Plant antivirus-related miR159a and application thereof in improving virus resistance of plants

Abstract

The invention discloses miR159a related to plant antivirus and application of the miR159a in improvement of virus resistance of plants, and belongs to the technical field of genetic engineering. The miR159a related to the plant antivirus is nbe-miR159a, and the nucleotide sequence of the miR159a related to the plant antivirus is shown as SEQ ID NO: 1. The target gene of the nbe-miR159a is a CPR5 gene, an overexpression vector of the nbe-miR159a is constructed, the overexpression vector is converted into a tobacco plant, the expression quantity of the nbe-miR159a in tobacco is increased, and the relative expression quantity of CPR5 can be reduced, so that the resistance of the tobacco to the potato Y virus is improved. Therefore, the nbe-miR159a has an important application prospect in prevention and treatment of plant virus diseases and improvement of virus resistance of plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to miR159a related to plant antiviral resistance and its application in improving plant resistance to viruses. Background Art

[0002] Plant viral diseases are a major biological threat to global food security, causing economic losses exceeding US$30 billion annually. Among them, Begomoviruses, Tospoviruses, and Potyviruses are the three most devastating virus groups, causing yield reductions or even complete failure in major crops such as cassava, peanuts, and potatoes. For example, Potato virus Y (PVY) has a wide host range and is transmitted by aphids. Under extreme conditions, it can cause an 80% yield loss. Its single-stranded RNA genome encodes a multifunctional protein that interacts complexly with the host immune system during infection, making it difficult to effectively contain with traditional control measures.

[0003] The core mechanisms of plant antiviral immunity include pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). CPR5 (Constitutive expressor of pathogenesis-related genes 5), a component of the nuclear pore complex, plays a dual role in the immune regulatory network: on the one hand, it negatively regulates the ETI signaling pathway by binding to RNA recognition motifs (RRMs), inhibiting programmed cell death (PCD); on the other hand, it participates in pre-mRNA splicing, cell cycle regulation, and endoplasmic reticulum stress response, and its loss-of-function mutants can activate the expression of immune-related genes. Studies have shown that CPR5 dynamically regulates the balance between immunity and development by forming complexes with cyclin-dependent kinase inhibitors (CKIs), splicing activators NTC, and polyadenylation factor CPSF, but the specific molecular mechanisms remain to be elucidated.

[0004] MicroRNAs (miRNAs), highly conserved non-coding regulatory factors in eukaryotes, can precisely regulate immune-related genes by targeting mRNA for degradation or inhibiting translation. In virus-host interactions, host miRNAs can enhance resistance by silencing viral genes or host negative regulatory factors. Viruses can also encode miRNAs that hijack host regulatory networks, such as the Epstein-Barr virus miR-BART2, which maintains latent infection by targeting viral genes.

[0005] At present, traditional chemical control methods are inefficient in preventing and controlling plant viral diseases and are prone to causing environmental problems. The development of a miRNA related to plant antiviral resistance will not only help deepen our understanding of the host-virus game mechanism, but also provide new tools for the research and development of breakthrough plant disease resistance strategies, which is of great value to ensuring food security and sustainable agricultural development. Summary of the Invention

[0006] In view of the problems existing in the prior art, the object of the present invention is to provide miR159a related to plant antiviral activity and its application in improving plant resistance to viruses.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The miR159a related to plant antiviral activity is nbe-miR159a, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0008] The application of the above-mentioned miR159a related to plant antiviral resistance in improving plant resistance to viruses improves plant resistance to viruses by negatively regulating the expression of the CPR5 gene.

[0009] On the basis of the above scheme, the expression of CPR5 gene was negatively regulated by increasing the expression level of nbe-miR159a in plants.

[0010] On the basis of the above scheme, an overexpression vector of nbe-miR159a was constructed and transformed into plants, thereby increasing its expression level in the plants.

[0011] On the basis of the above scheme, the method of transforming the plant body is one of the following methods: Agrobacterium-mediated method, gene gun method, electric shock method, PEG method, and liposome method.

[0012] Based on the above scheme, the Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

[0013] Based on the above solution, the plant is tobacco.

[0014] Based on the above scheme, the virus is potato virus Y.

[0015] Advantages of the technical solution of the present invention The present invention provides a miR159a related to plant antiviral resistance, wherein the miR159a is nbe-miR159a, and its nucleotide sequence is shown in SEQ ID NO: 1. The CPR5 gene is a negative immune regulatory factor of plants. By inhibiting the expression of the CPR5 gene, the resistance of tobacco to potato virus Y can be improved. However, the negative immune regulatory mechanism of the CPR5 gene is complex, and direct gene editing will affect the growth and development of the plant. The target gene of nbe-miR159a is the CPR5 gene. An overexpression vector of nbe-miR159a is constructed and transformed into tobacco plants to increase the chronological expression of nbe-miR159a in tobacco, which can reduce the relative expression of CPR5, thereby improving the resistance of tobacco to potato virus Y. Therefore, nbe-miR159a has important application prospects in the prevention and control of plant viral diseases and improving plant resistance to viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a map of the nucleic acid mutation sites of three homozygous cpr5 mutation lines; Figure 2 The phenotypes of cpr5-KO plants and wild-type WT plants 7 days after PVY infection; Figure 3 The relative PVY RNA accumulation in cpr5-KO plants and wild-type WT plants was detected by qRT-PCR; Figure 4 Western blotting was used to detect the expression of PVY coat protein in cpr5-KO plants and wild-type WT plants; Figure 5 The phenotypes of CPR5-OE plants and wild-type WT plants 7 days after PVY infection; Figure 6 The relative expression of CPR5 in CPR5-OE plants and wild-type WT plants was detected by qRT-PCR; Figure 7 The relative PVY RNA accumulation in CPR5-OE plants and wild-type WT plants was detected by qRT-PCR; Figure 8 Western blotting was used to detect the expression of PVY coat protein in CPR5-OE plants and wild-type WT plants; Figure 9 Comparison of the growth of two CPR5-OE strains and two cpr5-KO strains of Nicotiana benthamiana (scale bar: 5 cm); Figure 10 Predict target gene expression for qRT-PCR detection; Figure 11 Figure 1: Fluorescein in vitro activity imaging assay. Figure 12Dual-luciferase reporter gene assay was used to verify the target genes of nbe-miR159a; Figure 13 Verify target gene binding sites for 5'RLM-RACE assay; Figure 14 Electropherograms for 5'RLM-RACE assay to verify target gene binding sites (A is the full-length CPR5 CDS; B is the length after cleavage); Figure 15 The phenotypes of p35S:nbe-miR159a plants and control group NC plants 7 days after PVY infection; Figure 16 The relative levels of nbe-miR159a in p35S:nbe-miR159a plants and control NC plants were detected by qRT-PCR; Figure 17 The relative PVY RNA accumulation in p35S:nbe-miR159a plants and control NC plants was detected by qRT-PCR; Figure 18 Western blotting was used to detect the expression of PVY coat protein in p35S:nbe-miR159a plants and control NC plants; Figure 19 Phenotypes of TRV-miR159a plants and TRV:00 control plants 7 days after PVY infection (scale bar: 3 cm); Figure 20 The relative levels of nbe-miR159a in TRV-miR159a plants and TRV:00 control plants were detected by qRT-PCR; Figure 21 The relative PVY RNA accumulation in TRV-miR159a plants and TRV:00 control plants was detected by qRT-PCR; Figure 22 Western blotting was used to detect the expression of PVY coat protein in TRV-miR159a plants and TRV:00 control group plants.

[0017] In the above figures, “*” indicates p < 0.05; “**” indicates p < 0.01; “***” indicates p < 0.001; “****” indicates p < 0.0001; and “ns” indicates no significant difference. DETAILED DESCRIPTION

[0018] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. Below, in conjunction with specific examples, the present invention will be further described in detail with reference to data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0019] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. The experimental materials, reagents, and drugs used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0020] The Nicotiana benthamiana and plant virus sources used in the following examples were all cultured by the Plant Protection Research Group of the Tobacco Research Institute. Nicotiana benthamiana was cultured in a greenhouse under conditions of 55% ± 5% humidity, 25°C ± 1°C, and a photoperiod of 16 h / 8 h.

[0021] Plasmid pRS300 was provided by Addgene Beijing Zhongyuan Company; pC1300s-GFP, Escherichia coli with the pGWC entry vector, and the Gateway100 expression vector were deposited by the Tobacco Research Institute, Chinese Academy of Agricultural Sciences; pTRV1, pTRV2, and pTRV2-PDS were provided by Professor Liu Yule's laboratory at Tsinghua University.

[0022] Example 1 A single-stranded small molecule RNA, wherein the small molecule RNA is nbe-miR159a, and its nucleic acid sequence is shown in SEQ ID NO: 1.

[0023] nbe-miR159a:5'-AAGCCTGCCGACCTATGGATTCC-3' (SEQ ID NO: 1).

[0024] Example 2 Improving plant resistance to PVY infection by regulating the expression of the CPR5 gene 1. Effect of targeted knockout of the CPR5 gene on plant resistance to PVY infection CRISPR-Cas9 gene editing technology was used to target and knock out the CPR5 gene in Nicotiana benthamiana, successfully creating a gene knockout mutant (cpr5-KO). The nucleic acid sequence of the Nicotiana benthamiana CPR5 gene is shown in SEQ ID NO: 2. Three homozygous mutant lines (4-3, 6-7, and 19-15) were obtained through T2 generation genetic segregation screening. The genomic target site sequencing results showed the characteristics of frameshift mutations after the deletion of 4 bases, the deletion of 7 bases, and the insertion of 1 base, respectively ( Figure 1 ).

[0025] PVY infection test: PVY-infected tobacco leaves (systemic infection stage) were homogenized in pre-chilled 0.03 M potassium phosphate buffer (pH 7.2) or PBS buffer containing 1% (w / v) sodium sulfite (1:30, w / v). The mixture was centrifuged at 12,000 × g for 10 min at 4°C to remove tissue debris. The supernatant was used as the crude virus extract. The OD value of the crude virus extract was measured by UV spectrophotometry. 260 value, adjust the final concentration of the inoculum to 0.8-1.2OD 260 / mL. The dilution ratio is usually 1:30 (infected leaf weight / buffer volume), ensuring that the per unit area (cm 2 The inoculum volume was approximately 10 μL. PVY virus solution was inoculated into leaves of healthy wild-type (WT) and cpr5-KO Nicotiana benthamiana plants using mechanical friction inoculation. The experiment was repeated three times for each group, and the disease progression was observed.

[0026] Compared with the wild type (WT), the cpr5-KO plants showed milder disease severity 7 days after PVY infection, with no obvious new leaf deformity symptoms ( Figure 2 ). The results of qRT-PCR detection of relative PVY RNA accumulation in cpr5-KO plants and wild-type plants WT showed that the relative expression of PVY gene in cpr5-KO plants was significantly lower than that in WT 3d, 5d and 7d after PVY infection ( Figure 3 ), WB was used to further analyze the expression of PVY coat protein in cpr5-KO plants and wild-type plants WT, and β-Actin was used as an internal reference protein. The results are as follows Figure 4 The data were normalized using the PVY / β-Actin ratio, and the results are shown in Table 1. 7 days after PVY infection, the accumulation of PVY virus particle protein in cpr5-KO plants was significantly reduced compared with that in wild-type plants WT.

[0027] The above results indicate that CPR5 functional loss can effectively enhance the host's antiviral ability.

[0028] Table 1 PVY / β-Actin ratio data homogenization SEQ ID NO:2 (5'→3') 2. Effect of overexpression of the CPR5 gene on plant resistance to PVY infection Using Nicotiana benthamiana cDNA as a template, primers (Super-1300-F and Super-1300-R) were used to amplify the CPR5 gene sequence to obtain the target fragment. The pC1300s-GFP overexpression vector was double-digested with KpnI and BamHI and the linear fragment was recovered. It was recombined with the CPR5 amplified product and the recombinant product was transformed into Escherichia coli. The monoclonal strain was selected and shaken, and the positive clones were verified by colony PCR using primers and sequenced. The one that was verified correctly was the successfully constructed CPR5-GFP fusion overexpression vector. The CPR5-GFP fusion overexpression vector was sent to Wuhan Tianwen Biological Company for tissue culture experiments to complete genetic transformation. After obtaining the T0 generation, its stable genetic expression was verified. Three Nicotiana benthamiana stable genetic transformation strains (CPR5 1 -OE, CPR5 2 -OE, CPR5 3 -OE).

[0029] The primer sequences used are as follows: Super-1300-F: 5'-CTGCAGGGGCCCGGGGTCGACATGCTTGGTGTACCACAAACTCC -3' (SEQ ID NO: 3); Super-1300-R: 5'-GCCCTTGCTCACCATGGTACCGTATTCAACTGGAGTTATAAAAGCG TC-3' (SEQ ID NO: 4); CPR5 gene overexpression plants (CPR5-OE) and wild-type WT plants were infected with PVY using the same method as above. After 7 days of PVY infection, the phenotypes of CPR5-OE plants and wild-type WT plants were observed. Figure 5 As shown, CPR5-OE plants showed a typical disease-susceptible phenotype: the new leaves at the top showed obvious curling.

[0030] The relative expression levels of CPR5 in three CPR5-OE lines and wild-type WT plants were detected by qRT-PCR. Figure 6 As shown, the relative expression levels of CPR5 in the three CPR5-OE lines were significantly higher than that in the WT control group.

[0031] qRT-PCR was used to detect the relative PVY RNA accumulation in CPR5-OE strains and wild-type WT plants after PVY infection. Figure 7 As shown in the figure, 3d, 5d and 7d after PVY infection, the relative PVY RNA accumulation in CPR5-OE plants was significantly higher than that in wild-type WT plants.

[0032] Western blotting was used to further analyze the expression of PVY protein in CPR5-OE strains and wild-type plants WT, and β-Actin was used as an internal reference protein. Figure 8 The data were normalized using the PVY / β-Actin ratio, and the results are shown in Table 2. After 5 days and 7 days of PVY infection, the accumulation of PVY virus particle protein in CPR5-OE plants was significantly increased compared with the wild-type plants WT.

[0033] Table 2 PVY / β-Actin ratio data homogenization The above results show that overexpression of CPR5 significantly weakens the host defense response. CPR5 acts as a host factor to negatively regulate the plant antiviral immune response. CPR5 coordinates the plant's immune response and normal growth and development by affecting RNA alternative splicing. In addition, by comparing the growth of cpr5-KO plants and CPR5-OE plants, it can be found that complete knockout of CPR5 (cpr5 19 -KO, cpr5 6 -KO) affects the growth of Nicotiana benthamiana ( Figure 9 ).

[0034] Example 3 Construction of nbe-miR159a transient overexpression vector (p35S:nbe-miR159a) based on p35S promoter (1) Using plasmid pRS300 as a template, the ath-MIR319a sequence in the pBluescript SK backbone was replaced by the nbe-miR159a precursor backbone sequence through overlapping PCR, while retaining the oligonucleotide sequence that forms the stem-loop structure protrusion in the MIR319a precursor. In the first set of PCR, the 5' arm (fragment a), the central loop (fragment b), and the 3' arm (fragment c) were amplified, and the anbe-miR159a* sequence was used to replace amiR319a. The three fragments were then fused in a PCR reaction (reaction d). The reaction procedure is shown in Table 3: Table 3 Overlap PCR reaction program The primer sequences used are as follows: Table 4 Primer sequences used in overlapping PCR reactions (2) The product (fragment d) after the reaction in step (1) above was subjected to agarose gel electrophoresis and gel recovery to obtain anbe-miR159a. The gel recovery method was referred to the instructions of the EasyPure® Quick Gel Extraction Kit DNA gel rapid purification kit.

[0035] (3) Extract the pGWC entry vector from E. coli harboring the pGWC entry vector. Refer to the instructions of the EasyPure® Plasmid MiniPrep Kit for the plasmid extraction method.

[0036] (4) The pGWC entry vector plasmid was digested with the restriction endonuclease AhdI (BioLabs) to obtain a linearized pGWC entry vector. The linear fragment of the pGWC entry vector was recombined with the anbe-miR159a fragment using a one-step cloning method to obtain the pGWC-anbe-miR159a recombinant vector. E. coli was transformed and sequenced to obtain the correct vector. The one-step cloning method was performed according to the instructions of the ClonExpress II One Step Cloning Kit.

[0037] (5) The pGWC-anbe-miR159a vector with the correct sequencing results was subjected to LR reaction with the Gateway100 expression vector to construct the p35S:nbe-miR159a expression vector.

[0038] Method for obtaining p35S:nbe-miR159a-LBA4404 recombinant bacteria: Plasmids were extracted from the p35S:nbe-miR159a expression vectors with correct sequencing results, and Agrobacterium transformation was performed. The Agrobacterium transformation procedure was based on the instructions for LBA4404 Agrobacterium competent cells (Bomaide Biotechnology) to obtain p35S:nbe-miR159a-LBA4404 recombinant bacteria.

[0039] Example 4 An infection solution for improving plant resistance to PVY virus: (1) Centrifuge the p35S:nbe-miR159a-LBA4404 recombinant bacterial solution at 8000 rpm for 4 min to collect the cells; (2) Add about 5 mL of resuspension solution to the collected bacteria, wash away the antibiotics, resuspend to dissolve the precipitate, centrifuge at 8000 rpm for 4 min, and discard the supernatant; Resuspension preparation (1 L): 100 mL, 0.1 mol / L MES; 10 mL, 1 mol / L MgCl2; 1 mL acetosyringone (As).

[0040] (3) Add the resuspension solution to the bacteria in step (2) and resuspend the bacteria to an OD of 600 The value is about 0.8-1.0; the infection solution is obtained and stored away from light.

[0041] Example 5 Target genes of nbe-miR159a and their molecular interaction mechanism Based on the molecular mechanism by which miRNAs mediate target mRNA degradation or translational repression through sequence complementarity, we focused on functional validation and target gene identification of nbe-miR159a. Using psRNATarget software, we predicted and screened nbe-miR159a target genes (score > 160), identifying five candidate target genes (Niben101Scf00319g00006.1, Niben101Scf10749g00007.1, Niben101Scf01182g01020.1, Niben101Scf07296g01002.1, and Niben101Scf01269g05002.1), all of which had binding sites located in the coding regions (CDS).

[0042] Nicotiana benthamiana was injected (transiently infiltrated) with the infection solution of the p35S:nbe-miR159a-LBA4404 recombinant bacteria and the LBA4404 (control) bacteria containing the empty vector. After 48 hours of treatment, the relative expression levels of the five candidate target genes in the different treatment groups were detected. qRT-PCR was used to detect the relative expression levels of the five candidate target genes in the p35S:nbe-miR159a overexpressing plants and the control plants. The results are shown in Figure 2. Figure 10 As shown in the results, differential expression analysis showed that only the CPR5 gene (Niben101Scf00319g00006.1) was significantly downregulated in p35S:nbe-miR159a overexpressing plants (p<0.001), and thus this gene was considered to be a functional target of nbe-miR159a.

[0043] To elucidate the molecular interaction mechanism between nbe-miR159a and CPR5, a CPR5-luciferase reporter system was constructed. Using an Agrobacterium-mediated transient co-expression system, Agrobacterium carrying the reporter vector and Agrobacterium carrying the p35S:nbe-miR159a vector were co-infiltrated into Nicotiana benthamiana leaves. The empty vector served as the control (NC), and Renilla luciferase was used as the internal control. The dual-luciferase in vivo imaging results are shown in Figure 2. Figure 11and Figure 12 As shown in the figure, the luciferase activity in the treated group was significantly lower than that in the control group (p<0.001), indicating that nbe-miR159a can inhibit the expression of CPR5. To confirm the specificity of the binding site, a CPR5 binding domain mutant (CPR5m) was designed based on the predicted binding domain, and a CPR5m-luciferase reporter system was constructed. The CPR5m-luciferase reporter system was co-infiltrated with Agrobacterium carrying the p35S:nbe-miR159a vector into Nicotiana benthamiana leaves. The results are shown in the figure. Figure 11 and Figure 12 As shown in the figure, mutant detection found that nbe-miR159a completely eliminated the inhibitory effect of the mutant reporter vector (p>0.05), confirming that targeted binding depends on the integrity of the predicted site. By 5'RLM-RACE technology, a specific splicing site was detected in p35S:nbe-miR159a overexpressing plants ( Figure 13 ), the electrophoresis results of the target gene binding site were verified by 5'RLM-RACE test. Figure 14 As shown, its location is consistent with the predicted binding domain. The above results systematically confirmed that nbe-miR159a mediates post-transcriptional silencing by precisely targeting the CDS region of CPR5 mRNA.

[0044] Example 6 Application of nbe-miR159a in enhancing plant resistance to PVY PVY infection test: The infection solution of the p35S:nbe-miR159a-LBA4404 recombinant bacteria (p35S:nbe-miR159a) prepared by the method of Example 4 and the bacterial solution of LBA4404 transformed with an empty vector (control NC) were respectively injected (transient infiltration) into Nicotiana benthamiana. After 24 hours, the PVY virus solution (the preparation method of the PVY virus solution is the same as above) was frictionally inoculated onto the two largest expanded leaves of Nicotiana benthamiana. The two upper systemic leaves of Nicotiana benthamiana were tested 3d, 5d, and 7d after PVY infection.

[0045] Seven days after PVY infection, the disease progression of the p35S:nbe-miR159a-LBA4404-treated plants and the empty vector control plants was observed. Figure 15 As shown, compared with the empty vector control (NC), the p35S:nbe-miR159a-LBA4404 plants showed a significant resistance phenotype: new leaves did not show typical curling symptoms, and the systemic infection process was significantly delayed.

[0046] qRT-PCR was used to detect the relative levels of nbe-miR159a and relative PVY RNA accumulation in p35S:nbe-miR159a-LBA4404-treated plants and empty vector control plants (NC) after PVY infection. Figure 16 and Figure 17As shown in Figure 2, the relative level of nbe-miR159a in plants treated with p35S:nbe-miR159a-LBA4404 was significantly higher than that in the NC control group; the relative expression of PVY in plants treated with p35S:nbe-miR159a-LBA4404 was significantly lower than that in the control group. Western blotting was used to further analyze the expression of PVY protein in plants treated with p35S:nbe-miR159a-LBA4404 and empty vector control plants (NC), using β-Actin as an internal reference protein. Figure 18 The data were normalized using the PVY / β-Actin ratio, and the results are shown in Table 5. 3d, 5d, and 7d after PVY infection, the accumulation of PVY virus particle protein in the p35S:nbe-miR159a-LBA4404 plants was significantly inhibited compared with the control plants (NC), indicating that nbe-miR159a can effectively enhance the host's antiviral ability.

[0047] Table 5 PVY / β-Actin ratio data homogenization Example 7 Method for constructing the nbe-miR159a silencing system (TRV2-miR159a) using short tandem target mimic (STTM) technology: The STTM-nbe-miR159a sequence (SEQ ID NO: 1) was designed using the PmiREN website (https: / / www.pmiren.com / tooloverexpress) and fully synthesized by Shanghai Sangon Biotechnology Co., Ltd. This sequence was homologously recombined into the pTRV2 vector (with restriction enzyme sites EcoR I and Kpn I) to generate the nbe-miR159a silencing vector (TRV2-miR159a).

[0048] Plasmids from the TRV2-miR159a silencing vectors with correct sequencing results were extracted and transformed with Agrobacterium. The pTRV1, pTRV2, and pTRV2-PDS vectors were also transformed into Agrobacterium. For Agrobacterium transformation procedures, refer to the instructions for LBA4404 Agrobacterium competent cells (Bomaide Biotech).

[0049] Example 8 Application of nbe-miR159a in reducing plant resistance to PVY PVY infection test: The correctly sequenced Agrobacterium carrying pTRV2-miR159a, pTRV1, pTRV2, and pTRV2-PDS were incubated in a shaking incubator at 28°C overnight. The bacteria were harvested on the second day and resuspended in infiltration buffer [10 mM MES (pH 5.6), 10 mM MgCl2, and 200 μM AS] until the OD 600 The pTRV1 Agrobacterium suspension was mixed with pTRV2, pTRV2-PDS, and pTRV2-miR159a Agrobacterium suspensions at a 1:1 volume ratio. The mixture was allowed to stand at room temperature in the dark for 3 hours. The leaves were then infiltrated with a needleless syringe for VIGS. Around 13 days after the new leaves of the TRV-PDS control (positive control, a mixture of pTRV1 and pTRV2-PDS Agrobacterium) showed complete bleaching, leaves of N. benthamiana treated with TRV-miR159a (a mixture of pTRV1 and pTRV2-miR159a) and TRV:00 (negative control, a mixture of pTRV1 and pTRV2 Agrobacterium) were inoculated with PVY at the same position (PVY virus solution preparation method was the same as above). Two upper systemic leaves of N. benthamiana were collected for testing 3, 5, and 7 days after PVY infection.

[0050] Seven days after PVY infection, the disease incidence of TRV-miR159a infected plants and TRV:00 control plants was observed. Figure 19 As shown, compared with TRV:00, TRV-miR159a-infected plants showed typical disease phenotype: the newly grown leaves at the top showed obvious curling.

[0051] The relative levels of nbe-miR159a in TRV-miR159a-infected plants and TRV:00 control plants were detected by qRT-PCR. Figure 20 As shown, the relative level of nbe-miR159a in TRV-miR159a plants was significantly lower than that in TRV:00 control plants.

[0052] The relative PVY RNA accumulation in TRV-miR159a infected plants and TRV:00 control plants after PVY infection was detected by qRT-PCR. Figure 21 As shown in Figure 2, the relative expression of PVY in TRV-miR159a infected plants was significantly higher than that in the TRV:00 control plants at 5 and 7 days after PVY infection. WB was used to further analyze the expression of PVY protein in TRV-miR159a infected plants and TRV:00 control plants, and β-Actin was used as an internal reference protein. The results are shown in Figure 2. Figure 22The data were normalized using the PVY / β-Actin ratio, and the results are shown in Table 6. 5d and 7d after PVY infection, the accumulation of PVY virus particle protein in TRV-miR159a-infected plants was significantly increased compared with that in the TRV:00 control group, confirming that the inhibition of nbe-miR159a function significantly enhanced the host's susceptibility to PVY.

[0053] Table 6 PVY / β-Actin ratio data homogenization The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. miR159a associated with plant antiviral resistance, characterized in that The miR159a is nbe-miR159a, and its nucleotide sequence is shown in SEQ ID NO:

1.

2. The use of miR159a related to plant antiviral activity according to claim 1 in improving plant resistance to viruses, characterized in that: By negatively regulating the expression of the CPR5 gene, the plant's resistance to viruses can be improved.

3. The use of plant antiviral-related miR159a in improving plant resistance to viruses according to claim 2, characterized in that: By increasing the expression level of nbe-miR159a in plants, the expression of the CPR5 gene is negatively regulated.

4. The use of plant antiviral-related miR159a in improving plant resistance to viruses according to claim 3, characterized in that: An nbe-miR159a overexpression vector is constructed and transformed into a plant, thereby increasing its expression level in the plant.

5. The use of plant antiviral-related miR159a in improving plant resistance to viruses according to claim 4, characterized in that: The method of transforming the plant body is one of the following: Agrobacterium-mediated method, gene gun method, electric shock method, PEG method, and liposome method.

6. The use of plant antiviral-related miR159a in improving plant resistance to viruses according to claim 5, characterized in that: The Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

7. The use of the plant antiviral-related miR159a according to any one of claims 2 to 6 in improving plant resistance to viruses, characterized in that: The plant is tobacco.

8. The use of plant antiviral-related miR159a in improving plant resistance to viruses according to claim 7, characterized in that: The virus is potato virus Y.

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