Application of rice BTB structural domain-containing protein OsFIP2 in rice antivirus

Abstract

The invention relates to a rice BTB structural domain-containing protein OsFIP2 and application thereof in resisting rice stripe virus and southern rice black-streaked dwarf virus, and after an OsFIP2 overexpression vector is transferred into plant rice, a stably inherited OsFIP2-ox transgenic plant is obtained.

Description

Technical Field

[0001] The invention relates to the field of plant disease prevention and control and transgenic technology, and mainly to a rice OsFIP2 gene and an application thereof in rice resistance to viral diseases. Background Art

[0002] Rice stripe virus (RSV), also known as rice stripe leaf blight virus, is a typical representative of the genus Tenuivirus. The virus is mainly transmitted by Laodelphax striatellus. After infecting rice, it causes dwarfing of plants, discontinuous yellow stripes on leaf veins, heart leaf death and other symptoms, resulting in reduced or no heading of diseased plants. Rice stripe leaf blight occurs in rice-growing areas in many provinces of my country, among which the most serious cases are in rice fields in Zhejiang, Jiangsu, Henan and Shandong, causing serious impacts on my country's food safety production. In recent years, domestic and foreign scholars have conducted a lot of research on the molecular biological functions of RSV, including genomics, proteomics, and the interaction between viral proteins and host factors. It was found that the genome of RSV consists of four single-stranded RNA fragments with a total size of about 17kb, which are named RNA1, RNA2, RNA3 and RNA4 according to their size, encoding a total of 7 proteins. Each viral protein has different functions to promote the infection and replication of the virus.

[0003] Southern rice black-streaked dwarf virus (SRBSDV) belongs to the genus Fijivirus of the family Reoviridae. The virus can be transmitted by the vector white-backed planthopper. It is not transmitted through eggs but can be persistently transmitted. After infecting plant hosts, it can cause abnormal growth of plants. The main symptoms are dwarfing of plants, increased tillering, dark green leaves, and white tumor-like protrusions on the back of leaves and stems. The virus mainly harms rice in areas south of the Yangtze River in my country, causing rice black-streaked dwarf disease, and harms corn in areas north of the Yangtze River, causing corn rough dwarf disease. The mature virus is spherical, icosahedral, and has a double-layer capsid structure with a diameter of 70 to 75 nm. The virus exists in the phloem cells of susceptible host plants. Its genome consists of 10 dsRNAs, encoding 7 structural proteins and 6 non-structural proteins.

[0004] At present, the main method of controlling rice virus diseases in agriculture is to spray chemical agents. Although this method is highly efficient, it will cause serious environmental pollution in the long run, which is not conducive to the sustainable development of green agriculture and ecological balance. Therefore, exploring resistance genes and cultivating resistant varieties are conducive to better controlling rice virus diseases. Based on current research, cultivating virus-resistant rice plant strains is currently the most effective means of controlling rice virus diseases.

[0005] The BTB domain is a highly conserved protein-protein interaction domain that was originally discovered in conserved sequence elements in the developmentally regulated Drosophila proteins Broad-complex, Tramtrack, and Bric-abrac. This domain contains approximately 120 amino acids and is found in more than 600 different proteins in organisms ranging from yeast to humans. In plants, the BTB domain plays an important role in growth and development. For example, AtBTB2 and AtBTB3 in Arabidopsis regulate the development of Arabidopsis gametophytes, and overexpression of AtBTB2 can actively promote lateral root development in Arabidopsis. In apple, MdBT2 and MdBHLH93 interact to regulate leaf senescence by degrading chlorophyll in apple. In rice, the Cullin3-based RING E3 ubiquitin ligase CRL3, Cullin3 (CUL3), and BTB proteins together form a ubiquitin proteasome system. The receptor protein NPR1 of the salicylic acid (SA) pathway is a known protein containing a BTB domain. OsCUL3a negatively regulates cell death and immunity by degrading OsNPR1 in rice. These studies have shown that proteins containing the BTB domain play an important role in rice's resistance to pathogen infection. However, there are currently no reports on the relationship between rice viruses and proteins containing the BTB domain. Summary of the invention

[0006] Based on the defects and shortcomings of the prior art, the present invention relates to a rice BTB domain protein encoding gene OsFIP2 and its application in resisting rice viruses;

[0007] In one aspect, a rice gene encoding a BTB domain protein, OsFIP2, has nucleotide and protein sequences as follows: The nucleotide sequence of OsFIP2:

[0008] SEQ ID No:1

[0009] ATGGAGTCGCCGCCGGAGTCCTCCTCCTCCTCCCCGGTCCTCCTCAACATCGGGGGCAAGAGGTACG

[0010] CGACCACCGTCGAGACGCTCACGCAGCGGGAGCCCAGCTCCATGCTCGCCGCCATGTTCAGCGGGAG

[0011] GCACACGCTCCCCCGGCACCCGGACACGGGCATGGTGTTTCGTGGACAGGGACGGGAAGCACTTCAG

[0012] GCATGTCCTCAACTGGCTCAGGGACGGCGCCGTCCCAGACATGTCGGAGTCTGAGTACCAGCAGCTC

[0013] CTGCGAGAAGCGGAGTACTACCAGTTGCTCGGCTTGGCTGATTGCATAAATGATAGATTGGGTTGGAA

[0014] AAATGATGAGAATTTCTCGGAGGCCGAATTGACACGCAAAGATGTGATCAAGTGCATCCAAGCTCCG

[0015] AGAGTCAGATTTCGTGGTGTAAATCTATCTGGCCTTGATCTATCCAAACTTGATTTATCAGAGGTGGAC

[0016] TTCAGCTATGCATGCATAAAGAATGCCAATTTTTCCTCTGCTTATCTTCGTAAAGCAAAGTTCAGGCTG

[0017] ACAGAAGCTACTTGTTCTTCATTCCAAAGTGCAAATTTACATGAGTGTGAGCTTATTGGAGCAAATCTT

[0018] GAAGGATCTGTTCTAGATAAAGCAAACCTTCAAAGTGCAAACCTACAGGATGCATGCTTAAAGCAATG

[0019] TTGCTTCATTGAAACGGATCTTCGTTCTGCTCATCTACAGGGTGCTAATCTTATGGGCGCCAACTTGAG

[0020] TGGAGCTAATCTTGAAGGGGCCAATCTCAAGGGAGCTAAGTTGGCCGGTACAAACTTGGAATGTGCA

[0021] AATCTTCAGCGAGCCTACTTGCGAGAAGTTGATTTGCGCGAAACTCATTTAACTGGTGCTAAGCTTGG

[0022] AGGTGCAAATCTGCTTGGAGCTATCAGATAA

[0023] Amino acid sequence of OsFIP2:

[0024] SEQ ID No:2

[0025] MESPPESSSSSPVLLNIGGKRYATTVETLTQREPSSMLAAMFSGRHTLPRHPDTGMVFVDRDGKHFRHVL

[0026] NWLRDGAVPDMSESEYQQLLREAEYYQLLGLADCINDRLGWKNDENFSEAELTRKDVIKCIQAPRVRFR

[0027] GVNLSGLDLSKLDLSEVDFSYACIKNANFSSAYLRKAKFRLTEATCSSFQSANLHECELIGANLEGSVLDK

[0028] ANLQSANLQDACLKQCCFIETDLRSAHLQGANLMGANLSGANLEGANLKGAKLAGTNLECANLQRAYLREVDLRETHLTGAKLGGANLLGAIR*

[0029] In some embodiments, the biological material related to the protein encoded by the rice gene OsFIP2 provided by the present invention is any one of the following A1) to A12):

[0030] A1) a nucleic acid molecule encoding an OsFIP2 gene protein;

[0031] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0032] A3) a recombinant vector containing the nucleic acid molecule described in A1);

[0033] A4) a recombinant vector containing the expression cassette described in A2);

[0034] A5) a recombinant microorganism containing the nucleic acid molecule described in A1);

[0035] A6) a recombinant microorganism containing the expression cassette described in A2);

[0036] A7) a recombinant microorganism containing the recombinant vector described in A3);

[0037] A8) a recombinant microorganism containing the recombinant vector described in A4);

[0038] A9) a transgenic plant cell line containing the nucleic acid molecule described in A1);

[0039] A10) a transgenic plant cell line containing the expression cassette described in A2);

[0040] A11) a transgenic plant cell line containing the recombinant vector described in A3);

[0041] A12) A transgenic plant cell line containing the recombinant vector described in A4).

[0042] On the other hand, the present invention relates to the use of the rice BTB domain protein encoding gene OsFIP2 in the breeding of Gramineae food crops resistant to Tenuivirus and Fijivirus;

[0043] In some embodiments, the Tenuivirus genus includes Ricestripe virus (RSV), Maize stripe virus (MSpV), preferably Ricestripe virus (RSV);

[0044] In some embodiments, the Fiji virus genus includes Maize Rough Dwarf Virus (MRDV), Rice black streaked dwarf virus (RBSDV), Southern rice black streaked dwarf virus (SRBSDV); preferably Southern rice black streaked dwarf virus;

[0045] In some embodiments, the gramineous food crops are preferably rice, corn, wheat, oats and barley; more preferably rice, and most preferably Oryza;

[0046] In another aspect, the present invention relates to a method for preparing a transgenic plant resistant to rice stripe virus and / or southern rice black-streaked dwarf virus, the steps of which include:

[0047] (1) Cloning of the rice OsFIP2 gene;

[0048] (2) Construction of overexpression vector;

[0049] (3) Agrobacterium transformation and callus induction culture;

[0050] (4) Positive identification of transgenic plants;

[0051] In some embodiments, the cloning of the rice OsFIP2 gene comprises the following steps:

[0052] 1) Design primers OsFIP2-F and OsFIP2-R based on the open reading frame (ORF) of OsFIP2

[0053] ATGGAGTCGCCGCCGGAGTCC(SEQ ID No:3);

[0054] TTATCTGATAGCTCCAAGCA(SEQ ID No:4);

[0055] 2) Carry out PCR reaction, recover PCR product, connect to pMD18-T vector, select single clone, send for testing, and obtain correct pMD18-T-OsFIP2 recombinant plasmid.

[0056] In some embodiments, the construction of the overexpression vector comprises the following steps:

[0057] 1) Design primers with GTCACC and CCGTCG restriction sites to amplify the OsFIP2 gene, and use the pMD18-T-OsFIP2 recombinant plasmid that has been sequenced and verified to be correct as a template; double-digest the pCV1300 vector with ApaI enzyme;

[0058] 2) After the PCV1300 vector is digested, the PCR products are recovered and connected to the vectors respectively; positive clones are selected and sequenced to confirm that the PCV1300-OsFIP2 overexpression vector has been successfully constructed;

[0059] The primer sequences are as follows:

[0060] PCV-OsFIP2-F CGACGACAAGACCGTCACCATGGAGTCCGCCGCCGGAGTCC (SEQ ID No: 5);

[0061] PCV-OsFIP2-R GAGGAGAAGAGCCGTCGTCTGATAGCTCCAAGCAGAT (SEQ ID No: 6);

[0062] In some embodiments, the Agrobacterium transformation and callus induction culture steps include:

[0063] (1) Add the constructed overexpression plasmid vector to the competent cells, mix well, add to the electrode cup precooled at 4°C, transform with 2200V voltage, add 500μL of LB without resistance, culture on a shaker at 28°C for 2-3h, centrifuge at 5000rpm for 1min, discard the supernatant, spread the remaining precipitate on a plate containing 50μg / ml Kan and 50μg / ml Rif, and culture in a 28°C incubator for 3d; dehull the mature rice seeds, soak them in 75% alcohol for 5min, rinse them with ddH2O, soak them in 30% sodium hypochlorite solution for 30min, and soak them in ddH2O for 30min; place the seeds in the induction medium with tweezers, culture them in a 28°C light incubator for 3 weeks, clip the grown callus tissue into the subculture medium with pre-sterilized tweezers, and subculture them in a 28°C light incubator for 1 week;

[0064] (2) Pick a single clone of GV3101 and inoculate it into LB liquid culture medium. Cultivate OD 600 = about 0.6; soak the induced callus in Agrobacterium suspension culture medium for 5 minutes; take out the callus, put it in co-culture medium, and culture it in a 26℃ light incubator for 2.5 days; grow the callus on a culture medium containing hygromycin for 30-45 days, put the selected callus in rooting medium, culture it under light to produce green plants with roots, and culture it for 2 weeks to obtain transgenic plants.

[0065] In some embodiments, the positive identification step of the transgenic plants comprises:

[0066] Total RNA of positive transgenic plants was extracted and reverse transcribed into cDNA, with the OsUBQ5 gene of rice as an internal reference; the quantitative primers are shown in SEQ ID No: 7-10:

[0067] qRT-OsFIP2-F: CGCCAACTTGAGTGGAGCTA (SEQ ID No: 7);

[0068] qRT-OsFIP2-R: GCGCAATCAACTTCTCGCA (SEQ ID No: 8);

[0069] OsUBQ5-F:ACCACTTCGACCGCCACTACT(SEQ ID No:9);

[0070] OsUBQ5-R: ACGCCTAAGCCTGCTGGTT (SEQ ID No: 10);

[0071] In some embodiments, the induction medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, pH=5.8.

[0072] The subculture culture medium comprises: 24.1 g / L N6 culture medium, 2 mg / L 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, and pH=5.8.

[0073] The co-culture medium comprises: 24.1 g / L N6 culture medium, 2 mg / L 2,4-D, 200 μmol / L acetosyringone, and pH=5.2.

[0074] The rooting medium comprises: 1 / 2MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, and pH=5.8.

[0075] On the other hand, the present invention also relates to a method for detecting resistance of rice to rice stripe virus and / or southern rice black-streaked dwarf virus, the steps of which include:

[0076] 1) The total RNA of the tested rice and the control Nipponbare rice was extracted using the TRIzol method, and the RNA was reverse transcribed into cDNA; the relative expression level of the OsFIP2 gene was detected using quantitative primers, and the quantitative primers were as follows:

[0077] qRT-OsFIP2-F: CGCCAACTTGAGTGGAGCTA (SEQ ID No: 7);

[0078] qRT-OsFIP2-R: GCGCAATCAACTTCTCGCA (SEQ ID No: 8);

[0079] OsUBQ5-F:ACCACTTCGACCGCCACTACT(SEQ ID No:9);

[0080] OsUBQ5-R: ACGCCTAAGCCTGCTGGTT (SEQ ID No: 10);

[0081] According to the above gene expression detection results, it is judged whether the rice plant to be tested is resistant rice. If the relative expression level of the OsFIP2 gene in the rice plant to be tested is significantly higher than that of the control, it is resistant rice.

[0082] The present invention uses plant transgenic technology to transform the full-length OsFIP2 gene into rice, and then screens and obtains transgenic strains with high expression levels. Experiments show that after RSV or SRBSDV infects rice, the resistance of the OsFIP2 overexpressing transgenic to virus infection is significantly increased compared with the control wild type. OsFIP2 plays a positive regulatory role in the antiviral process of rice.

[0083] The present invention mainly adopts the following scheme to achieve the above invention objectives:

[0084] The present invention takes OsFIP2 in rice as the object, constructs a recombinant expression vector pCV1300-OsFIP2 of the OsFIP2 gene, transforms the constructed expression vector into mature rice Zhonghua 11 (ZH11) embryo by using an Agrobacterium-mediated genetic transformation method, and finally induces callus formation. The selected Agrobacterium strain is GV3101. After obtaining transgenic rice seedlings, transgenic plants are identified. The expression amount of the OsFIP2 gene in rice is detected by real-time fluorescence quantitative PCR technology (qRT-PCR), and homozygous transgenic rice with stable inheritance of the T3 generation is obtained. By artificially inoculating RSV or SRBSDV, the resistance of OsFIP2 transgenic rice is evaluated. The results show that compared with the control wild type, the transgenic rice plant overexpressing OsFIP2 significantly enhances the resistance of rice to RSV or SRBSDV infection (including the symptoms of the diseased plant and the amount of virus carried). This indicates that OsFIP2 overexpression transgenic rice can significantly enhance rice resistance to RSV or SRBSDV infection. The research results not only show that OsFIP2 affects the infection efficiency of RSV or SRBSDV, but also lay the foundation for the study of rice-virus interaction.

[0085] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0086] (1) The present invention obtains stably inherited overexpressed transgenic rice OsFIP2 by overexpressing OsFIP2, and analyzes the resistance of the transgenic rice to rice stripe leaf blight and southern rice black-streaked dwarf virus, which helps to deepen our understanding and knowledge of rice viruses and plant host factors and enrich the molecular mechanism of the interaction between viruses and rice.

[0087] (2) The present invention studies the mechanism of action of OsFIP2 in virus infection, thereby discovering the interaction between plants and viruses, laying a foundation for further research on disease resistance theory. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 :Experimental results of relative expression level of OsFIP2 in OsFIP2 overexpressing transgenic rice.

[0089] Figure 2 : Comparison of symptoms between OsFIP2 overexpressing transgenic and wild type control after RSV infection.

[0090] Figure 3 : Experimental results of virus content of OsFIP2 overexpressing transgenic and wild-type control after RSV infection.

[0091] Figure 4: Experimental results of disease symptoms of OsFIP2 overexpressing transgenic and wild type control after SRBSDV infection.

[0092] Figure 5 : Experimental results of virus content of OsFIP2 overexpressing transgenic and wild type control after SRBSDV infection. DETAILED DESCRIPTION

[0093] The rice varieties used in this series of experiments are: Oryza.Sativa L.spp.japonica

[0094] The relevant culture medium components are as follows:

[0095] Induction medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., catalog number: HBZ0601)

[0096] 24.1g / L, 2.5mg / L 2,4-D, PH=5.8.

[0097] 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.

[0098] Co-culture medium: N6 culture medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2.5 mg / L 2,4-D, 200 μmol / L acetosyringone, pH = 5.8.

[0099] Rooting medium: 1 / 2MS (Manufacturer: Haibo Biotechnology Co., Ltd., Product No.: HB8469-6)

[0100] 39.45g / L, 0.5mg / L NAA, 50mg / L hygromycin, PH=5.8.

[0101] Example 1: Construction of rice OsFIP2 overexpression vector

[0102] (1) Cloning of the rice OsFIP2 gene

[0103] Primers OsFIP2-F and OsFIP2-R were designed based on the open reading frame (ORF) of OsFIP2. The primer sequences are as follows:

[0104] Table 1: Primer sequences involved in gene cloning

[0105] OsFIP2-F ATGGAGTCGCCGCCGGAGTCC(SEQ ID No:3); OsFIP2-R TTATCTGATAGCTCCAAGCA(SEQ ID No:4);

[0106] PCR amplification system: total volume 50 μL, including 2×PCR Buffer for KOD FX Neo 25 μL, upstream and downstream primers (10 μM) 1.5 μL each, dNTP Mix (2.5 mM) 5 μL, cDNA template 1 μL, KOD FX Neo 1 μL, ddH2O 15 μL.

[0107] PCR amplification program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 3 min, 40 cycles; final extension at 72°C for 10 min.

[0108] The PCR product was recovered, connected to the pMD18-T vector, single clones were selected, and sent for testing to obtain the correct pMD18-T-OsFIP2 recombinant plasmid, which was sent to Hangzhou Youkang Biological Company for sequencing. A sequence with a length of 912 bp encoding 303 amino acids was obtained.

[0109] (2) Construction of overexpression vector

[0110] Primers with BamHI and SacI restriction sites were designed to amplify OsFIP2. The pMD18-T-OsFIP2 plasmid with correct sequencing was used as the template. The pCV1300 vector was double-digested with BamHI and SacI enzymes for the construction of the OsFIP2 gene binary expression vector PCV1300. The primer sequences are as follows:

[0111] Table 2: Primer sequences involved in the construction of overexpression vectors

[0112]

[0113] PCR amplification system: The reaction system (50 μL) was as follows: 2×PCR KOD FX Neo Buffer 25 μL, 2 mM dNTPs, KOD FX Neo 1 μL, upstream primer 1.5 μL, downstream primer 1.5 μL, cDNA 1 μL, H2O 15 μL, total volume 50 μL.

[0114] PCR program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, 35 cycles; final extension at 72°C for 10 min.

[0115] After the PCV1300 vector was digested by enzymes, the PCR products were recovered and connected to the vectors respectively; positive clones were selected and sequenced to confirm that the PCV1300-OsFIP2 expression vector had been successfully constructed.

[0116] Example 2: Rice genetic transformation

[0117] (1) Agrobacterium transformation and callus induction culture: The plasmid containing the target vector was transformed into Agrobacterium GV3101. The specific steps include: taking 2 μL of plasmid and adding it to 50 μL of competent medium, mixing it, adding it to a pre-cooled electrode cup at 4°C, electroporating it with a voltage of 2200V, adding 500 μL of LB without resistance, shaking and culturing it at 28°C and 200rpm for 2-3 hours, centrifuging it at 5000rpm for 1 minute, discarding part of the supernatant, and coating the remaining precipitate on a plate containing 50 μg / ml Kan and 50 μg / ml Rif, and culturing it in an incubator at 28°C for 3 days. Mature rice seeds were shelled, soaked in 75% alcohol for 5 minutes, rinsed several times with ddH2O, soaked in 30% sodium hypochlorite solution for 30 minutes, and soaked in ddH2O for 30 minutes. Use tweezers to place the seeds into the induction medium and culture them in a 28°C light incubator for 3 weeks. Use pre-sterilized tweezers to clip the grown callus into the subculture medium and culture them in a 28°C light incubator for 1 week.

[0118] (2) Agrobacterium-transfected callus and screening and cultivation of resistant callus: A single clone of GV3101 was selected and inoculated into LB liquid culture medium, and the culture OD600 was about 0.6. The induced callus was placed in the Agrobacterium suspension culture medium and soaked for 5 min. The callus was taken out and placed in the co-culture medium and cultured in a 26°C light incubator for 2.5 days. The callus was placed on a culture medium containing hygromycin and grown for 30-45 days. The selected callus was placed in a rooting medium and cultured under light to produce green plants with roots. After culturing for 2 weeks, the OsFIP2-ox transgenic lines OsFIP2-3# and OsFIP2-4# overexpressing the OsFIP2 gene were obtained.

[0119] Example 3: Positive identification of transgenic plants

[0120] Total RNA of positive transgenic plants was extracted and reverse transcribed into cDNA, and the OsUBQ5 gene of rice was used as an internal reference. The quantitative primers are shown in SEQ ID No: 7-10. The relative expression levels of OsFIP2-ox transgenic lines OsFIP2-3# and OsFIP2-4# overexpressing the OsFIP2 gene were significantly higher than those of the control ( Figure 1 ).

[0121] Table 3: Primers involved in positive identification of transgenic plants

[0122] qRT-OsFIP2-F CGCCAACTTGAGTGGAGCTA(SEQ ID No:7); qRT-OsFIP2-R GCGCAAATCAACTTCTCGCA(SEQ ID No:8); OsUBQ5-F ACCACTTCGACCGCCACTACT(SEQ ID No:9); OsUBQ5-R ACGCCTAAGCCTGCTGGTT(SEQ ID No:10);

[0123] Example 4: Artificial inoculation of RSV

[0124] The OsFIP2 transgenic rice 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 cup and 3 biological replicates, and cultured in an artificial climate chamber at 25℃, 16h light and 8h dark. 1-2-year-old non-toxic Laodelphax striatellus was used, and after 3-5 days of infection on RSV-infected rice seedlings, it was transferred to healthy rice seedlings for the transition period (10-12 days).

[0125] The infection rate of insects was detected, and the number of insects inoculated per seedling was calculated based on the infection rate. The infected / non-infected Laodelphax striatellus was inoculated on rice seedlings at the three- to four-leaf stage (seedlings of about 15 days). All insects were swept out 3 days after infection, and the seedlings were placed in a 30℃ greenhouse for growth. After 30 days, the symptoms of the plants were observed and the disease status of the rice was determined by statistically analyzing the incidence rate. The two OsFIP2-ox transgenic lines (#3 and #4) of OsFIP2 showed obvious resistance to RSV, such as Figure 2 Further testing of the virus content showed that the virus content of the susceptible transgenic strain was significantly lower than that of the wild type, as shown in Figure 3 shown.

[0126] The sequences of the quantitative primers are as follows:

[0127] qRSV-CP-F AGGCAATCAATGACATCTCC; SEQ ID NO:11

[0128] qRSV-CP-R ATCTCTCACAAAGCCAGTGC; SEQ ID NO:12

[0129] Example 5: Artificial inoculation of SRBSDV

[0130] The OsFIP2 transgenic and control rice seed materials were soaked in a 37°C incubator for 2-3 days. After the seeds turned white, they were sown in 1L glass beakers, with 30-35 seedlings per cup and 3 biological replicates. They were cultured in an artificial climate chamber at 25°C with 16h light and 8h darkness.

[0131] The 1-2-year-old non-virus white-backed planthoppers were infected with SRBSDV for 3-5 days and then transferred to healthy rice seedlings for the transition period (10-12 days). The virus-carrying rate of the insects was tested, and the number of insects inoculated per seedling was calculated based on the virus-carrying rate. The virus-carrying / non-virus-carrying gray planthoppers were inoculated on rice seedlings at the three- to four-leaf stage (seedlings of about 15 days). All the insects were swept out 3 days after infection, and the seedlings were placed in a 30℃ greenhouse for growth. After 30 days, the disease symptoms of the plants were observed and the disease status of the rice was determined by statistically analyzing the incidence rate. The two OsFIP2-ox transgenic lines (#3 and #4) of OsFIP2 showed obvious disease resistance phenotypes to SRBSDV, such as Figure 4Further testing of the virus content showed that the virus content of the susceptible transgenic strain was significantly lower than that of the wild type, as shown in Figure 5 Therefore, the present invention successfully obtains RSV and SRBSDV infection-resistant transgenic rice through transgenic means, achieving unexpected technical effects.

[0132] The sequences of the quantitative primers are as follows:

[0133] qSRB-S2-F:CATCGACCAAGTTCAACCCG; SEQ ID NO:13

[0134] qSRB-S2-R:AAGAAGTCTGCGGGTGAAGA; SEQ ID NO:14

[0135] It should be understood that the specific examples and schemes described in the present invention are only given as examples for illustrative purposes and are not to be construed as limitations of the present invention. Various further modifications or changes within the spirit and scope of the present invention are to be considered as being included within the scope of the disclosure of the present invention.

Claims

1. An application of a rice protein OsFIP2 containing a BTB domain in breeding of gramineous food crops resistant to Fiji virus and / or Tenuivirus; the nucleotide sequence of the OsFIP2 gene is shown in SEQ ID No:

1.

2. The use according to claim 1, wherein the amino acid sequence of the OsFIP2 gene is shown in SEQ ID No:

2.

3. The use according to any one of claims 1 to 2, wherein the Fiji virus genus includes Maize Rough Dwarf Virus (MRDV), Rice black streaked dwarf virus (RBSDV), and Southern rice black streaked dwarf virus (SRBSDV); preferably Southern rice black streaked dwarf virus.

4. The use according to any one of claims 1 to 3, wherein the Tenuivirus genus includes Rice stripe virus (RSV) and Maize stripe virus (MSpV); preferably Rice stripe virus.

5. The use according to any one of claims 1 to 4, wherein the gramineous crops are selected from rice, corn, wheat, oats and barley; preferably rice, more preferably japonica rice varieties such as Huaidao No. 5, Nipponbare, Wuyujing No. 3, and most preferably Nipponbare.

6. A method for preparing resistant transgenic rice, comprising the following steps: transferring an OsFIP2 overexpression vector into a starting rice to obtain resistant transgenic rice, wherein the resistant transgenic rice has resistance to Rice stripe virus (RSV) and Southern rice black-streaked dwarf virus (SRBSDV) compared with the starting rice, and the nucleotide sequence of the OsFIP2 gene is shown in SEQ ID No:

1.

7. A method for identifying rice resistant to rice stripe virus and / or southern rice black-streaked dwarf virus, comprising the following steps: extracting total RNA from the rice plant to be tested, reverse transcribing it into cDNA, using the OsUBQ5 gene of rice as an internal reference, using OsFIP2 and OsUBQ5 specific primers for detection, and judging whether the rice plant to be tested is resistant rice based on the gene expression detection results.

8. The method of claim 7, wherein the specific primer sequence is as follows: qRT-OsFIP2-F CGCCAACTTGAGTGGAGCTA(SEQ ID No:7) qRT-OsFIP2-R GCGCAAATCAACTTCTCGCA(SEQ ID No:8) OsUBQ5-F ACCACTTCGACCGCCACTACT(SEQ ID No:9) OsUBQ5-R ACGCCTAAGCCTGCTGGTT (SEQ ID No: 10).

9. The method according to any one of claims 7-8, wherein the determination method is: if the relative expression level of the OsFIP2 gene in the rice plant to be detected is significantly higher than that in the control, the rice plant is resistant rice.

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