACCATGGCGTCCTAGGTTACAGCCCCCTCGTCCTCGACCACCACCACCCAACCACCGCCGCCGCCTCCTCGCACGCGCCCTCTCCTCCCACCCTCCACCACCACCACGGCGGCGGCCTCCACGCCGCCGCGCCTCCTCCGAGATCGTCGCCGCCGCATCCATGGTCTTGCGAGGAAGGCGATCATCGAGGGAGGCCGCAGATGGGGAATAAGGGCGAGGCGGCGGCGGCGATGGGCGCCATGGGGATCAACGACGCCGGCAACAACACGGCGGCGGCGGCGGCGGCGCAGCATCATCTGGGGGTTGGCGCGGTGAGGATGAAGAAGGTTGGGGGAGGAGGAGGAGGAGGAGGGAAGGCGCGGCGGAAGGTGCGGGAGCCGAGATTCTGCTTCAAGACGATGAGCGACGTGGACGTGCTCGACGACGGCTACAAGTGGCGCAAGTACGGCCAGAAGGTCGTCAAGAACACCCAGCACCCAAGGAGCTATTACCGGTGCACGCAGGACAACTGCCGGGTGAAGAAGCGGGTGGAGCGGCTGGCGGAGGACCCCCGCATGGTGATCACCACCTACGAGGGCCGCCACGTCCACTCCCCCTCCCGCGACGACGACGACGCCGCCCGCGCCTCCGCCGAGATGACCTTCATCTGGTAG

[0012] In some embodiments, the amino acid sequence of the OsWRKY102 gene (SEQ ID NO: 2) is as follows:

[0013] MFSPSPGRAVMALGHHGAARQPPTTMAAAASSSTTSAAAAPATATTTVAFSFQHPTPTPSHHHHHHGVLGYSPLVLDHHHPTTAAASSHAPSPPTLHHHHGGGLHAAAPPPRSSPPHPWSCEEGDHRGRPQMGNKGEAAAA MGAMGINDAGNNTAAAAAAQHHLGVGAVRMKKVGGGGGGGGKARRKVREPRFCFKTMSDVDVLDDGYKWRKYGQKVVKNTQHPRSYYRCTQDNCRVKKRVERLAEDPRMVITTYEGRHVHSPSRDDDDAARASAEMTFIW

[0014] On the other hand, the present invention relates to the application of the OsWRKY102 gene in the breeding of crops resistant to the genus Cervus, especially in the breeding of gramineous food crops resistant to Cervus.

[0015] In some embodiments, the Tenuiviruses include Echinochloa hoja blanca virus (EHBV), Maize stripe virus (MSpV), Rice grassy stunt virus (RGSV), Rice hoja blanca virus (RHBV), Rice stripe virus (RSV), and Urochloa hojablanca virus (UHBV).

[0016] In some implementations, the Fibraviirvirus is preferably rice stripe virus.

[0017] In some implementations, the preferred gramineous food crops are rice, maize, wheat, oats, and barley; more preferably rice, and most preferably Nipponbare.

[0018] On the other hand, the present invention relates to a method for preparing a transgenic plant resistant to rice stripe virus blight, the steps of which include:

[0019] 1) Construction of rice OsWRKY102 gene knockout vector;

[0020] 2) Rice genetic transformation, Agrobacterium-mediated transformation and callus induction culture

[0021] 3) Identification of positive transgenic plants;

[0022] In some embodiments, the rice OsWRKY102 gene knockout vector construction step further includes:

[0023] Primers were designed using the CRISPR-GE website (http: / / skl.scau.edu.cn / ) based on the OsWRKY102 gene sequence shown in SEQ ID No:1. The sgRNA fragment was cloned from rice, and the PCR product was recovered from the gel. Using the pYLsgRNA-OsU3 vector as a template, the target sequence was ligated to the U3 promoter and gRNA scaffold, respectively. The two PCR products were mixed and used as a template to ligate the U3 promoter, target sequence, and gRNA scaffold together using overlapping PCR to form an sgRNA expression cassette. The gRNA expression cassette product and the uncut pYLCRISPR / Cas9Pubi-H plasmid were mixed and digested with BsaI at 37°C for 15 min. After digestion, 1.5 μl of 10×DNA Ligase Buffer and 35 U T4 ligase were added, and the digestion and ligation were performed using a variable-temperature cycling program for 15 cycles: 37°C for 5 min; 10°C for 5 min; 20°C for 5 min. Positive clones were selected and sequenced to confirm the successful construction of the expression vector YL-Hu-OsWRKY102. The recombinant plasmid was obtained, and its quality was assessed by 1% agarose gel electrophoresis.

[0024] In some embodiments, the steps of rice genetic transformation, Agrobacterium transformation, and callus induction culture further include:

[0025] 1) Callus induction: First, select mature rice seeds, peel off the husks, pour them into centrifuge tubes, add 75% ethanol for 1 minute for disinfection, then discard the ethanol, rinse three times with sterile water, then add 15mL of 30% sodium hypochlorite for 20 minutes for disinfection, then discard the sodium hypochlorite and rinse 5-6 times with sterile water; use a pipette to remove excess water, transfer the seeds to the induction medium, and incubate in a 28℃ light incubator for 3 weeks. The callus tissue that grows is then transferred to the subculture medium with pre-sterilized forceps and subcultured in a 28℃ light incubator for 1 week.

[0026] 2) Transformation and culture of Agrobacterium: The plasmid containing the target vector was transformed into Agrobacterium rhizogenes GV3101 using the following steps: 5 μL of plasmid was added to 100 μL of competent Agrobacterium GV3101 cells, and the mixture was mixed by pipetting. The mixture was then added to a sterile electrode cup that had been pre-cooled to 4°C. Electroporation was performed at 220V. LB liquid medium without antibiotics was added, and the mixture was cultured in a shaker at 28°C for about 3 hours. The mixture was then evenly spread on LB solid medium containing 50 μg / ml Kan and 50 μg / ml Rif, and cultured in the dark at 28°C for 2 days until single colonies appeared.

[0027] 3) Agrobacterium transfection: Use a pipette to aspirate the infection solution and wash off the Agrobacterium on the plate to obtain the Agrobacterium suspension for co-culture transformation of rice. Select a sufficient number of callus tissues and place them in a 100ml sterile Erlenmeyer flask. Add an appropriate amount of Agrobacterium suspension and incubate at room temperature for 20 minutes, shaking occasionally. Discard the bacterial solution, place the callus tissues on sterile filter paper to absorb excess bacterial solution, and then transfer them to a solid co-culture medium lined with a layer of sterile filter paper. Incubate at 26℃ in the dark for 3 days.

[0028] 4) Screening Culture: After 3 days of co-culture, the callus tissue needs to be cleaned. Using a 1ml blue pipette tip, the callus on the co-culture medium is transferred to a sterilized Erlenmeyer flask. Sterile water is added to rinse both sides. For the third rinse, sterile water containing 500ul / L carbenicillin is used. After removing excess water with a pipette, the callus is transferred to sterile filter paper and dried using the air blower in a laminar flow hood for about 30 minutes. After the callus is dried, it is transferred to a medium containing hygromycin B for screening culture. The culture conditions are 28-30℃, dark culture; the screening time is 3-4 weeks.

[0029] 5) Seedling rooting: When the differentiated seedlings grow to about 2-3cm and have obvious roots, they can be transferred to rooting medium to allow them to grow. The rooting medium should be poured into a relatively tall bottle or tube so that the rooted seedlings have enough space to grow tall. The rooting culture conditions are 28-30℃, sterile light culture, and transgenic plants are obtained through screening.

[0030] In some embodiments, the identification of the positive transgenic plants further includes: taking young leaves from T0 and T1 generation plants, extracting DNA, amplifying the sgRNA target site using target-specific primers, detecting the amplified products by 1% gel electrophoresis, recovering the specific and correctly sized fragments from the gel, and sequencing them. These were named Oswrky102-ko (Oswrky102-7# and Oswrky102-17#), demonstrating the successful construction of the transgenic rice line.

[0031] In some embodiments, the identification step of the positive transgenic plant further includes:

[0032] The resistance of the Oswrky102-ko transgenic line overexpressing RSV to rice stripe virus was tested by artificial inoculation.

[0033] In some embodiments, the induction medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, pH 5.8.

[0034] In some embodiments, the subculture medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, pH 5.8.

[0035] In some embodiments, the rooting medium comprises: 1 / 2 MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, pH 5.8.

[0036] In some embodiments, the co-culture medium comprises: 24.1 g / L N6 medium, 2 mg / L 2,4-D, 200 μmol / L acetylsylgenone, and pH 5.2.

[0037] In some embodiments, the method for preparing a rice stripe virus (RSV) resistant transgenic rice includes the following steps: gene editing of starting rice to obtain transgenic rice; the gene editing is performed using a Crispr / Cas9 system; the target of the Crispr / Cas9 system is shown in SEQ ID NO:3.

[0038] On the other hand, this application also relates to an sgRNA whose target sequence binding region is shown in SEQ ID NO:3.

[0039] On the other hand, this application also relates to the application of the rice OsWRKY102 gene in rice virus-resistant crop breeding, the gene sequence of which is shown in SEQ ID NO:1.

[0040] In some embodiments, the protein sequence encoding the OsWRKY102 gene is shown in SEQ ID NO:2.

[0041] In some embodiments, the rice virus is selected from the group consisting of rice stripe virus (RSV), southern rice black-streaked dwarf virus (SRBSDV), and rice stripe mosaic virus (RSMV).

[0042] On the other hand, this application also relates to a method for improving the resistance of rice to rice viruses, which utilizes CRISPR / Cas9 technology to target and edit the rice OsWRKY102 gene. The nucleotide sequence of the OsWRKY102 gene is shown in SEQ ID NO:1. The rice virus is selected from the group consisting of rice stripe virus (RSV), southern rice black-streaked dwarf virus (SRBSDV), and rice stripe mosaic virus (RSMV).

[0043] On the other hand, this application also relates to a method for preparing virus-resistant transgenic rice, comprising the following steps: gene editing of starting rice to obtain transgenic rice; the gene editing is performed through a Crispr / Cas9 system; the target of the Crispr / Cas9 system is shown in SEQ ID NO:3.

[0044] In some embodiments, the rice virus is selected from the group consisting of rice stripe virus (RSV), southern rice black-streaked dwarf virus (SRBSDV), and rice stripe mosaic virus (RSMV).

[0045] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0046] This invention constructs the YL-Hu-OsWRKY102 vector and introduces it into the rice variety (Nipponbare) using Agrobacterium-mediated transformation, obtaining two transgenic rice varieties that can be stably inherited, named Oswrky102-ko#7 and Oswrky102-ko#17, respectively. These varieties are resistant to three typical rice viruses: RSV, SRBSDV, and RSMV. Furthermore, the analysis of their resistance to rice stripe virus helps to deepen our understanding of rice viruses and enrich our knowledge of the molecular mechanisms of interaction between rice and RSV, SRBSDV, or RSMV. Attached Figure Description

[0047] Figure 1 Sequencing results of the OsWRKY102 gene in Oswrky102-ko mutant transgenic rice.

[0048] Figure 2Schematic diagram of the symptoms of RSV infection in transgenic Oswrky102-ko mutant and control Nipponbare.

[0049] Figure 3 Results of the experiment on the detection of virus content in transgenic Oswrky102-ko mutant and control Nipponbare after RSV infection.

[0050] mock: healthy rice; RSV: rice infected with rice stripe virus (RSV); NIP: Nipponbare rice.

[0051] Figure 4 : Schematic diagram of the symptoms of disease in transgenic Oswrky102-ko mutant after SRBSDV infection and control Nipponbare.

[0052] Figure 5 Results of the experiment on the detection of virus content in transgenic Oswrky102-ko mutant and control Nipponbare after SRBSDV infection.

[0053] mock: healthy rice; SRBSDV: rice infected with SRBSDV; NIP: Nipponbare rice.

[0054] Figure 6 Schematic diagram of the symptoms of disease in transgenic Oswrky102-ko mutant and control Nipponbare after RSMV infection.

[0055] Figure 7 Results of the experiment on the detection of virus content in transgenic Oswrky102-ko mutant and control Nipponbare after RSMV infection.

[0056] mock: healthy rice; RSMV: rice infected with RSMV; NIP: Nipponbare rice.

[0057] The following examples are provided to better illustrate the content of the present invention, but are not intended to limit the scope of the invention to the illustrated examples. Non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above description are still within the protection scope of the present invention. Examples: Detailed Implementation

[0058] The rice variety used in this series of experiments is: Nipponbare.

[0059] The relevant culture medium components are as follows:

[0060] Induction medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1g / L, 2mg / L 2,4-D, pH=5.8.

[0061] 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 / mL cephalosporin, pH=5.8.

[0062] 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=5.2.

[0063] Rooting medium: 1 / 2 MS (Manufacturer: Haibo Biotechnology Co., Ltd., Product No.: HB8469-6)

[0064] 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, pH = 5.8.

[0065] Example 1: Construction of rice YL-Hu-OsWRKY102 vector

[0066] The CRISPR / Cas9 gene editing vector was constructed using overlapping PCR. Primers were designed on the CRISPR-GE website (http: / / skl.scau.edu.cn / ) based on the OsWRKY102 sequence shown in SEQ ID No:1 to clone the sgRNA fragment from rice. The target sequence was CAACCGCCGACCACCATGG (SEQ ID NO:3). The PCR primer sequence was: YL-Hu-OsWRKY102-F.

[0067] CAGTGGTCTCATGCACGATCAACCGCCGACCACCATGG; SEQ ID NO:4YL-Hu-OsWRKY102-R:

[0068] CAGTGGTCTCAAAACCCATGGTGGTCGGCGGTTG SEQ ID NO:5

[0069] After PCR product recovery and purification, the target sequence was ligated to the U3 promoter and gRNA scaffold using the pYLsgRNA-OsU3 vector as a template. The two PCR products were mixed and used as a template to ligate the U3 promoter, target sequence, and gRNA scaffold together using overlapping PCR to form an sgRNA expression cassette. The gRNA expression cassette product and undigested pYLCRISPR / Cas9Pubi-H plasmid were mixed and digested with BsaI at 37℃ for 15 min. After digestion, 1.5 μl of 10×DNA Ligase Buffer and 35 U T4 ligase were added, and the digestion and ligation were performed using a variable-temperature cycling program of 15 cycles: 37℃ for 5 min; 10℃ for 5 min; 20℃ for 5 min. Positive clones were selected and sequenced to confirm the successful construction of the expression vector YL-Hu-OsWRKY102. The recombinant plasmid was obtained, and its quality was checked by 1% agarose gel electrophoresis. The recombinant vector with the correct sequence was transformed into Agrobacterium GV3101.

[0070] Example 2: Genetic transformation of rice

[0071] 1) Callus induction and subculture: Select newly harvested mature rice seeds, remove the husks, pour into 50ml centrifuge tubes, add 15mL of 75% ethanol for 1 minute for sterilization, discard the ethanol, rinse three times with sterile water, discard the ethanol, then add 15mL of 30% sodium hypochlorite for 20 minutes for sterilization, discard the sodium hypochlorite, and rinse 5-6 times with sterile water. Use a pipette to remove excess water, transfer the seeds to induction medium, and incubate in a 28℃ light incubator for 3 weeks. Transfer the grown callus tissue to subculture medium using pre-sterilized forceps, and subculture in a 28℃ light incubator for 1 week.

[0072] 2) Transformation and culture of Agrobacterium: The plasmid containing the target vector was transformed into Agrobacterium rhizogenes GV3101 (manufacturer: Shanghai Weidi, catalog number: AC1003S) using the following steps: 5 μL of plasmid was added to 100 μL of competent Agrobacterium GV3101 cells, and the mixture was mixed by pipetting. The mixture was then added to a sterile electrode cup that had been pre-cooled to 4°C. Electroporation was performed at 220V. LB liquid medium without antibiotics was added, and the mixture was cultured in a shaker at 28°C for about 3 hours. The culture was then evenly spread on LB solid medium containing 50 μg / ml Kan and 50 μg / ml Rif, and cultured in the dark at 28°C for 2-3 days until single colonies appeared.

[0073] 3) Agrobacterium transfection of callus: Use a pipette to aspirate the infection solution and wash off the Agrobacterium from the plate to prepare the Agrobacterium suspension needed for rice transformation. Select a sufficient number of callus tissues (the callus should be in good condition, bright yellow in color, round and firm in texture, and the particle diameter should be about 3 mm), place them in a 100 ml sterile Erlenmeyer flask, add an appropriate amount of Agrobacterium suspension (ensuring sufficient contact between the bacterial solution and the material), and incubate at room temperature for 20 minutes, shaking occasionally. Discard the bacterial solution, place the callus tissues on sterile filter paper to absorb excess bacterial solution, and then transfer them to a solid co-culture medium lined with a layer of sterile filter paper. Incubate at 26°C in the dark for 3 days.

[0074] 4) Screening Culture: After 3 days of co-culture, the callus tissue needs to be cleaned. Using a 1ml blue pipette tip, transfer the callus from the co-culture medium to a sterile Erlenmeyer flask and rinse twice with sterile water. Rinse a third time with sterile water containing 500ul / L carbenicillin. After aspirating excess water with a pipette, transfer the callus to sterile filter paper and dry it using the airflow from a laminar flow hood for approximately 30 minutes. Once dry, transfer the callus to screening medium for screening culture at 28-30℃ in the dark for 3-4 weeks.

[0075] 5) Seedling Rooting: When the seedlings grow to about 2-3 cm and have a noticeable root system, they can be transferred to rooting medium to continue growth. The rooting medium should be poured into taller bottles or tubes so that the seedlings have enough space to grow upwards. The optimal rooting conditions are aseptic light culture at 28-30 degrees Celsius. Transgenic plants Oswrky102-ko mutants (Oswrky102-ko#7 and Oswrky102-ko#17) were obtained through screening.

[0076] Example 3: Identification of positive transgenic plants

[0077] Young leaves from T0 and T1 generation plants were collected, and DNA was extracted using the CTAB method. sgRNA targeting sites were amplified using target-specific primers. The amplification products were detected by 1% gel electrophoresis. Fragments with specific bands and correct sizes were recovered from the gel and sequenced. The sequencing primers are as follows:

[0078] SEQ ID NO:6

[0079] OsWRKY102-F:CAAGACAGCGAAAATAATCAG

[0080] SEQ ID NO:7

[0081] OsWRKY102-R:GGAGAGGGCGCGTGCGA

[0082] Experimental results are as follows Figure 1 As shown, sequencing results indicate that Oswrky102-ko#7 has a deletion of a base A at the target site, causing OsWRKY102 to terminate prematurely at the 24th amino acid site, while Oswrky102-ko#17 has an insertion of a base at the target site, causing OsWRKY102 to terminate prematurely at the 25th amino acid site. This demonstrates the successful construction of the transgenic rice line.

[0083] Example 4: Artificial inoculation of RSV

[0084] (1) After soaking and germinating the Oswrky102-ko transgenic plants and the control Nipponbare rice material for 2-3 days, the seeds were sown in 1L beakers after they turned white, with about 30 seedlings per beaker. Three biological replicates were made and cultured at 30℃ under 16h light and 8h dark conditions.

[0085] (2) Virus inoculation experiment was conducted using 3-4 instar RSV-carrying and healthy planthoppers. At a ratio of 2-3 planthoppers per seedling, RSV-carrying and virus-free planthoppers were inoculated onto rice plants at the 3-4 leaf stage. After feeding for 3 days, all insects were swept out.

[0086] (3) After 30 days, observe the symptoms of diseased rice and determine the virus-carrying status of rice by qRT-PCR.

[0087] Example 5: Artificial inoculation with SRBSDV

[0088] The Oswrky102-ko transgenic and control rice seed materials were soaked in a 37℃ incubator for 2-3 days. After the seeds turned white, they were sown in 1L glass beakers with 30-35 seedlings per beaker and 3 biological replicates. The beakers were then placed in an artificial climate chamber at 25℃ with 16 hours of light and 8 hours of darkness for cultivation.

[0089] The non-toxic white-backed planthoppers, aged 1-2 years, acquire the virus on SRBSDV-infected rice seedlings for 3-5 days, and then transfer to healthy rice seedlings to complete the cycle (10-12 days).

[0090] The virus-carrying rate of the insects was detected, and the number of insects to be 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 (about 15 days old). After 3 days of inoculation, all the insects were removed and the seedlings were placed in a 30℃ greenhouse for growth.

[0091] After 30 days, the symptoms of diseased rice were observed and the virus-carrying status of rice was determined by qRT-PCR.

[0092] Example 6: Artificial inoculation of RSMV

[0093] The Oswrky102-ko transgenic and control rice seed materials were soaked in a 37℃ incubator for 2-3 days. After the seeds turned white, they were sown in 1L glass beakers with 30-35 seedlings per beaker and 3 biological replicates. The beakers were then placed in an artificial climate chamber at 25℃ with 16 hours of light and 8 hours of darkness for cultivation.

[0094] First- and second-instar electric leafhoppers acquire the virus on RSMV-infected rice seedlings for 3-5 days, then transfer to healthy rice seedlings to complete the cycle (10-12 days).

[0095] The virus-carrying rate of the insects was detected, and the number of insects to be inoculated per seedling was calculated based on the virus-carrying rate. Virus-carrying / non-virus-carrying electric leafhoppers were inoculated onto rice seedlings at the three- to four-leaf stage (about 15 days old). After 3 days of inoculation, all the insects were removed and the seedlings were placed in a 30℃ greenhouse for growth.

[0096] After 30 days, the symptoms of diseased rice were observed and the virus-carrying status of rice was determined by qRT-PCR.

[0097] Example 7: Resistance analysis of rice after RSV inoculation

[0098] Thirty days after transplanting, RSV-infected rice exhibited mosaic symptoms. Compared to the control Nipponbare NIP, the two mutant transgenic lines OsWRKY102-ko (Oswrky102-7# and Oswrky102-17#) showed mild mosaic symptoms, while the control NIP showed obvious mosaic symptoms. Figure 2 As shown. Mixed samples were taken from diseased rice plants, with three biological replicates per group. The expression level of the RSV CP gene was detected using qRT-PCR. Figure 3 As shown, the expression level of the CP gene in the transgenic lines was significantly lower than that in the control. These results indicate that the resistance of transgenic plants to RSV infection is affected by the OsWRKY102 gene, and that mutation of OsWRKY102 in rice can significantly enhance the resistance to RSV infection.

[0099] The quantitative primer sequences are as follows:

[0100] qRSV-CP-F AGGCAATCAATGACATCTCC; SEQ ID NO:8

[0101] qRSV-CP-R ATCTCTCACAAAGCCAGTGC; SEQ ID NO:9

[0102] Example 8: Resistance analysis of rice after inoculation with SRBSDV

[0103] Thirty days after transplanting, SRBSDV-infected rice exhibited mosaic symptoms. Compared to the control Nipponbare NIP, the two mutant transgenic lines OsWRKY102-ko (Oswrky102-7# and Oswrky102-17#) showed mild dwarfing symptoms, while the control NIP showed significant dwarfing and increased tillering. Figure 4 As shown. Diseased rice plants were sampled in a pooled manner, with three biological replicates per group. The expression level of the SRBSDV-S10 virus gene was detected using qRT-PCR. Figure 5 As shown, the expression level of the S10 gene in the transgenic line was significantly lower than that in the control. These results indicate that the resistance of transgenic plants to SRBSDV infection is affected by the OsWRKY102 gene, and that mutation of OsWRKY102 in rice can significantly enhance the resistance to SRBSDV infection.

[0104] The quantitative primer sequences are as follows:

[0105] qSRBSDV-S10-F ACAAACATGGAGCGGAGTCT; SEQ ID NO:10

[0106] qSRBSDV-S10-RCGGTCTTACGCAACGATGAA; SEQ ID NO:11

[0107] Example 9: Resistance analysis of rice after RSMV inoculation

[0108] Thirty days after transplanting, RSMV-infected rice exhibited mosaic symptoms. Compared to the control Nipponbare NIP, the two mutant transgenic lines OsWRKY102-ko (Oswrky102-7# and Oswrky102-17#) showed mild mosaic symptoms, while the control NIP showed obvious mosaic and nodular protrusion symptoms, such as... Figure 6 As shown. Diseased rice plants were sampled in a pooled manner, with three biological replicates per group. The expression level of the RSMV-M viral gene was detected using qRT-PCR. Figure 7 As shown, the expression level of the M gene in the transgenic lines was significantly lower than that in the control. These results indicate that the resistance of transgenic plants to RSMV infection is affected by the OsWRKY102 gene, and that mutation of OsWRKY102 in rice can significantly enhance the resistance to RSMV infection.

[0109] The quantitative primer sequences are as follows:

[0110] qRSMV-MF TGCACAGACGTTAGTGAGTTAC; SEQ ID NO:12

[0111] qRSMV-MRTCCGTCTTTCATAGCCTTCAG; SEQ ID NO:13

[0112] In summary, the experimental results show that, compared with the control NIP, the Oswrky102-ko mutant transgenic rice plants significantly enhance the resistance of rice to RSV, SRBSDV, and RSMV infections (including phenotypic symptoms and viral load in diseased plants). Therefore, this invention successfully obtained transgenic rice resistant to RSV, SRBSDV, and RSMV infections through gene editing, which is of great significance and has achieved unexpected technical results.

Claims

1. Application of the rice OsWRKY102 gene in breeding rice virus-resistant crops, the gene sequence of which is shown in SEQ ID NO:

1.

2. The application according to claim 1, wherein the protein sequence encoding the OsWRKY102 gene is shown in SEQ ID NO:

2.

3. The application according to any one of claims 1-2, wherein the rice virus is selected from the group consisting of rice stripevirus (RSV), southern rice black-streaked dwarf virus (SRBSDV), and rice stripe mosaic virus (RSMV).

4. The application according to any one of claims 1-3, wherein the crop is preferably rice, corn, wheat, oats and barley; more preferably rice, and most preferably Nipponbare.

5. A method for improving rice resistance to rice viruses, comprising using CRISPR / Cas9 technology to target and edit the rice OsWRKY102 gene, wherein the nucleotide sequence of the OsWRKY102 gene is shown in SEQ ID NO:1, and the rice virus is selected from the group consisting of rice stripe virus (RSV), southern rice black-streaked dwarf virus (SRBSDV), and rice stripe mosaic virus (RSMV).

6. A method for preparing transgenic rice resistant to rice virus, comprising the following steps: gene editing of starting rice to obtain transgenic rice; wherein the gene editing is performed using a CRISPR / Cas9 system; and the target of the CRISPR / Cas9 system is shown in SEQ ID NO:

3.

7. The preparation method according to claim 6, wherein the rice virus is selected from the group consisting of rice stripevirus (RSV), southern rice black-streaked dwarf virus (SRBSDV), and rice stripe mosaic virus (RSMV).

8. An sgRNA having a target sequence binding region as shown in SEQ ID NO:3.

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