P3IP1 protein mutant and application of P3IP1 protein mutant in regulation and control of resistance of plants to rice straw dwarf virus

Abstract

The invention discloses a P3IP1 protein mutant and application of the P3IP1 protein mutant in regulation and control of resistance of plants to rice straw dwarf viruses, the protein mutant is named as OsP3IP1T499A, the 499-site amino acid of the OsP3IP1T499A is formed by threonine mutation alanine with the amino acid sequence shown as SEQ ID NO.1, the amino acid sequence of the OsP3IP1T499A is shown as SEQ ID NO.2, and the nucleotide sequence of the coding gene of the OsP3IP1T499A is shown as SEQ ID NO.3. The invention further discloses a preparation method of the P3IP1 protein mutant. The invention further discloses application of the OsP3IP1T499A in regulating and controlling the resistance of plants to the water straw dwarf virus, experiments prove that the resistance to the water straw dwarf virus can be improved by over-expression of the OsP3IP1T499A in rice, and the OsP3IP1T499A has important significance in research of the resistance to the water straw dwarf virus and cultivation of anti-virus rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a P3IP1 protein mutant and application thereof in regulating plant resistance to rice grassy stunt virus. Background Art

[0002] The ubiquitin proteasome system (UPS) has been widely studied in animals and plants. Taking the virus-host interaction system as a research model, UPS is a double-edged sword. On the one hand, UPS can affect the virus's infection, replication and movement in the host by directly targeting the virus-encoded pathogenic factors or the endogenous host factors that assist the virus infection process, thus becoming an important antiviral defense strategy for the host; on the other hand, UPS can also modify key proteins in the host's antiviral signaling pathway, affecting the host's antiviral defense system, thereby promoting the virus's infection process.

[0003] Viral proteins can promote infection by targeting the UPS and stabilizing host proteins. For example, the satellite protein βC1 encoded by cotton leaf curl multan virus (CLCuMuV) promotes its spread by regulating the plant ubiquitination pathway. CLCuMuB βC1 interacts with NbSKP1 (S-phase kinase-associated protein) and prevents the interaction of NbSKP1s with NbCUL1 (Cullin1). Silencing of NbSKP1s or NbCUL1 enhances the accumulation of CLCuMuV genomic DNA and causes severe symptoms in plants. βC1 impairs the integrity of the SCFCOI1 (SKP1 / CUL1 / F-box) complex and the stability of GAI, a substrate of SCFSYL1, which inhibits JA and gibberellin (GA) responses. Therefore, CLCuMuV βC1 inhibits the ubiquitination function of SCFE3 ligase by interacting with NbSKP1s, thereby enhancing CLCuMuV infection and symptom occurrence in plants. The C2 protein encoded by the tomato yellow leaf curl virus TYLCV promotes infection by interacting with plant ubiquitin and inhibits the degradation of the JAZ1 protein, thereby inhibiting the expression of terpene synthase genes regulated by jasmonic acid and MYC2. The p22 protein encoded by the tomato chlorosis virus (ToCV) inhibits the degradation of NbAux / IAA and inhibits auxin signal transduction by binding to the C-terminal domain of NbSKP1, interfering with the formation of the SCF TIR1 complex. The coat protein P2 encoded by the rice dwarf virus RDV binds to OsIAA10 (auxin / indole-3-acetic acid), blocks the interaction between OsIAA10 and OsTIR1 (Transport inhibitor response 1), and inhibits the 26S proteasome-mediated degradation of OsIAA10. OsIAA10 Knockout mutants of the gene enhance resistance to RDV. The C2 protein encoded by beet curly top virus (BSCTV) interacts with SAMDC1

[138] . C2 protein can inhibit the degradation of SAMDC1. SAMDC1 Loss of function resulted in plant resistance to BSCTV and reduced viral accumulation, similar to the BSCTV C2 mutant. The C2 mutant increased DNA methylation in plant viral genomes, suggesting that BSCTV C2 attenuates 26S proteasome-mediated degradation of SAMDC and interferes with DNA methylation-mediated gene silencing. 2+The interaction between PCaP1 and P3N-PIPO of Turnip mosaic virus TuMV regulates the intercellular movement of TuMV. TuMV-P3N-PIPO recruits PCaP1 to plasmodesmata, and the actin activity of PCaP1 is required for the intercellular movement of TuMV. REM1.2 competes with PCaP1 for binding and negatively regulates the intercellular movement of TuMV. As a countermeasure, VPg (viral protein genome) encoded by TuMV interacts with REM1.2 through autophagy and UPS pathways to achieve systemic infection, thereby mediating the degradation of REM1.2. The P2 protein encoded by rice stripe virus RSV promotes the degradation of OsNPR1 by enhancing the binding of OsNPR1 to OsCUL3a. OsNPR1 induces JA signaling by disrupting the formation of the OsJAZ-OsMYC complex and enhancing the transcriptional activation of OsMYC2, thereby synergistically regulating rice antiviral immunity. In the monocot rice, the negative-sense single-stranded RNA virus Rice Grassy Dwarf Virus (RGSV) can induce the UPS pathway mediated by the U-box type E3 ubiquitin ligase P3IP1 (P3-inducible protein 1) by encoding the pathogenic factor P3 protein, affecting the protein stability of the core component of the RNA-dependent DNA methylation pathway (RdDM), NUCLEAR RNA POLY MERASED 1a (NRPD1a or Pol IVa), leading to the formation of disease symptoms such as dwarfing and increased tillering of rice plants. However, no studies have yet proven what effect a mutation in one amino acid in P3IP1, namely threonine at position 499, would have on the Rice Grassy Dwarf Virus. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a threonine mutation at position 499 of a P3IP1 protein in regulating plant resistance to rice grassy stunt virus.

[0005] To achieve the above object, the present invention adopts the following technical solution: Application of a P3IP1 protein mutant in regulating plant resistance to rice grassy stunt virus, wherein the protein mutant is named OsP3IP1 T499A , which is formed by the amino acid sequence of the protein shown in SEQ ID NO.1, in which the 499th amino acid is mutated to alanine by threonine, and its amino acid sequence is shown in SEQ ID NO.2.

[0006] A gene encoding the P3IP1 protein mutant as described above, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0007] The application of the above-mentioned P3IP1 protein mutant in controlling plant resistance to rice grassy stunt virus.

[0008] Furthermore, the plant is a monocotyledonous plant.

[0009] Furthermore, the monocotyledonous plant is rice.

[0010] Furthermore, overexpression of OsP3IP1 T499A Improve resistance to rice strawy stunt virus.

[0011] Application of the above-mentioned P3IP1 protein mutant in breeding virus-resistant rice varieties.

[0012] A method for improving resistance to rice strawy stunt virus, comprising the steps of overexpressing the above-mentioned P3IP1 protein mutant.

[0013] The significant advantages of the present invention are: E3 ubiquitin ligase in rice is closely related to the expression induced by pathogen infection, but there is no report proving that the mutation of one amino acid in E3 ubiquitin ligase can achieve the effect of resisting rice grassy stunt virus. At present, there is no relevant functional report on the role of threonine mutated to alanine at position 499 of P3IP1 in antiviral. The present invention provides the use of threonine mutated to alanine at position 499 of P3IP1 in regulating plant resistance to rice grassy stunt virus. The present invention finds for the first time that rice expressed after mutating threonine at position 499 of P3IP1 to alanine can improve resistance to rice grassy stunt virus. Therefore, the mutation of threonine at position 499 of P3IP1 is of great significance for studying resistance to rice grassy stunt disease and cultivating virus-resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 :change OsP3IP1 T499A Identification of rice.

[0015] Figure 2 : OsP3IP1 T499A qRT-PCR detection of RGSV virus-related genes in rice and wild-type rice (Zhonghua 11) after RGSV infection.

[0016] Figure 3 : OsP3IP1 T499A Statistical graph of the incidence of rice and wild-type rice (Zhonghua 11) after RGSV infection; Figure 4 : OsP3IP1 T499A Symptom diagram of rice and wild-type rice (Zhonghua 11) in different rice strains infected with RGSV. DETAILED DESCRIPTION

[0017] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0019] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0020] In this patent, the protein obtained by mutating the threonine at position 499 of the OsP3IP1 protein to alanine is referred to as OsP3IP1 T499A express.

[0021] Example 1: Rice OsP3IP1 T499A Obtaining proteins and their encoding genes 1. Rice OsP3IP1 T499A Acquisition of proteins and their encoding genes The sequences of rice OsP3IP1 protein and its encoding gene are publicly available in the Rice Genome Annotation Database (http: / / rice.plantbiology.msu.edu / ) and can be retrieved through LOC_Os02g33680. OsP3IP1 The nucleotide sequence starting from the ATG initial codon of the gene CDS sequence has the 499th threonine mutated to alanine. The protein amino acid sequence is shown in SEQ ID NO.2, and the corresponding nucleotide sequence is shown in SEQ ID NO.3.

[0022] 2. Rice OsP3IP1 T499A Cloning of genes and obtaining recombinant vectors containing the fragments Primers were designed according to the sequences shown in SEQ ID NO. 2~3, and the required restriction sites were added to both ends of the primers to obtain the primer sequences: OsP3IP1 T499A -5'SfiI:5'-GGCCATTACGGCCATGGACCCCTCATCCTCCTCC-3'; OsP3IP1 T499A -3'Sfi I: 5'-3'GGCCGAGGCGGCCTTACTCAGAAAGGCATGATGTGGGTGG-3'.

[0023] According to the instructions, total RNA from Oryza sativa L. japonica cv. Zhonghua11 (Xu Yu et al., "Cloning of the Glutelin Gt1 Gene of Zhonghua11 Rice and Construction of the Gt1 Gene Expression Vector Guided by the Wax Gene Promoter", Journal of Shanghai Normal University (Natural Science Edition), Vol. 39, No. 2, April 2010, p. 204, available to the public from Peking University) was extracted using TRIzol Reagent from Invitrogen Company, and reverse transcribed using SuperScript II reverse transcriptase from the company to obtain cDNA. The primer used for reverse transcription was a 16-nucleotide Oligod (T) primer.

[0024] The cDNA obtained by reverse transcription was used as template and the above primers were used to OsP3IP1 T499A -5'Sfi I and OsP3IP1 T499A -3'Sfi I was used to perform PCR (Polymerase Chain Reaction) reaction to obtain a 1518 bp PCR product having a nucleotide sequence shown in SEQ ID NO.3.

[0025] After the above PCR product is recovered, it is digested with restriction endonuclease Sfi I to recover the target gene PCR product with sticky ends; the vector pPR3-N (Dualsystems Biotech, product catalog number: P01601-P01629) is digested with restriction endonuclease Sfi I to recover the vector digestion product; the above target gene PCR product with sticky ends and the vector digestion product are connected with T4 DNA ligase, and transformed into Escherichia coli strain DH5α to obtain transformants. The plasmid extracted from the transformant is sent for sequencing to obtain the recombinant vector pPR3-N- OsP3IP1 T499A . Recombinant vector pPR3-N- OsP3IP1 T499A The rice shown in SEQ ID NO.3 OsP3IP1 T499A The gene was inserted into the Sfi I restriction site of the vector pPR3-N.

[0026] Example 2: Overexpression OsP3IP1 T499A Genetic material preparation 1. Construction of expression vector 1) Design primers according to the restriction site sequence and obtain the primer sequence as follows: OsP3IP1 T499A-5':5'-ggtacccggggatcctctagaATGGACCCCTCATCCTCCTCC-3', OsP3IP1 T499A -3':5'-cagcttctgctccatgtcgacGAAAGGCATGATGTGGGTGG-3' 2) Using the intermediate vector as a template, OsP3IP1 T499A -5', OsP3IP1 T499A -3' primer for PCR amplification to obtain the expression cassette with the linker; 3) After amplification, the final product was purified and recovered by gel excision and named pActin-OsP3IP1 T499A .

[0027] 2. Overexpression OsP3IP1 T499A Obtaining genetically modified rice 1) Callus induction culture The seeds of Zhonghua 11 rice (hereinafter also referred to as wild-type rice) were shelled, soaked in 70% ethanol for 10 minutes, and then soaked in 0.1% mercuric chloride for 30 minutes; the surface was sterilized. The solution on the surface of the seeds was washed off with a large amount of sterile water, and the moisture on the surface of the seeds was absorbed with sterile filter paper. The seeds were placed on a plate of mature embryo callus induction medium, the edge of the plate was sealed with Parafilm film, and cultured in a 26°C incubator away from light. After about 15 days, the grown callus was carefully removed and transferred to the mature embryo subculture medium, and cultured under the same conditions. Subculture is required every two weeks. When used for transformation, it is necessary to select granular callus that has been subcultured for about 5 days and is light yellow.

[0028] 2) Cultivation of Agrobacterium pActin-OsP3IP1 T499A Electrotransformed into Agrobacterium EHA105 to obtain recombinant bacteria EHA105 / pActin-OsP3IP1 T499A .

[0029] The recombinant bacteria EHA105 / pActin-OsP3IP1 T499A Streak on LB plates containing antibiotics (50 mg / L Kanamycin, 50 mg / L R Fampicin) and culture at 28°C for 2 days. Pick a single colony and inoculate it into liquid LB medium and culture at 28°C with shaking until OD 600 The value was about 0.5, and acetosyringone was added to a final concentration of 100 mM to obtain an Agrobacterium suspension for transforming rice callus.

[0030] 3) Co-culture of rice callus and Agrobacterium Place the subcultured callus in a sterilized conical flask and pour in the Agrobacterium suspension used to transform rice callus to submerge the callus. Leave at room temperature for 20 minutes and gently shake from time to time to allow the callus to fully contact the bacterial solution. Gently remove the callus with sterile tweezers, place it on a sterile filter paper to absorb the excess bacterial solution, and transfer it to a co-culture medium plate covered with a layer of sterile filter paper. Culture in the dark at 28°C for 3 days to obtain co-cultured callus.

[0031] 4) Screening and differentiation of resistant callus The co-cultivated callus tissue is washed with an appropriate amount of sterile water to remove the residual Agrobacterium on the surface, placed on the screening medium, and cultured in the dark at 26°C for screening. After two weeks, it is transferred to a new screening medium and continues to be screened for two weeks. Select the callus tissue that is in good condition after two rounds of screening, transfer it to the differentiation medium plate, culture it in the dark for 3 days, and then transfer it to a light incubator (15hr / day) for light culture. Differentiated seedlings can be seen after one month. When the differentiated seedlings grow to about 2cm, transfer them to the rooting medium in a conical flask and continue to culture for about two weeks. Select seedlings with good growth and developed root system, wash the culture medium on the roots with tap water, and transplant them into the soil. Collect the seeds to obtain the T1 generation overexpressing OsP3IP1 T499A Rice seeds, sowed to obtain T1 generation OsP3IP1 transgenic T499A Rice.

[0032] The T1 rice seeds were preliminarily screened by hygromycin or G418 (pCambia2300 vector carries the G418 resistance screening gene). The germinated seeds indicated that the vector was transferred into rice. The germinated seeds were planted in the soil. After growing for 2 weeks, 0.1 g of leaves were taken and ground into powder with liquid nitrogen for genomic DNA extraction. The specific method was referred to the High-Efficiency Plant Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Cat. No.: DP350). OsP3IP1 T499A Add 200 μl of protein extraction buffer (0.25M Tris-HCl, pH 6.8, 8% SDS, 8% β-mercaptoethanol, 20% glycerol) to the leaf powder of the over-expressed transgenic rice line, incubate on ice for 10 min, boil at 100°C for 10 min, centrifuge at 4°C, 12,000 rpm for 10 min, take the supernatant, perform SDS-PAGE, and use Western detection after transfer. SDS-PAGE and Western Blot were performed according to known methods and product instructions. The antibody used was anti-MYC-HRP (sigma), and the antibody anti-Actin was used to detect the endogenous Actin protein of rice as an internal reference. Figure 1, those with a band at 50 KDa were positive, indicating gene transfer and protein expression. Two strains #1 and #2 (#1 and #2 were duplicate strains) were selected for subsequent disease resistance analysis experiments ("WT" in the figure is the wild-type Zhonghua 11 rice, used as a negative control).

[0033] Example 3: OsP3IP1 T499A Can improve rice resistance to RGSV 1) Identify the infection of RGSV by identifying the expression of RGSV CP by fluorescence quantitative PCR (qRT-PCR) Inoculation of T1 OsP3IP1 with brown planthoppers carrying RGSV (the pathogen is Rice Grassy Stunt Virus) T499A Overexpressing transgenic rice and wild-type rice Zhonghua 11 were used, and 30 plants of each type of rice were inoculated. The temperature was 30 degrees during the day, 22 degrees at night, and the humidity was 60%. Each plant was inoculated with 5 brown planthoppers. The brown planthoppers were caught after biting for three days, and the bitten rice was cultured in a sunny greenhouse (natural light, temperature. The experiment was repeated 3 times and the results were averaged).

[0034] Four weeks after infection, T1 generation OsP3IP1 was extracted T499A The rice leaf powder of overexpressing transgenic rice and wild-type rice Zhonghua 11 after infection was added with Trizol reagent (Invitrogen) and RNA was extracted according to the instructions. Then, the genomic DNA in the RNA was digested with RQ1 DNase (Promega, catalog number: M610A) according to Table 1 below: Table 1 Digestion system Take 2 μg of digested RNA for reverse transcription qRT-PCR. For specific methods, refer to invitrogen M-MLVReverse Transcriptase (Cat. No.: 28025-021). OsEF1a As an internal control, the primers for internal control were EF1a-F: 5′-GCACGCTCTTCTTGCTTTCACTCT-3′ and EF1a-R: 5′-AAAGGTCACCACCATACCAGGCTT-3′, and to detect the expression of RGSVCP, the primers for RGSV CP were CP-F: 5′-AGAGCAGTTTCCTGTAGTCCC-3′, and CP-R: 5′-CCAGTTCGGCTGTTCAGATTAG-3′.

[0035] The results are as follows Figure 2 As shown, it can be seen that OsP3IP1 T499AThe accumulation of RGSV CP in the overexpressing transgenic rice was lower than that in the wild-type rice Zhonghua11 (the asterisks in the figure indicate significant differences). T499A Overexpression can improve rice resistance to RGSV.

[0036] 2) Determine RGSV infection rate by phenotype Four weeks after infection, 30 T1-generation OsP3IP1-transfected strains were observed. T499A Symptoms of overexpressing rice and 30 wild-type rice Zhonghua 11 (those infected with RGSV showed dwarfing, increased tillering, and yellow and elongated leaves, while those not infected with RGSV showed no dwarfing, increased tillering, and yellow and elongated leaves). Figure 3 As shown, the number of symptomatic plants was counted and the virus rate was calculated = (number of phenotypic plants / total number of plants) × %.

[0037] The results are shown in Table 2. T499A The overexpression rice had a relatively high susceptibility to disease.

[0038] Table 2 Statistical results of virus infection rate of transgenic rice after virus infection In addition, we took photos of different strains of diseased rice, such as Figure 4 Shown is OsP3IP1 4 weeks after infection T499A Disease symptoms of overexpressing transgenic rice. T499A Overexpressing transgenic rice showed stronger disease symptoms, with a stronger degree of dwarfing and significantly yellow and elongated leaves ( Figure 4 WT in the figure represents wild-type Zhonghua 11 rice, OsP3IP1 T499A OsP3IP1 T499A Compared with wild-type rice, OsP3IP1 T499A Overexpressing transgenic rice is more disease-resistant.

[0039] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Application of a P3IP1 protein mutant in regulating plant resistance to rice grassy stunt virus, characterized in that: The protein mutant was named OsP3IP1 T499A , which is formed by the amino acid sequence of the protein shown in SEQ ID NO.1, in which the 499th amino acid is mutated to alanine by threonine, and its amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding the P3IP1 protein mutant according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. Use of the P3IP1 protein mutant as claimed in claim 1 in controlling plant resistance to rice grassy stunt virus.

4. The use according to claim 3, characterized in that: The plant is a monocotyledonous plant.

5. The use according to claim 3, characterized in that: The monocotyledonous plant is rice.

6. The use according to claim 3, characterized in that: Overexpression of OsP3IP1 T499A Improve resistance to rice strawy stunt virus.

7. Use of the P3IP1 protein mutant according to claim 1 in breeding virus-resistant rice varieties.

8. A method for improving resistance to rice grassy stunt virus, characterized in that: Overexpression of the P3IP1 protein mutant according to claim 1.

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