P3ip1 protein mutant and application thereof in regulating resistance of plants to rice grassy stunt virus

Abstract

The application discloses a P3IP1 protein mutant and application of the P3IP1 protein mutant in regulating rice against rice grassy stunt virus, wherein the protein mutant is named as OsP3IP1 T499A , the OsP3IP1 T499A is formed by mutating threonine at the 499th amino acid of a protein with an amino acid sequence shown in SEQ ID NO. 1 into alanine, has an amino acid sequence shown in SEQ ID NO. 2, and has a nucleotide sequence of a coding gene shown in SEQ ID NO. 3. The application also discloses application of the OsP3IP1 T499A in regulating rice against rice grassy stunt virus, and experiments prove that overexpression of the OsP3IP1 T499A in rice can improve the resistance of the rice against the rice grassy stunt virus, and has important significance for studying the resistance of the rice grassy stunt virus and cultivating rice varieties resistant to the virus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a P3IP1 protein mutant and its application in regulating plant resistance to rice straw dwarf virus. Background Technology

[0002] The ubiquitin-proteasome system (UPS) has been extensively studied in plants and animals. Using the virus-host interaction system as a research model, UPS is a double-edged sword. On the one hand, UPS can directly target viral-encoded pathogenic factors or endogenous host factors that assist viral infection, thus affecting viral infection, replication, and movement within the host, and 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 pathways, affecting the host's antiviral defense system and thereby promoting viral infection.

[0003] Viral proteins can promote infection by targeting UPS and stabilizing host proteins. For example, the satellite protein βC1 encoded by cotton leaf curl multan virus (CLCuMuV) facilitates transmission by regulating plant ubiquitination pathways. CLCuMuV βC1 interacts with NbSKP1 (S-phase kinase-associated protein), preventing the interaction of NbSKP1s with NbCUL1 (Cullin1). Silencing of NbSKP1s or NbCUL1 enhances the accumulation of CLCuMuV genomic DNA and leads to 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 enhances CLCuMuV infection and symptom development in plants by inhibiting the ubiquitination function of the SCFE3 ligase through interaction with NbSKP1s. The C2 protein encoded by Tomato Yellow Leaf Roll Virus (TYLCV) promotes infection by interacting with plant ubiquitin and inhibits the degradation of JAZ1 protein, thereby suppressing the expression of jasmonic acid and MYC2-regulated terpene synthase genes. The p22 protein encoded by Tomato Yellow Wilt Virus (ToCV) inhibits the degradation of NbAux / IAA and suppresses auxin signal transduction by binding to the C-terminal domain of NbSKP1, interfering with the formation of the SCF TIR1 complex. The P2 capsid protein encoded by Rice Dwarf Virus (RDV) binds to OsIAA10 (auxin / indole-3-aceticacid), blocking the interaction between OsIAA10 and OsTIR1 (Transport Inhibitor Response 1) and inhibiting 26S proteasome-mediated OsIAA10 degradation. OsIAA10 Gene knockout mutants enhance resistance to RDV. The C2 protein encoded by beetroot scintillator virus (BSCTV) interacts with SAMDC1.

[138] The C2 protein can inhibit the degradation of SAMDC1. SAMDC1 Loss of function leads to plant resistance to BSCTV, with reduced virus accumulation, similar to the BSCTV C2 mutant. The C2 mutant enhances DNA methylation in the plant viral genome. This indicates that BSCTV C2 weakens 26S proteasome-mediated SAMDC degradation and interferes with DNA methylation-mediated gene silencing. Plasma membrane-associated calcium-binding protein 1 (pMMR) is involved. 2+The interaction between PCaP1 (a binding protein 1) 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 essential for TuMV intercellular movement. REM1.2 competitively binds to PCaP1, negatively regulating TuMV intercellular movement. As an antagonistic strategy, the VPg (viral protein genome) encoded by TuMV interacts with REM1.2 via autophagy and UPS pathways to achieve systemic infection, thereby mediating REM1.2 degradation. The P2 protein encoded by rice stripe virus (RSV) promotes OsNPR1 degradation by enhancing the binding of OsNPR1 to OsCUL3a. OsNPR1 induces JA signaling by disrupting OsJAZ-OsMYC complex formation and enhancing OsMYC2 transcriptional activation, thereby synergistically regulating antiviral immunity in rice. In monocotyledonous rice, the negative-sense single-stranded RNA virus rice straw 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. This affects the protein stability of NUCLEAR RNA POLY MERASED 1a (NRPD1a or Pol IVa), a core component of the RNA-dependent DNA methylation pathway (RdDM), leading to disease symptoms such as dwarfing and increased tillering in rice plants. However, no studies have yet demonstrated the effect of a single amino acid mutation in P3IP1, specifically threonine at position 499, on rice straw dwarf virus. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the threonine mutation at position 499 of the P3IP1 protein in regulating plant resistance to rice straw dwarf virus.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of the P3IP1 protein mutant in regulating plant resistance to rice straw-like dwarf virus; the protein mutant is named OsP3IP1. T499A It is formed by mutating threonine to alanine at amino acid position 499 of the protein, as shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.

[0007] A gene encoding a mutant of the P3IP1 protein as described above, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0008] The above-mentioned P3IP1 protein mutant was used to control plant resistance to rice straw dwarf virus.

[0009] Furthermore, the plant is a monocotyledonous plant.

[0010] Furthermore, the monocotyledonous plant is rice.

[0011] Furthermore, overexpression of OsP3IP1 T499A Improve resistance to rice straw dwarf virus.

[0012] The application of the aforementioned P3IP1 protein mutant in the breeding of virus-resistant rice varieties.

[0013] A method for improving resistance to rice straw-like dwarf virus, comprising the step of overexpressing the above-mentioned P3IP1 protein mutant.

[0014] The significant advantages of this invention are:

[0015] E3 ubiquitin ligase in rice is closely related to pathogen infection-induced expression, but there are no reports demonstrating that a single amino acid mutation in the E3 ubiquitin ligase can achieve resistance to rice straw dwarf virus. Currently, there are no reports on the role of the P3IP1 threonine-alanine mutation in antiviral activity. This invention provides the application of the P3IP1 threonine-alanine mutation in regulating plant resistance to rice straw dwarf virus. This invention is the first to discover that rice expressing the P3IP1 threonine-alanine mutation can improve resistance to rice straw dwarf virus. Therefore, the P3IP1 threonine-alanine mutation at position 499 is of great significance for studying resistance to rice straw dwarf virus and for breeding virus-resistant rice varieties. Attached Figure Description

[0016] Figure 1 :change OsP3IP1 T499A Identification of rice.

[0017] Figure 2 : OsP3IP1 T499A qRT-PCR detection of RGSV virus-related genes in rice and wild-type rice (Zhonghua 11) after RGSV infection.

[0018] Figure 3 : OsP3IP1 T499A Statistical chart of RGSV infection incidence in rice and wild-type rice (Zhonghua 11);

[0019] Figure 4 : OsP3IP1 T499ASymptoms of rice and wild-type rice (Zhonghua 11) in different rice lines infected with RGSV. Detailed Implementation

[0020] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] In this patent, the protein obtained by mutating alanine to threonine at position 499 of the OsP3IP1 protein uses OsP3IP1. T499A express.

[0024] Example 1: Rice OsP3IP1 T499A Acquisition of proteins and their encoding genes

[0025] I. Rice OsP3IP1 T499A Acquisition of proteins and their encoding genes

[0026] The sequences of the 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 using LOC_Os02g33680. (Excerpt from rice...) OsP3IP1 The nucleotide sequence starting from the initial codon of ATG in the gene CDS sequence is modified by mutating threonine at position 499 to alanine. The amino acid sequence of the protein is shown in SEQ ID NO.2, and its corresponding nucleotide sequence is shown in SEQ ID NO.3.

[0027] II. Rice OsP3IP1 T499A Gene cloning and obtaining recombinant vectors containing the fragment

[0028] Primers were designed based on the sequences shown in SEQ ID NO. 2~3, and the required restriction enzyme sites were added to both ends of the primers to obtain the primer sequences as follows:

[0029] OsP3IP1 T499A -5'SfiI:5'-GGCCATTACGGCCATGGACCCCTCATCCTCCTCC-3';

[0030] OsP3IP1 T499A-3'Sfi I: 5'-3'GGCCGAGGCGGCCTTACTCAGAAAGGCATGATGTGGGTGG-3'.

[0031] According to the instructions, total RNA was extracted from Zhonghua 11 rice (Oryzasativa L. japonica cv. Zhonghua 11, Xu Yu et al., "Cloning of Glutelin Gt1 Gene in 'Zhonghua 11' Rice and Construction of Gt1 Gene Expression Vector Guided by Waxy Gene Promoter", Journal of Shanghai Normal University (Natural Science Edition), April 2010, Vol. 39, No. 2, p. 204, publicly available from Peking University) using Invitrogen's SuperScript II reverse transcriptase to obtain cDNA. The primers used for reverse transcription were 16-nucleotide Oligod(T) primers.

[0032] Using the cDNA obtained from reverse transcription as a template, and the above primers OsP3IP1 T499A -5'Sfi I and OsP3IP1 T499A A PCR (Polymerase Chain Reaction) was performed using -3'Sfi I to obtain a 1518bp PCR product with the nucleotide sequence shown in SEQ ID NO.3.

[0033] After recovering the PCR products, the samples were digested with restriction endonuclease Sfi I to recover the PCR product containing the sticky ends of the target gene. The vector pPR3-N (Dualsystems Biotech, catalog number: P01601-P01629) was digested with restriction endonuclease Sfi I, and the digestion product was recovered. The PCR product containing the sticky ends of the target gene and the digestion product were ligated using T4 DNA ligase and transformed into *E. coli* strain DH5α to obtain transformants. The plasmids of the transformants were extracted and sequenced to obtain the recombinant vector pPR3-N- OsP3IP1 T499A Recombinant vector pPR3-N- OsP3IP1 T499A To make the rice shown in SEQ ID NO.3 OsP3IP1 T499A The gene was obtained by inserting it between the Sfi I restriction sites of the vector pPR3-N.

[0034] Example 2: Overexpression OsP3IP1 T499A Genetic material preparation

[0035] I. Construction of the Expression Carrier

[0036] 1) Primers were designed based on the restriction enzyme site sequence, and the primer sequences were obtained as follows: OsP3IP1 T499A -5':5'-ggtacccggggatcctctagaATGGACCCCTCATCCTCCTCC-3', OsP3IP1 T499A -3':5'-cagcttctgctccatgtcgacGAAAGGCATGATGTGGGTGG-3'

[0037] 2) Using the intermediate carrier as a template, respectively... OsP3IP1 T499A -5', OsP3IP1 T499A PCR amplification was performed using the -3' primer to obtain the expression cassette with adapter;

[0038] 3) After amplification, the final product was purified by gel extraction and named pActin-OsP3IP1. T499A .

[0039] II. Overexpression OsP3IP1 T499A The acquisition of genetically modified rice

[0040] 1) Induction and culture of callus tissue

[0041] Zhonghua 11 rice (hereinafter also known as wild-type rice) seeds were dehulled, soaked in 70% ethanol for 10 minutes, and then soaked in 0.1% mercuric chloride for 30 minutes for surface sterilization. The seeds were then rinsed with plenty of sterile water to remove the solution from the seed surface, and the moisture was absorbed with sterile filter paper. The seeds were placed on mature embryo callus induction medium plates, the edges of the plates were sealed with Parafilm, and cultured in a 26°C incubator in the dark. After approximately 15 days, the callus tissue was carefully removed and transferred to mature embryo subculture medium, and cultured under the same conditions. Subculture was performed every two weeks. For transformation, pale yellow granular callus tissue that had been subcultured for about 5 days was selected.

[0042] 2) Culture of Agrobacterium

[0043] pActin-OsP3IP1 T499A Electroporation into Agrobacterium EHA105 yielded recombinant strain EHA105 / pActin-OsP3IP1 T499A .

[0044] Recombinant bacteria EHA105 / pActin-OsP3IP1 T499AStreak the bacteria on LB agar plates containing antibiotics (50 mg / L Kanamycin, 50 mg / L Ifampicin) and incubate at 28°C for 2 days. Inoculate single colonies into liquid LB medium and incubate at 28°C with shaking until OD reaches 100%. 600 The concentration was approximately 0.5. Acetyleugenol was added to a final concentration of 100 mM to obtain an Agrobacterium suspension for transforming rice callus.

[0045] 3) Co-culture of rice callus and Agrobacterium

[0046] Subcultured callus tissue was placed in a sterilized Erlenmeyer flask, and Agrobacterium suspension used for transforming rice callus was poured in to submerge the callus tissue. The flask was incubated at room temperature for 20 minutes, gently agitating occasionally to ensure adequate contact between the callus tissue and the bacterial suspension. The callus tissue was then carefully removed using sterile forceps, placed on sterile filter paper to absorb excess bacterial suspension, and transferred to a co-culture medium plate lined with sterile filter paper. The plate was incubated in the dark at 28°C for 3 days to obtain co-cultured callus tissue.

[0047] 4) Screening and differentiation of resistant callus

[0048] The co-cultured callus tissue was washed with an appropriate amount of sterile water to remove residual Agrobacterium on the surface. It was then placed on selection medium and incubated at 26°C in the dark for two weeks. After two weeks, it was transferred to a new selection medium for another two weeks of selection. Callus tissue in good condition after two rounds of selection was selected and transferred to differentiation medium plates. It was first incubated in the dark for 3 days, and then transferred to a light incubator (15 hours / day) for further light cultivation. Differentiated plantlets were visible after one month. When the differentiated plantlets grew to approximately 2 cm, they were transferred to rooting medium in Erlenmeyer flasks and cultured for about two weeks. Plantlets with good growth and well-developed root systems were selected, and the culture medium was washed off the roots with tap water before transplanting them into soil. Seeds were harvested to obtain the T1 generation transgenic OsP3IP1. T499A Rice seeds, when sown, yield T1 generation OsP3IP1. T499A Rice.

[0049] T1 generation rice seeds underwent preliminary screening using hygromycin or G418 (the pCambia2300 vector carries the G418 resistance selection gene). Germinated seeds indicated vector transfer into rice. Germinated seeds were planted in soil, and after two weeks of growth, 0.1g of leaves were collected, ground into powder using liquid nitrogen, and used for genomic DNA extraction. Specific methods were described using a high-efficiency plant genomic DNA extraction kit (Tiangen Biotech Co., Ltd., catalog number: DP350). OsP3IP1 T499A200 μl of protein extraction buffer (0.25 M Tris-HCl, pH 6.8, 8% SDS, 8% β-mercaptoethanol, 20% glycerol) was added to leaf powder from overexpressed transgenic rice lines. The mixture was incubated on ice for 10 min, boiled at 100°C for 10 min, and centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected and subjected to SDS-PAGE. After transfer to a membrane, Western blotting was performed. SDS-PAGE and Western blotting were performed according to known methods and product instructions. The antibody used was anti-MYC-HRP (Sigma), and the anti-Actin antibody was used as an internal control to detect endogenous Actin protein in rice. Figure 1 The presence of a band at 50 kDa indicates a positive result, signifying gene transfer and protein expression. Two lines, #1 and #2 (#1 and #2 are duplicate lines), were selected for subsequent disease resistance analysis experiments (in the figure, "WT" represents wild-type Zhonghua 11 rice, used as a negative control).

[0050] Example 3: OsP3IP1 T499A It can improve rice's resistance to RGSV.

[0051] 1) RGSV infection was identified by measuring the expression level of RGSV CP using quantitative real-time PCR (qRT-PCR).

[0052] Inoculate T1 generation OsP3IP1 with brown planthoppers carrying RGSV (the pathogen is Rice Grassy Stunt Virus). T499A Transgenic rice and wild-type rice Zhonghua 11 were overexpressed. Thirty plants of each type were inoculated and cultured at 30 degrees Celsius during the day and 22 degrees Celsius at night with 60% humidity. Five brown planthoppers were inoculated on each plant. After feeding on the plants for three days, the planthoppers were removed. The fed rice plants were then cultured in a sunny greenhouse (natural light and temperature. The experiment was repeated three times, and the average value was taken).

[0053] Four weeks after exposure, T1 generation OsP3IP1 was extracted. T499A RNA was extracted from leaf powder of transgenic rice and wild-type rice (Zhonghua 11) after infection, using Trizol reagent (Invitrogen) and following the manufacturer's instructions. Then, the genomic DNA in the RNA was digested using RQ1 DNase (Promega, catalog number: M610A) according to Table 1 below.

[0054] Table 1 Digestive System

[0055]

[0056] Take 2 μg of digested RNA for reverse transcription qRT-PCR. For specific methods, refer to invitrogen M-MLVReverse Transcriptase (catalog number: 28025-021). OsEF1a As internal controls, the primers for internal controls were EF1a-F: 5'-GCACGCTCTTCTTGCTTTCACTCT-3' and EF1a-R: 5'-AAAGGTCACCACCATACCAGGCTT-3'. To detect the expression level of RGSVCP, the primers for RGSVCP were CP-F: 5'-AGAGCAGTTTCCTGTAGTCCC-3' and CP-R: 5'-CCAGTTCGGCTGTTCAGATTAG-3'.

[0057] The results are as follows Figure 2 As shown, it can be seen that OsP3IP1 T499A The accumulation of RGSV CP in overexpressed transgenic rice was lower than that in wild-type rice Zhonghua 11 (asterisks in the figure indicate significant differences). Therefore, OsP3IP1 T499A Overexpression can enhance rice resistance to RGSV.

[0058] 2) Determining RGSV infection rate through phenotype

[0059] Four weeks after inoculation, 30 T1 strains transformed into OsP3IP1 were observed. T499A Symptoms of overexpression in rice and 30 wild-type rice plants of variety Zhonghua 11 were observed (those infected with RGSV virus exhibited stunted growth, increased tillering, and yellowing, elongated leaves; those not infected with RGSV virus did not exhibit these symptoms). Results are as follows: Figure 3 As shown, the number of symptomatic plants was counted, and the virus-carrying rate was calculated as (number of phenotypic plants / total number of plants) × 100%.

[0060] The results are shown in Table 2 below. It can be seen that the conversion to OsP3IP1... T499A Overexpression of the rice has a relatively high disease susceptibility rate.

[0061] Table 2. Statistical results of virus-carrying rate in transgenic rice after viral infection.

[0062]

[0063] In addition, we photographed different strains of susceptible rice, such as... Figure 4 As shown, this is OsP3IP1 4 weeks after infection. T499A A diagram showing the disease symptoms of transgenic rice overexpressing OsP3IP1. It can be seen that OsP3IP1... T499A Overexpression of transgenic rice resulted in stronger disease symptoms, including greater stunting and significantly yellowing and elongated leaves. Figure 4In the diagram, WT represents wild-type Zhonghua 11 rice, OsP3IP1 T499A Represents OsP3IP1 T499A (Overexpression of transgenic rice). Compared with wild-type rice, OsP3IP1 T499A Overexpression of genetically modified rice makes it more resistant to disease.

[0064] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. The application of P3IP1 protein mutants in regulating plant resistance to rice straw-like dwarf virus, characterized by: The protein mutant was named OsP3IP1. T499A It is formed by mutating threonine to alanine at amino acid position 499 of the protein shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2; the plant is rice, which overexpresses OsP3IP1. T499A Improve resistance to rice straw dwarf virus.

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

3.

3. The application of the P3IP1 protein mutant in the breeding of virus-resistant rice varieties, characterized by: The protein mutant was named OsP3IP1. T499A Its amino acid sequence is shown in SEQ ID NO.2; the virus is a rice straw-like dwarf virus, generated by overexpression of OsP3IP1. T499A To improve the resistance of rice to grass dwarf virus.

4. A method for improving resistance to rice straw dwarf virus, characterized in that: Overexpression of the P3IP1 protein mutant OsP3IP1 in rice T499A To improve resistance to rice straw dwarf virus, the OsP3IP1 T499A The amino acid sequence is shown in SEQ ID NO.2.

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