Application of histone deacetylase gene in resisting rice stripe virus

Abstract

The invention relates to a rice histone deacetylase coding gene and application thereof in resisting rice stripe virus.

Description

Technical Field

[0001] The invention relates to the field of transgenic technology and plant disease prevention and control, and in particular to a rice histone deacetylase encoding gene OsHDA706 and an application thereof in resistance to rice stripe virus. Background Art

[0002] Rice stripe virus (RSV) is a typical representative of the genus Tenuivirus. Its virus particles are non-enveloped filaments with a diameter of 3-8nm and varying lengths. It is mainly transmitted by the gray leafhopper (Laodelphaxstriatellus), causing serious losses to my country's rice production. RSV virus is a negative-sense single-stranded RNA (-ss RNA) virus. Its genome is named RNA1, RNA2, RNA3 and RNA4 in descending order of molecular weight, encoding 7 proteins. Among them, the largest RNA1 encodes a 337kDa RNA-dependent RNA polymerase on the complementary strand. The other three RNAs each have a bidirectional coding strategy, with the open reading frame at the 5' end of the RNA encoding proteins P2-P4, and the ORF on the complementary strand encoding proteins pc2-pc4. PC2 participates in vector transmission by overcoming insect barriers. The P2 protein functions as a silencing suppressor and can target key modules of the jasmonic acid (JA) pathway to inhibit the JA pathway. PC3 is a nucleocapsid protein and is also believed to play a key role in the spread of rice stripe virus through insect ovaries. P3 is a silencing suppressor protein that competes with host proteins for double-stranded and single-stranded siRNA to prevent the formation of silencing complexes. PC4 (32 kDa) is mainly distributed near or within the cell wall and can support the intercellular transport of the movement-defective potato virus X, so it is considered to be a movement protein. P4 is a non-structure-specific protein.

[0003] Under natural conditions, rice stripe virus (RSV) only infects grasses, but under laboratory conditions, rice stripe virus can infect Nicotiana benthamiana through mechanical inoculation. Rice infected with rice stripe virus usually shows yellowing and necrosis on newly developed leaves, followed by plant stunting, heading deformity and few fruits, which can lead to death in severe cases. However, most of the disease-resistant rice varieties currently promoted in production have the problems of few resistance genes and single loci. In addition, the use of chemical agents will cause pesticide residue problems. Therefore, the most economical and effective measure to prevent and control rice stripe leaf blight is to cultivate resistant varieties. In recent years, more and more transgenic rice resistant materials have provided important theoretical and practical significance for crop disease resistance breeding.

[0004] Jasmonic acid (JA) and its derivatives are a class of lipid-based oxygen-containing hormones that are ubiquitous in the plant kingdom. They play key roles in plant development and responses to abiotic stresses and pathogens. Numerous studies have shown that JA actively regulates plant antiviral defense in Arabidopsis and rice. As a natural antiviral defense system in rice, rewiring of the JA response network has been proposed for engineering plant disease resistance. However, manipulation of specific components of the JA pathway may increase resistance to one pathogen or pest, but when the balance of hormone signaling in plants is altered, it often leads to increased susceptibility to other pathogens or pests. Therefore, in areas susceptible to damage by specific insects or pathogens, the use of specially bred varieties combined with disease prediction may be a useful strategy for future disease control. In addition, further research work is needed to determine how to achieve broad-spectrum resistance to pathogens.

[0005] Histone deacetylases (HDACs) are a class of proteases that play an important role in the structural modification of chromosomes and the regulation of gene expression. In general, the acetylation of histones is conducive to the dissociation of DNA from the histone octamer, resulting in the relaxation of the nucleosome structure, so that various transcription factors and co-transcription factors can specifically bind to the DNA binding site and activate gene transcription. However, the deacetylation of histones plays the opposite role. The basic unit of the nucleosome is the histone octamer (H2A, H2B, H3 and H4) and the DNA molecule. The histone H3 binding sites are K9, K14, K18 and K23, and the histone H4 binding sites are K5, K8, K12, K16 and K20. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the acetylation and deacetylation of histones, respectively, to regulate gene expression. This process is crucial for regulating various plant cell processes, including stress response, hormone signaling, and growth and development. In addition to histones, an increasing number of studies have shown that acetylation and deacetylation of non-histone proteins also play an important role in various cellular processes in eukaryotes.

[0006] The rice genome contains at least 19 HDACs genes, and except for a few genes, the functions of most genes have not been studied. For example, OsHDA714 deacetylates ribosomal-associated protein and OsHDA7 14 knockout mutant increases the frequency of rice ribosome arrest; OsHDA701 negatively regulates rice innate immunity by regulating histone H4 acetylation levels and the expression of defense genes (Li et al., 2021). However, the role of OsHDA706 in resistance to rice stripe virus infection remains unclear.

[0007] Based on the above-mentioned defects of the prior art, the present invention constructs an overexpression vector of OsHDA706, and introduces it into a rice variety (Nipponbare) by Agrobacterium-mediated method to obtain two transgenic rices that can be stably inherited, respectively named OsHDA706-OE#1 and OsHDA706-OE#2. The content of jasmonic acid is determined by liquid chromatography-tandem mass spectrometry, and it is found that overexpression of OsHDA706 gene in rice can significantly increase the jasmonic acid content in rice. By artificial inoculation of rice stripe virus (RSV), it is found that overexpression of OsHDA706 gene in rice can significantly enhance the resistance of rice to RSV infection, which is manifested as reduced symptoms of plant disease, reduced virus content, etc. The present invention provides new ideas for breeding resistant plants for rice stripe virus and breeding new rice antiviral varieties, and provides technical guarantee for ensuring the continuous increase in production of agricultural rice crops in my country. Summary of the invention

[0008] The invention relates to a rice histone deacetylase encoding gene OsHDA706 and an encoded protein thereof.

[0009] On the one hand, the nucleotide sequence of the OsHDA706 gene is shown in SEQ ID NO.1.

[0010] On the other hand, the amino acid sequence of the protein encoded by the OsHDA706 gene is shown in SEQ ID NO.2.

[0011] In some embodiments, the CDS sequence of the rice histone deacetylase encoding gene OsHDA706 (SEQ ID NO: 1) is as follows:

[0012]

[0013] The amino acid sequence of the rice histone deacetylase encoding gene OsHDA706 (SEQ ID NO: 2) is as follows:

[0014] MASSAPSAAAGATPPDPLRRDRILSSKLYLDVPGSKAPVVYSPAYDIAFLGIEKLHPFDSSKWGRICKFLTKEGHLEKNRVVEPLEATKDDLLVVHSESYLNSLKSSLKVASIVELPPVAFIPNWLVQQKLLYPFRKQVGGSILSAKLALERGWAINVGGGFHHCSAEQGGGFCAY ADISLCIQFAFVRLNISRVMIIDLDAHQGNGHEKDFANDGRVYTLDMYNAGIYPYDHVAKRYIDQKVELVSGTKTEDYLDQLDKALKVAESRFQPQLIVYNAGTDILDGDPLGRLKISPQGVVIRDEKVFRFAKDQSIPLLMLTSGGYMKSSARVIADSIINLSNKNLIELGSQLG.

[0015] On the other hand, the present invention relates to the use of the rice histone deacetylase encoding gene OsHDA706 in the breeding of Tenuivirus-resistant Gramineae food crops.

[0016] In some embodiments, the Tenuivirus is preferably Rice stripe virus (RSV) or Maize stripe virus (MSpV); most preferably Rice stripe virus.

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

[0018] In another aspect, the present invention relates to a method for preparing a rice stripe virus resistant transgenic plant, the steps comprising:

[0019] 1) Construction of rice OsHDA706 expression vector;

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

[0021] 3) Identification of positive transgenic plants;

[0022] In some embodiments, the rice OsHDA706 expression vector construction step further comprises:

[0023] Primers were designed according to the sequence of OsHDA706 shown in SEQ ID No: 1, and the full-length sequence of OsHDA706 was cloned from rice cDNA. The PCR product was recovered from gel; the pCAMBIA1300 vector containing the MYC tag was digested with BamHI and SacI, and the above PCR products were connected to the vectors respectively, positive clones were selected, and sequencing was performed to confirm that the pCAMBIA1300-OsHDA706 expression vector had been successfully constructed.

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

[0025] Callus induction: Select mature rice seeds, peel off the husk, pour into a centrifuge tube, add 75% ethanol to disinfect for 1 minute, pour out the ethanol, rinse with sterile water once, pour out, add 30% sodium hypochlorite to disinfect for 20 minutes, pour out the sodium hypochlorite and rinse with sterile water 5-6 times; use a pipette to absorb excess water, transfer the seeds to the induction medium, place in a 28°C light incubator for 3 weeks, clip the grown callus with pre-sterilized tweezers to the subculture medium, and subculture in a 28°C light incubator for 1 week;

[0026] Transformation and culture of Agrobacterium: The plasmid containing the target vector was transformed into Agrobacterium tumefaciens GV3101 using the following steps: 5 μL of plasmid was added to 100 μL of Agrobacterium GV3101 competent medium, mixed by pipetting, added to a sterile electrode cup precooled at 4°C, and transformed by electric shock at 220 V, added to resistant LB liquid culture medium, and cultured on a constant temperature shaker at 28°C for about 3 hours, evenly spread on LB solid culture 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] Agrobacterium infection: Prepare the infection solution, use a pipette to absorb the infection solution to wash off the Agrobacterium on the plate, which is the Agrobacterium suspension for co-culture and rice transformation. Select a sufficient number of callus tissues, put them into a 100ml sterile triangular flask, add an appropriate amount of Agrobacterium suspension, and infect at room temperature for 20 minutes, shaking from time to time. Pour out the bacterial solution, place the callus tissue on a sterile filter paper to absorb the excess bacterial solution, and then transfer it to a solid co-culture medium covered with a layer of sterile filter paper, and culture it in the dark at 26℃ for 3 days.

[0028] Screening and culture: After 3 days of co-culture, the callus tissue needs to be cleaned. Use a 1ml blue pipette tip to sow the callus on the co-culture medium into a sterilized conical flask, add sterile water to rinse both sides, and rinse once with sterile water containing 500ul / L carbenicillin for the third time. After the excess water is sucked off by a pipette, the callus is transferred to sterile filter paper and the water on the callus is blown dry by the wind from the clean bench. The blowing time is controlled at about 30min. After the callus is dried, it is transferred to the culture medium containing hygromycin B for screening and culture. The culture conditions are 28-30℃ and dark culture; the screening time is 3-4 weeks.

[0029] Rooting of seedlings: When the differentiated seedlings grow to about 2-3cm and have obvious root systems, they can be transferred to rooting medium to allow them to grow. The rooting medium should be poured into a relatively high bottle or tube so that the rooted seedlings have enough space to grow taller. The rooting culture conditions are 28-30℃ and sterile light culture.

[0030] In some embodiments, the step of identifying positive transgenic plants further comprises:

[0031] TRIzol reagent was used to extract total RNA from positive transgenic plants and reverse transcribed into cDNA, with rice OsUBQ5 as the internal reference gene. The quantitative primers are shown in SEQ ID No: 7-10. The relative expression levels of OsHDA706 in the two transgenic lines overexpressing the OsHDA706 gene were significantly higher than those in the control group, and they were named OsHDA706-OE#1 and OsHDA706-OE#2, respectively, proving that the transgenic rice lines were successfully constructed.

[0032] In some embodiments, the step of identifying positive transgenic plants further comprises:

[0033] The jasmonic acid (JA) content in transgenic lines overexpressing OsHDA706 was measured by liquid chromatography-tandem mass spectrometry.

[0034] In some embodiments, the step of identifying positive transgenic plants further comprises:

[0035] The resistance of transgenic lines overexpressing OsHDA706 to rice stripe virus was tested by artificial inoculation with RSV.

[0036] In some embodiments, the induction medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, pH=5.8.

[0037] 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.

[0038] In some embodiments, the rooting medium comprises: 1 / 2MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, PH=5.8.

[0039] In some embodiments, the co-culture medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, 200 μmol / L acetosyringone, pH=5.2.

[0040] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0041] 1) The present invention constructs an overexpression vector of OsHDA706 and introduces it into a rice variety (Nipponbare) by Agrobacterium-mediated method to obtain two transgenic rices with stable inheritance, which are named OsHDA706-OE#1 and OsHDA706-OE#2. The jasmonic acid (JA) content in the transgenic strains overexpressing OsHDA706 and the resistance analysis to rice stripe virus are measured, which helps to deepen our understanding and knowledge of the interaction between rice virus and JA and enrich the molecular mechanism of the interaction between rice and RSV.

[0042] 2) The present invention uses liquid chromatography-tandem mass spectrometry to measure the jasmonic acid content in transgenic strains overexpressing OsHDA706, and finds that a histone deacetylase OsHDA706 can effectively promote the accumulation of JA in rice, which greatly enriches the research direction of rice disease resistance theory.

[0043] 3) The present invention studies the mechanism of action of rice histone deacetylase OsHDA706 in RSV infection, thereby discovering the interaction between rice and the virus, laying a foundation for further theoretical research on disease resistance and breeding of resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : Relative expression level of OsHDA706 in OsHDA706 overexpressing transgenic rice.

[0045] Figure 2 :Jasmonic acid (JA) content in OsHDA706 overexpressing transgenic rice.

[0046] Figure 3 : Pathogenic symptoms of OsHDA706 overexpressing transgenic and control Nipponbare after RSV infection.

[0047] Figure 4 : Detection of virus content in OsHDA706 overexpressing transgenic and control Nipponbare after RSV infection.

[0048] mock: healthy rice; RSV: rice infected with rice stripe virus (RSV); NIP: Nipponbare rice. DETAILED DESCRIPTION

[0049] The rice varieties used in this series of experiments are: Nipponbare

[0050] The relevant culture medium components are as follows:

[0051] Induction medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2 mg / L 2,4-D, PH=5.8.

[0052] 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.

[0053] Co-culture medium: N6 culture medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2 mg / L 2,4-D, 200 μmol / L acetosyringone, pH = 5.2.

[0054] Rooting medium: 1 / 2MS (Manufacturer: Haibo Biotechnology Co., Ltd., Product No.: HB8469-6)

[0055] 39.45g / L, 0.5mg / L NAA, 50mg / L hygromycin, PH=5.8.

[0056] Example 1: Construction of rice OsHDA706 plant expression vector

[0057] 1) Cloning of Rice OsHDA706 Gene

[0058] Primers OsHDA706-F and OsHDA706-R were designed based on the sequence of OsHDA706 (SEQ ID No: 1). The primer sequences are as follows:

[0059] OsHDA706-F:ATGGCCTCTTCCGCGCCGTC (SEQ ID No: 3);

[0060] OsHDA706-R:TCAGCCAAGCTGGCTACCCAA (SEQ ID No: 4);

[0061] The total volume was 50 μL, including 25 μL of 2×KOD buffer, 1.5 μL of upstream and downstream primers (10 μM), 5 μL of dNTP Mix (2.5 mM), 1 μL of cDNA template, 1 μL of KOD Taq enzyme (5 U / μL), and 15 μL of ddH2O.

[0062] 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.

[0063] The PCR product was recovered, connected to the pMD18-T vector (Takara Biotech, catalog number D101A), cloned, and sent for testing to confirm that the pMD18-T-OsHDA706 recombinant plasmid was successfully obtained.

[0064] 2) Construction of OsHDA706 overexpression vector

[0065] Primers for connecting restriction sites were designed for the construction of the OsHDA706 gene binary expression vector pCAMBIA1300 (Beijing Dingguo Changsheng Biotechnology Co., Ltd., MCV033). After the pCAMBIA1300 vector was cut with BamHI and SacI, the PCR products were connected to the vectors respectively. Positive clones were selected for sequencing. The primer sequences used are as follows:

[0066] PCV-OsHDA706-F:

[0067] GTTCCAGATTACGCTGGATCCATGGCCTCTTCCGCGCCGTC (SEQ ID No: 5);

[0068] PCV-OsHDA706-R:

[0069] ATCGGGGAAATTCGAGCTCTCAGCCAAGCTGGCTACCAA (SEQ ID No: 6);

[0070] Sequencing confirmed that the pCAMBIA1300-OsHDA706 expression vector had been successfully constructed.

[0071] Example 2: Rice genetic transformation

[0072] 1) Callus induction and subculture: Select mature rice seeds (preferably newly harvested in the current year), peel off the husk, pour into a 50 ml centrifuge tube, add 75% ethanol to disinfect for 1 min, pour out the ethanol, rinse once with sterile water, pour out, add 30% sodium hypochlorite to disinfect for 20 min, pour out the sodium hypochlorite and rinse with sterile water 5-6 times. Use a pipette to absorb excess water (sterilized filter paper can be used to absorb dry), transfer the seeds to the induction medium, place in a 28°C light incubator for 3 weeks, and use pre-sterilized tweezers to clamp the grown callus tissue into the subculture medium, and subculture in a 28°C light incubator for 1 week.

[0073] 2) Transformation and culture of Agrobacterium: The plasmid containing the target vector was transformed into Agrobacterium tumefaciens GV3101 (manufacturer: Shanghai Weidi, product number: AC1003S) using the following steps: 5 μL of plasmid was added to 100 μL of Agrobacterium GV3101 competent medium, mixed by pipetting, added to a sterile electrode cup precooled at 4°C in advance, and transformed by electroporation at a voltage of 220 V, added to LB liquid culture medium without resistance, and cultured on a constant temperature shaker at 28°C for about 3 hours, evenly spread on LB solid culture 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.

[0074] 3) Agrobacterium infection: Prepare the infection solution, use a pipette to absorb the infection solution and wash off the Agrobacterium on the plate, which is the Agrobacterium suspension for co-culture transformation of rice. Select a sufficient number of callus tissues (the callus is in good condition, bright yellow in color, round and hard in texture, and the particle diameter is about 3mm), put it into a 100ml sterile triangular flask, add an appropriate amount of Agrobacterium suspension (make sure there is enough bacterial liquid in contact with the material), and place it at room temperature for infection for 20 minutes, shaking it from time to time. Pour out the bacterial liquid, place the callus tissue on a sterile filter paper to absorb the excess bacterial liquid, and then transfer it to a solid co-culture medium covered with a layer of sterile filter paper, and culture it in the dark at 26℃ for 3 days.

[0075] 4) Screening and culture: After 3 days of co-culture, the callus tissue needs to be cleaned. Use a 1 ml blue pipette tip to sow the callus on the co-culture medium into a sterilized Erlenmeyer flask, add sterile water to rinse both sides, and rinse once with sterile water containing 500 ul / L carbenicillin for the third time. After the excess water is sucked off with a pipette, the callus is transferred to sterile filter paper and the water on the callus is blown dry by the wind from the clean bench. The blowing time is controlled at about 30 minutes. After the callus is dried, it is transferred to the screening medium for screening and culture. The culture conditions are 28-30°C and cultured in the dark. The screening time is 3-4 weeks.

[0076] 5) Rooting of seedlings: When the differentiated seedlings grow to about 2-3cm and have obvious root systems, they can be transferred to rooting medium to grow. The rooting medium should be poured into a relatively high bottle or tube so that the rooted seedlings have enough space to grow taller. The rooting culture conditions are 28-30℃ and sterile light culture.

[0077] Example 3: Identification of positive transgenic plants

[0078] 1) Extraction of total RNA from rice plants and synthesis of first-strand cDNA sequences

[0079] TRIzol was used to extract total RNA from positive transgenic and Nipponbare rice growing healthily for 15-30 days, and reverse transcription kit 5* was used to extract total RNA from positive transgenic and Nipponbare rice growing healthily for 15-30 days. III qRT Super Mix (manufacturer: Novozymes, catalog number: R323-01) was used to reverse transcribe the extracted RNA into cDNA.

[0080] 2) qRT-PCR verification and analysis of the expression changes of OsHDA706 gene

[0081] The above cDNA samples were used, and the rice OsUBQ5 gene was used as an internal reference. The quantitative primers used were qRT-OsHDA706-F and qRT-OsHDA706-R. The relative expression level of OsHDA706 was as follows: Figure 1 The quantitative primer sequences are as follows:

[0082] qRT-OsHDA706-F: CCTGGAGAAGAACCGAGTGG (SEQ ID No: 7);

[0083] qRT-OsHDA706-R: TGCCACAGGAGGAAGCTTCTA (SEQ ID No: 8);

[0084] OsUBQ5-F:ACCACTTCGACCGCCACTACT(SEQ ID No:9);

[0085] OsUBQ5-R:ACGCCTAAGCCTGCTGGTT(SEQ ID No:10);

[0086] The experimental results are as follows Figure 1 As shown, the relative expression level of OsHDA706 in the two transgenic lines overexpressing OsHDA706, OsHDA706-OE#1 and OsHDA706-OE#2, was significantly higher than that of NIP, indicating that the transgenic rice lines were successfully constructed.

[0087] Example 4: Determination of JA content in transgenic lines overexpressing OsHDA706

[0088] Leaves of OsHDA706 overexpressing transgenic lines and Nipponbare seedlings grown under normal conditions for 15-30 days were collected (at least three biological replicates were set, and each biological replicate contained at least 10 seedlings) and thoroughly ground in liquid nitrogen. 0.2 g of sample was added to 1.5 ml of extraction solution (methanol: water: formic acid = 7.9:2:0.1). Then, it was sonicated with low-speed vortex on ice for 30 min, centrifuged and mixed, immersed and extracted at 4°C for 12 h, and then centrifuged (at 4°C, 12000 rpm for 20 min, and then at 12°C for 20 min) to extract the supernatant. The supernatant was extracted by solid phase extraction column (Waters, USA) and dried and concentrated into powder. Each sample was reconstituted with 0.12 ml of the starting mobile phase (5% acetonitrile + 0.1% formic acid + 94.9 aqueous solution), mixed by rotation and transferred to a 0.2 μm nylon membrane, and then detected by liquid chromatography-tandem mass spectrometry (LC–MS). Original sample content (ng / g) = measured concentration × 0.12 mL / 0.2 g.

[0089] The results are as follows Figure 2 As shown, the JA content in the two transgenic lines overexpressing OsHDA706, OsHDA706-OE#1 and OsHDA706-OE#2, was significantly higher than that in NIP, indicating that OsHDA706 increased the accumulation of JA in rice.

[0090] Example 5: Artificial inoculation of RSV

[0091] 1) Two transgenic lines overexpressing OsHDA706, OsHDA706-OE#1, OsHDA706-OE#2, and NIP rice materials were soaked at 37°C for 2-3 days. After the seeds sprouted white buds, they were evenly sown in 1L glass beakers (with at least 3 replicates, 30 seedlings in each replicate), and cultured in an artificial climate chamber at 30°C, 16 h light and 8 h dark.

[0092] 2) The SBPHs were artificially reared to 1-2 years old, then infected on RSV-infected rice seedlings for 3-5 days and then transferred to healthy rice seedlings for the transition period (the time required for RSV to complete the infection cycle in the SBPH, 7-15 days).

[0093] 3) The RSV-carrying or RSV-free gray leaf hoppers were inoculated at a ratio of 3 per seedling onto the transgenic lines OsHDA706-OE#1, OsHDA706-OE#2 and NIP rice materials overexpressing OsHDA706 at the 3-4 leaf stage, and all insects were swept out 3 days after inoculation (the virus inoculation experiment was performed using RSV-carrying and healthy gray leaf hoppers of the same age).

[0094] 4) 30 days after transplanting the infected rice materials into the field, the disease symptoms of the plants were observed and the disease status of the rice was determined by qRT-PCR.

[0095] Example 6: Resistance analysis of transgenic lines overexpressing OsHDA706

[0096] Thirty days after rice was transplanted to the field, the disease symptoms were observed. Compared with the control NIP, the two overexpressing OsHDA706 transgenic lines OsHDA706-OE#1 and OsHDA706-OE#2 showed obvious resistance to RSV, such as Figure 3 As shown. The total RNA of rice material was extracted as shown in Example 3, reverse transcribed into cDNA, and the OsUBQ5 gene of rice was used as an internal reference. The expression level of RSV CP gene (virus capsid protein encoding gene) was further detected by qRT-PCR, as shown. Figure 4 As shown, the expression level of RSV CP gene in transgenic lines was significantly lower than that of NIP; the above results indicate that overexpression of OsHDA706 in rice can significantly enhance rice resistance to RSV infection. The quantitative primer sequences are as follows:

[0097] qRT-CP-F: AGGCAATCAATGACATCTCC (SEQ ID No: 11);

[0098] qRT-CP-R: ATCTCTCACAAAGCCAGTGC (SEQ ID No: 12);

[0099] In summary, the experimental results show that: compared with the control NIP, the transgenic rice plants overexpressing OsHDA706 can significantly increase the JA content in rice and enhance the resistance of rice to RSV infection (including phenotypic symptoms and viral load of diseased plants). Therefore, the present invention successfully obtained RSV infection-resistant transgenic rice through transgenic means, which is of great significance to agricultural development and national food security, and has achieved unexpected technical effects.

Claims

1. Use of a rice histone deacetylase OsHDA706 gene in rice breeding for resistance to rice stripe virus (RSV), wherein the nucleotide sequence of the OsHDA706 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, wherein the amino acid sequence of the OsHDA706 gene is shown as SEQ ID NO.

2.

3. A method for preparing a rice stripe virus-resistant transgenic plant, comprising the steps of: 1) Construction of rice expression vector Primers were designed according to the sequence of OsHDA706 shown in SEQ ID NO.1, and the full-length sequence of OsHDA706 was cloned from rice cDNA, and the PCR product was recovered from gel; the pCAMBIA1300 vector containing the MYC tag was digested with BamHI and SacI, and the above PCR products were connected to the vectors respectively, and positive clones were selected and sequenced to confirm that the pCAMBIA1300-OsHDA706 expression vector had been successfully constructed; 2) Rice genetic transformation, Agrobacterium transformation and callus induction culture Callus induction: Select mature rice seeds, peel off the husk, pour into a centrifuge tube, add 75% ethanol to disinfect for 1 minute, pour out the ethanol, rinse with sterile water once, pour out, add 30% sodium hypochlorite to disinfect for 20 minutes, pour out the sodium hypochlorite and rinse with sterile water 5-6 times; use a pipette to absorb excess water, transfer the seeds to the induction medium, place in a 28°C light incubator for 3 weeks, clip the grown callus with pre-sterilized tweezers to the subculture medium, and subculture in a 28°C light incubator for 1 week; Transformation and culture of Agrobacterium: The plasmid containing the target vector was transformed into Agrobacterium tumefaciens GV3101, and the following steps were adopted: 5 μL of plasmid was added to 100 μL of Agrobacterium GV3101 competent medium, and mixed by pipetting, and added to a sterile electrode cup precooled at 4°C in advance, and electroporated at a voltage of 220V, and non-resistant LB liquid culture medium was added, and cultured in a constant temperature shaker at 28°C for about 3 hours, and evenly coated on LB solid culture 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; Agrobacterium infection: prepare infection solution, use a pipette to suck the infection solution to wash off the Agrobacterium on the plate, which is the Agrobacterium suspension for co-culture transformation of rice; select a sufficient number of callus tissues, put them into a sterile triangular flask, add an appropriate amount of Agrobacterium suspension, and infect at room temperature for 20 minutes, pour out the bacterial solution, put the callus tissue on sterile filter paper to absorb the excess bacterial solution, and then transfer it to a solid co-culture medium covered with a layer of sterile filter paper, and culture it in the dark at 26℃ for 3 days; Screening culture: After 3 days of co-culture, the callus tissue needs to be cleaned. Use a 1ml blue pipette tip to sow the callus tissue on the co-culture medium into a sterilized triangular flask, add sterile water to rinse both sides, and rinse once with sterile water containing 500ul / L carbenicillin for the third time. After the excess water is sucked off by a pipette, the callus is transferred to a sterile filter paper and the water on the callus is blown dry by the wind of the clean bench. The blowing time is controlled at about 30 minutes. After the callus is blown dry, it is transferred to the screening culture medium for screening culture. The culture conditions are 28-30℃ and dark culture; the screening time is 3-4 weeks; Seedling rooting: When the differentiated seedlings grow to about 2-3cm and have obvious root systems, they can be transferred to the rooting medium to grow the seedlings. The seedlings are cultured under sterile light and the rooting culture conditions are 28-30℃. 3) Identification of positive transgenic plants TRIzol reagent was used to extract total RNA from positive transgenic plants and reverse transcribed into cDNA, with rice OsUBQ5 as the internal reference gene; the quantitative primers were shown in SEQ ID No: 9-10, and the relative expression levels of OsHDA706 in the two transgenic lines overexpressing the OsHDA706 gene were significantly higher than those in the control group, and they were named OsHDA706-OE#1 and OsHDA706-OE#2, respectively, indicating that the transgenic rice lines were successfully constructed.

4. The preparation method according to claim 3, wherein the induction medium comprises: 24.1 g / L N6 medium, 2 mg / L 2,4-D, and pH = 5.

8.

5. The preparation method according to claim 3, wherein 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.

6. The preparation method according to claim 3, wherein the rooting medium comprises: 1 / 2MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, and pH = 5.

8.

7. The preparation method according to claim 3, wherein 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.

8. A method for increasing the jasmonic acid content in rice, comprising transferring a rice histone deacetylase OsHDA706 gene into rice, wherein the nucleotide sequence of the OsHDA706 gene is shown in SEQ ID NO.

1.

9. A method for improving the resistance of rice to rice stripe virus, comprising transferring a rice histone deacetylase OsHDA706 gene into rice, wherein the nucleotide sequence of the OsHDA706 gene is shown in SEQ ID NO.1.

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