Application of rice transcription factor chaperonin TFDP in resisting rice stripe virus

Abstract

The invention relates to the technical field of plant disease prevention and control and transgenosis, and mainly relates to a rice OsTFDP gene and application thereof in rice stripe virus resistance of rice, and after an OsTFDP vector is transferred into plant rice, a stably inherited OsTFDP-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 OsTFDP gene and an application thereof in rice resistance to rice stripe virus. Background Art

[0002] Rice is the main food source for more than half of the world's population. With the continuous growth of the global population, the demand for food production is also increasing. Rice is infected by different plant pathogens during its growth, which seriously affects the quality and yield of rice. Rice stripe virus (RSV) is currently considered to be one of the most important rice viral pathogens in East Asia due to the increase in planting area, narrow genetic basis of varieties and susceptibility to debilitating diseases. Under natural conditions, RSV can infect several plants in the Poaceae family, including rice (Oryza sativa L.), wheat (Triticumaestivum L.), barley (Hordeum vulgare L.), oats (Avena sativa L.), millet (Setariaitalica L.), sorghum (Sorghum bicolor L.) and other important crops. After RSV infects rice, the plants usually show symptoms such as chlorosis, discontinuous yellow stripes, necrosis of new leaves and growth retardation. The vector of rice stripe virus is the gray leafhopper, which reproduces in insects in a persistent cycle and can also be transmitted through eggs. It not only carries the virus for life, but also can pass it on from generation to generation. Rice stripe virus RSV belongs to the genus Tenuivirus. Its genome consists of 4 single-stranded RNAs with a total size of about 17kb. They are named RNA1, RNA2, RNA3 and RNA4 according to their size, encoding a total of 7 proteins. Each viral protein has a different function to promote the infection and replication of the virus.

[0003] The cell cycle regulatory transcription factor DP-1 (TFDP-1) is a member of the E2F family, which includes three DP subunits and eight E2F subunits. Both typical and atypical E2F transcription factors are key components of the cell cycle protein D / RB / E2F pathway. Typical E2F proteins have a homologous DNA binding domain and form heterodimers with the dimerization part (DP) protein, binding to DNA through a leucine zipper in the dimerization region. In contrast, atypical E2F / DEL factors do not interact with DPs due to the lack of a dimerization region. However, they have a homologous DNA binding domain for replication, allowing them to bind to DNA autonomously. Yeast uses E2F homologs MCB-binding factor (MBF) and scbinding factor (SBF) proteins to regulate G1 / S transition and cell proliferation, and are widely used as an in vivo model to study cell cycle response mechanisms. It is reported that in Arabidopsis, auxin response proteins ARF7 and ARF19 can regulate DPa transcription, while protein kinase MAPK3 / 6 can phosphorylate DPa, increase DPa stability, maintain the characteristics of distal root stem cells, and promote plant growth. These studies have shown that TFDP proteins play an important role in the plant life cycle. However, there are currently no reports on the relationship between rice stripe virus and TFDP proteins.

[0004] At present, once rice virus disease occurs, there is no cure, and the only option is to burn the entire diseased plant or diseased area. Therefore, agriculture generally adopts a prevention-oriented control method, by spraying chemical agents to kill the virus vectors for control. Although chemical pesticides are highly efficient, long-term use will cause serious environmental pollution, 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 prevention and control of rice virus diseases. Based on current research, cultivating virus-resistant rice plant strains is currently the most effective means of preventing and controlling rice virus diseases. Summary of the invention

[0005] Based on the defects and shortcomings of the prior art, the present invention relates to a rice E2F transcription factor partner gene OsTFDP and its application in resistance to rice stripe virus;

[0006] In one aspect, a rice E2F transcription factor chaperone protein TFDP encoding gene OsTFDP, the nucleotide and protein sequences of which are as follows: The nucleotide sequence of OsTFDP:

[0007] SEQ ID NO:1

[0008] ATGGCGCCTCCCTGCGGCGATGCCGCGGCGGCTGCCTCCGCCGCGCCCGGCCTGGCCAACCTGCTCA

[0009] TTCGCGAAGGCGCCGGCCTCCCTTCGCGTCCCGAACGGTACCCGCCATTCCGGCCCTGTACTTCTGAT

[0010] TCCTTTGCTCCAATCTCTAGGGAAGGGGACGATATTCCCCCCCAAAAAAAATCGGTTTCTTTACGCAG

[0011] CGGCGGCGGCGGCAATGCGGCAGAGAGGGAGGAGGGCGGTGCGAACAGGAACGGGAAGAAGGAG

[0012] AAGACCGGGGCGCAGAGGATCACCGGGTGGGGGCTCCGTGAGTTCAGCAAGATAGTTTCTAAGAAA

[0013] GTTGAGGCCAAAGGAAGAACCACATATAATGAGGTTGCCGATGAGATTTTTGCGGAGCTGAAGTCCA

[0014] TTACGCAGAACGGTCTGGAGTTTGATGAGAAGAATATTAGGCGGAGGGTATATGATGCTTTCAATGTG

[0015] CTCATTGCAATTCGTGTTATTGCAAAAGATAAAAAGGAGATAAAGTGGATGGGCCTTACTAATTATAGA

[0016] TACGAAAAGATACAGAAGTTGGAGGAAGTTCACAAAGAACTCATCACCAGGATCAAGAATAAGAAG

[0017] AAGCTTCTCCAGGAAATTGAAAAGCAGTTTGATGACCTTCAGAATATTACATTACGCAACCAGGCTAG

[0018] TCAGAGGCCAGCAGAAAGTGTTAATGGCATCCTCCTTCCGTTCTTATTGATCAAGACATCCCGAAAAG

[0019] CAAGGGTGGAAATTGAGATTTCGGAAGATTCGAAGTTTGCACGGTTCGACTTCAACGGTGCACCATT

[0020] CACCATGCATGATGATGTATCAATCCTTGAAGCCATCAGGCGTAACAAAGGAAGAGCTGGCCTCTCCA

[0021] TTCACCCTTAA

[0022] Codon-optimized nucleotide sequence:

[0023] SEQ ID NO:2

[0024] ATGGCACCTCCATGTGGTGATGCAGCTGCAGCAGCCTCAGCAGCTCCAGGGCTGGCAAATTTGCTGAT

[0025] CAGGGAGGGGGCTGGACTTCCTTCAAGACCAGAGCGTTACCCTCCCTTCCGTCCATGCACCTCAGAC

[0026] TCATTTGCTCCCATATCTCGGGAAGGAGATGACATTCCACCTCAGAAGAAGAGTGTAAGTCTTAGAAG

[0027] CGGTGGCGGCGGAAATGCTGCGGAGAGAGAGGAAGGTGGTGCCAATAGAAATGGGAAGAAGGAGA

[0028] AGACTGGAGCTCAAAGGATAACAGGATGGGGTCTCCGAGAGTTCAGCAAGATTGTCTCCAAGAAAGT

[0029] TGAAGCTAAGGGAAGAACTACATATAATGAGGTTGCTGATGAGATATTTGCAGAATTGAAGAGCATAA

[0030] CGCAGAATGGTCTTGAATTTGATGAAAAGAATATACGTCGGAGAGTTTATGATGCTTTTAATGTTTTAA

[0031] TTGCTATCAGAGTGATTGCAAAAGATAAGAAAGAAATAAAATGGATGGGATTAACCAACTACAGGTAT

[0032] GAAAAAATCCAGAAATTGGAAGAAGTACATAAAGAGCTGATAACTAGAATTAAAAACAAAAAAAAG

[0033] CTCCTTCAAGAAATTGAGAAACAATTTGACGACTTACAAAATATTACTTTGCGAAACCAAGCATCTCA

[0034] GCGACCTGCTGAGAGTGTCAATGGAATTTTGTTACCATTTCTTCTCATCAAGACATCAAGGAAAGCAC

[0035] GCGTGGAAATTGAAATCTCTGAAGATTCTAAATTCGCCAGATTTGATTTCAACGGGGCGCCGTTCACA

[0036] ATGCATGATGATGTTTCGATTCTAGAAGCAATTAGGAGGAACAAGGGTCGTGCAGGCCTATCCATTCA

[0037] CCCTTGA

[0038] Amino acid sequence of OsTFDP:

[0039] SEQ ID NO:3

[0040] MAPPCGDAAAAASAAPGLANLLIREGAGLPSRPERYPPFRPCTSDSFAPISREGDDIPPQKKSVSLRSGGG

[0041] GNAAEREEGGANRNGKKEKTGAQRITGWGLREFSKIVSKKVEAKGRTTYNEVADEIFAELKSITQNGLEF

[0042] DEKNIRRRVYDAFNVLIAIRVIAKDKKEIKWMGLTNYRYEKIQKLEEVHKELITRIKNKKKLLQEIEKQFD

[0043] DLQNITLRNQASQRPAESVNGILLPFLLIKTSRKARVEIEISEDSKFARFDFNGAPFTMHDDVSILEAIRRNKGRAGLSIHP*

[0044] In some embodiments, the biological material related to the protein encoded by the rice gene TFDP provided by the present invention is any one of the following A1) to A12):

[0045] A1) a nucleic acid molecule encoding an OsTFDP gene protein;

[0046] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0047] A3) a recombinant vector containing the nucleic acid molecule described in A1);

[0048] A4) a recombinant vector containing the expression cassette described in A2);

[0049] A5) a recombinant microorganism containing the nucleic acid molecule described in A1);

[0050] A6) a recombinant microorganism containing the expression cassette described in A2);

[0051] A7) a recombinant microorganism containing the recombinant vector described in A3);

[0052] A8) a recombinant microorganism containing the recombinant vector described in A4);

[0053] A9) a transgenic plant cell line containing the nucleic acid molecule described in A1);

[0054] A10) a transgenic plant cell line containing the expression cassette described in A2);

[0055] A11) a transgenic plant cell line containing the recombinant vector described in A3);

[0056] A12) A transgenic plant cell line containing the recombinant vector described in A4).

[0057] On the other hand, the present invention relates to the use of the rice E2F transcription factor chaperone protein TFDP encoding gene OsTFDP in the breeding of Tenuivirus-resistant Gramineae food crops;

[0058] In some embodiments, the Tenuivirus includes rice stripe virus (RSV) and maize stripe virus (MSpV), preferably rice stripe virus (RSV);

[0059] In some embodiments, the gramineous crops are preferably rice, corn, wheat, oats and barley; more preferably rice, and most preferably Hordeum vulgare 11;

[0060] In another aspect, the present invention relates to a method for preparing a rice stripe virus resistant transgenic plant, the steps comprising:

[0061] (1) Cloning of the rice OsTFDP gene;

[0062] (2) Construction of overexpression vector;

[0063] (3) Agrobacterium transformation and callus induction culture;

[0064] (4) Positive identification of transgenic plants;

[0065] In some embodiments, the cloning of the rice OsTFDP gene comprises the following steps:

[0066] 1) Design primers OsTFDP-F and OsTFDP-R based on the optimized OsTFDP open reading frame (ORF)

[0067] ATGGCACCTCCATGTGGTG(SEQ ID NO:4);

[0068] TCAAGGGTGAATGGATAGGC (SEQ ID NO:5);

[0069] 2) Perform PCR amplification, recover PCR products by gel, connect to pMD18-T vector, pick single clones, send for testing, and identify to obtain pMD18-T-OsTFDP recombinant plasmid.

[0070] In some embodiments, the construction of the overexpression vector comprises the following steps:

[0071] 1) Design primers with GTCACC and CCGTCG restriction sites to amplify the OsTFDP gene, and use the pMD18-T-OsTFDP recombinant plasmid that has been sequenced and verified to be correct as a template; double-digest the pCV1300 vector with ApaI enzyme;

[0072] 2) After digesting the pCV1300 vector, the above PCR product was connected to the vector; positive clones were selected and sent to a biological company for sequencing to confirm that the pCV1300-OsTFDP overexpression vector had been successfully constructed;

[0073] The primer sequences are as follows:

[0074] pCV-OsTFDP-F CGACGACAAGACCGTCACCATGGCACCTCCATGTGGTG (SEQ ID NO: 6);

[0075] pCV-OsTFDP-R GAGGAGAAGAGCCGTCGAGGGTGAATGGATAGGC (SEQ ID NO: 7);

[0076] In some embodiments, the Agrobacterium transformation and callus induction culture steps include:

[0077] (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 700μL of LB without resistance, shake and incubate at 28°C, centrifuge at 5000rpm for 1min, discard the supernatant, resuspend the remaining supernatant and precipitate on the LB solid medium plate containing Kan and Rif, and incubate in a 28°C incubator for 2d; dehull the fully mature rice seeds, soak in 75% alcohol for 5min, rinse with sterile water, add 30% sodium hypochlorite for disinfection for 20min, and finally rinse thoroughly with sterile water for 5 to 6 times. The disinfected seeds are transferred to the induction medium and cultured in a 28°C light incubator for 3 weeks. When callus tissue grows, use pre-sterilized tweezers to clamp it into the subculture medium and culture it in a 28°C light incubator for 1 week.

[0078] (2) Pick a GV3101 clone transformed with the pCV-OsTFDP plasmid and inoculate it into LB liquid medium and culture until OD 600 = about 0.6; soak the induced callus in Agrobacterium suspension for 5 minutes; transfer the callus to symbiotic culture medium and culture it in a 26°C light incubator for 2.5 days; grow the callus on a culture medium containing hygromycin for 30-45 days, transfer the selected callus to rooting culture medium, culture it under light until the callus grows roots, and obtain transgenic plants.

[0079] In some embodiments, the positive identification step of the transgenic plants comprises:

[0080] 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:

[0081] qRT-OsTFDP-F:GAAGGAGATGACATTCCACC (SEQ ID NO:8);

[0082] qRT-OsTFDP-R: CTCCAGTCTTCTCCTTCTTC (SEQ ID NO: 9);

[0083] OsUBQ5-F:ACCACTTCGACCGCCACTACT(SEQ ID NO:10);

[0084] OsUBQ5-R: ACGCCTAAGCCTGCTGGTT (SEQ ID NO: 11);

[0085] In some embodiments, the induction medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, pH=5.8.

[0086] 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.

[0087] 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.

[0088] The rooting medium comprises: 1 / 2MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, and pH=5.8.

[0089] In another aspect, the present invention also relates to a method for detecting resistance of rice to rice stripe virus, the steps of which include:

[0090] 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 OsTFDP gene was detected using quantitative primers, and the quantitative primers were as follows:

[0091] qRT-OsTFDP-F:GAAGGAGATGACATTCCACC (SEQ ID NO:8);

[0092] qRT-OsTFDP-R: CTCCAGTCTTCTCCTTCTTC (SEQ ID NO: 9);

[0093] OsUBQ5-F:ACCACTTCGACCGCCACTACT(SEQ ID NO:10);

[0094] OsUBQ5-R: ACGCCTAAGCCTGCTGGTT (SEQ ID NO: 11);

[0095] 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 OsTFDP gene in the rice plant to be tested is significantly higher than that of the control, it is resistant rice.

[0096] The present invention uses plant transgenic technology to transform the full-length OsTFDP gene into rice, and then screens and obtains transgenic strains with high expression levels. Experiments show that after RSV infects rice, the resistance of the OsTFDP overexpressing transgenic to virus infection is significantly increased compared with the wild type control. OsTFDP plays a positive regulatory role in the process of rice resistance to rice stripe virus.

[0097] The present invention mainly adopts the following scheme to achieve the above invention objectives:

[0098] The present invention takes OsTFDP in rice as the object, constructs a recombinant expression vector pCV1300-OsTFDP, and transforms the constructed expression vector into rice Zhonghua 11 (ZH11) seeds by using an Agrobacterium-mediated genetic transformation method to induce callus formation. The selected Agrobacterium strain is GV3101. After obtaining transgenic rice seedlings, positive transgenic plants are identified. The expression amount of the OsTFDP 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, the resistance of OsTFDP transgenic rice is evaluated. The results show that compared with the wild-type control, the transgenic rice plants overexpressing OsTFDP significantly enhance the resistance of rice to RSV (including the symptoms of the diseased plants and the amount of virus). This shows that OsTFDP overexpressing transgenic rice can significantly enhance the resistance of rice to RSV infection. The research results not only show that OsTFDP affects the infection efficiency of RSV, but also lay the foundation for the study of the interaction between rice and viruses.

[0099] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0100] (1) The present invention obtains stably inherited transgenic rice overexpressing OsTFDP by overexpressing OsTFDP, and analyzes the resistance of the transgenic rice to rice stripe virus, which helps to deepen our understanding and knowledge of rice viruses and plant host factors and analyze the molecular mechanism of rice resistance to virus infection.

[0101] (2) The present invention studies the mechanism of action of OsTFDP in viral infection and discovers the interaction between plant host factors and viral proteins, laying a foundation for further studying the mechanism of plant and viral defense and counter-defense. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 :Experimental results of relative expression level of OsTFDP in OsTFDP overexpressing transgenic rice.

[0103] Figure 2 : Comparison of symptoms between OsTFDP overexpressing transgenic and wild type control after RSV infection.

[0104] Figure 3 : Experimental results of virus content of OsTFDP overexpressing transgenic and wild type control after RSV infection. DETAILED DESCRIPTION

[0105] The rice varieties used in this series of experiments are: Zhonghua 11 (ZH11)

[0106] The relevant culture medium components are as follows:

[0107] Induction medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., catalog number: HBZ0601)

[0108] 24.1g / L, 2.5mg / L 2,4-D, PH=5.8.

[0109] 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.

[0110] 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.

[0111] Rooting medium: 1 / 2MS (Manufacturer: Haibo Biotechnology Co., Ltd., Product No.: HB8469-6)

[0112] 39.45g / L, 0.5mg / L NAA, 50mg / L hygromycin, PH=5.8.

[0113] Example 1: Construction of rice OsTFDP overexpression vector

[0114] (1) Cloning of the rice OsTFDP gene

[0115] Primers OsTFDP-F and OsTFDP-R were designed based on the optimized OsTFDP gene open reading frame (ORF). The primer sequences are as follows:

[0116] Table 1: Primer sequences involved in gene cloning

[0117] OsTFDP-F ATGGCACCTCCATGTGGTG(SEQ ID NO:4); OsTFDP-R TCAAGGGTGAATGGATAGGC (SEQ ID NO:5);

[0118] 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, ddH 2 O 15 μL.

[0119] 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.

[0120] The PCR product was recovered, connected to the pMD18-T vector, transformed into Escherichia coli, spread on LB solid culture medium plates containing 50 mg / mL ampicillin, cultured at 37°C for 24 hours, single clones were picked, and sent to a biological company for sequencing to obtain the correct pMD18-T-OsTFDP recombinant plasmid.

[0121] (2) Construction of overexpression vector

[0122] Primers with BamHI and SacI restriction sites were designed to amplify OsTFDP. The pMD18-T-OsTFDP 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 OsTFDP gene binary expression vector PCV1300. The primer sequences are as follows:

[0123] Table 2: Primer sequences involved in the construction of overexpression vectors

[0124]

[0125] PCR amplification system: The reaction system (50 μL) is 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, H 2 O 15 μL, total volume 50 μL.

[0126] 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.

[0127] After the pCV1300 vector was digested with enzymes, the PCR product was recovered by gel and connected to the vector; Escherichia coli was transformed, positive clones were selected, and sent to a biological company for sequencing to confirm that the pCV1300-OsTFDP expression vector had been successfully constructed.

[0128] Example 2: Rice genetic transformation

[0129] (1) Agrobacterium transformation and callus induction culture: The pCV1300-OsTFDP expression vector plasmid was transformed into Agrobacterium GV3101. The specific steps include: taking 2-5 μL of plasmid and adding it to 100 μL of competent medium, gently mixing, transferring to an electrode cup precooled at 4°C, electroporating with 2200V voltage, adding 700 μL of LB liquid medium without resistance, shaking and culturing at 28°C, 200rpm for 2-3h, centrifuging at 5000rpm for 1min, discarding part of the supernatant, resuspending the remaining supernatant and the precipitate, coating on a LB solid medium plate containing 50 μg / ml Kan and 50 μg / ml Rif, and culturing in a 28°C incubator for 3d.

[0130] The rice seeds were dehulled and soaked in 75% alcohol for 5 min. 2 O rinse 3-4 times, soak in 30% sodium hypochlorite solution for 30 min, and ddHO 2 O for 30 minutes. Use tweezers to place the seeds in the induction medium and culture them at 28℃ for 3 weeks. Cut the grown callus and clamp it into the subculture medium with pre-sterilized tweezers. Subculture them at 28℃ for 1 week in a light incubator.

[0131] (2) Screening and culture of Agrobacterium-transfected callus and resistant callus: A single clone of GV3101 was selected and inoculated into LB liquid culture medium and cultured to a concentration of about OD600 = 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 a co-culture medium and cultured in a 26°C light incubator for 2-3 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 OsTFDP-ox transgenic lines OsTFDP-2# and OsTFDP-7# overexpressing the OsTFDP gene were obtained.

[0132] Example 3: Positive identification of transgenic plants

[0133] 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:8-11. The relative expression levels of OsTFDP-ox transgenic lines OsTFDP-2# and OsTFDP-7# overexpressing the OsTFDP gene were significantly higher than those of the control ( Figure 1 ).

[0134] Table 3: Primers involved in positive identification of transgenic plants

[0135] qRT-OsTFDP-F GAAGGAGATGACATTCCACC(SEQ ID NO:8); qRT-OsTFDP-R CTCCAGTCTTCTCCTTCTTC(SEQ ID NO:9); OsUBQ5-F ACCACTTCGACCGCCACTACT(SEQ ID NO:10); OsUBQ5-R ACGCCTAAGCCTGCTGGTT (SEQ ID NO: 11);

[0136] Example 4: Artificial inoculation of RSV

[0137] The OsTFDP transgenic rice and control rice seed materials were placed in a 37°C incubator for 2-3 days. After the seeds germinated, 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°C, 16h light and 8h dark. 1-2-year-old non-toxic gray planthoppers were used, and after 3-5 days of infection on RSV-infected rice seedlings, they were transferred to healthy rice seedlings for the transition period (10-12 days).

[0138] 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 plants 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 OsTFDP-ox transgenic lines (#3 and #4) 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.

[0139] The sequences of the quantitative primers are as follows:

[0140] qRSV-CP-F AGGCAATCAATGACATCTCC; SEQ ID NO:12

[0141] qRSV-CP-R ATCTCTCACAAAGCCAGTGC; SEQ ID NO:13

[0142] In summary, the experimental results show that compared with wild-type Zhonghua 11 rice, the transgenic rice plants overexpressing OsTFDP are more resistant to RSV infection. Therefore, the present invention successfully obtained RSV infection-resistant transgenic rice through transgenic means, adding valuable genetic resources to the rice germplasm resource bank, which is of great significance for the cultivation of resistant plants.

Claims

1. An application of a rice E2F transcription factor chaperone protein TFDP in the breeding of Gramineae food crops resistant to Tenuivirus; the nucleotide sequence of the rice E2F transcription factor chaperone protein TFDP encoding gene: OsTFDP gene is shown in any one of SEQ ID NO: 1-SEQ ID NO:

2.

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

3.

3. The use according to any one of claims 1 to 2, wherein the Tenuivirus genus includes Rice stripe virus (RSV) and Maize stripe virus (MSpV); preferably Rice stripe virus (RSV).

4. The use according to any one of claims 1 to 3, wherein the gramineous crops are selected from rice, corn, wheat, oats and barley; preferably rice, more preferably Zhonghua 11, Huaidao No. 5, Nipponbare, Wuyujing No. 3, and the most preferred is Zhonghua 11.

5. The use according to any one of claims 1 to 4, wherein the Gramineae crop is rice.

6. A method for preparing resistant transgenic rice, comprising the following steps: transferring an OsTFDP gene overexpression vector into a starting rice to obtain resistant transgenic rice, wherein the resistant transgenic rice has resistance to rice stripe virus (RSV) compared with the starting rice, and the nucleotide sequence of the OsTFDP gene is shown in any one of SEQ ID NOs: 1-2.

7. A method for identifying rice resistant to rice stripe 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 OsTFDP 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 according to claim 7, wherein the specific primer sequence is as follows: qRT-OsTFDP-F CGCCAACTTGAGTGGAGCTA(SEQ ID NO:8) qRT-OsTFDP-R GCGCAAATCAACTTCTCGCA(SEQ ID NO:9) OsUBQ5-F ACCACTTCGACCGCCACTACT(SEQ ID NO:10) OsUBQ5-R ACGCCTAAGCCTGCTGGTT (SEQ ID NO: 11).

9. The method according to any one of claims 7-8, wherein the determination method is: if the relative expression level of the OsTFDP 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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