MiR159a related to plant virus resistance and application thereof in improving plant resistance to virus

Abstract

The application discloses a plant virus resistance related miR159a and application thereof in improving plant virus resistance, and belongs to the technical field of genetic engineering. The plant virus resistance related miR159a is nbe-miR159a, and the nucleotide sequence 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 nbe-miR159a is transformed into a tobacco plant, the expression amount of the nbe-miR159a in the tobacco is increased, the relative expression amount of the CPR5 is reduced, and thus the resistance of the tobacco to a potato Y virus is improved. Therefore, the nbe-miR159a has an important application prospect in plant virus disease prevention and treatment and improvement of plant virus resistance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to miR159a related to plant antiviral and application thereof in improving plant resistance to viruses. BACKGROUND

[0002] Plant viral diseases are major biological disasters threatening global food security, causing more than 30 billion US dollars in economic losses each year. Among them, Begomoviruses, Tospoviruses and Potyviruses are the three most serious viral groups, which can cause cassava, peanut, potato and other major crops to reduce yield or even fail. For example, Potato virus Y (PVY) has a wide host range and can be transmitted by aphids, which can cause 80% yield loss under extreme conditions. The multifunctional protein encoded by the single-stranded RNA genome of PVY interacts with the host immune system in the infection process, and traditional control measures are difficult to effectively curb.

[0003] The core mechanism of plant antiviral immunity includes pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), and CPR5 (Constitutive expressor of pathogenesis-related genes 5) as a component of nuclear pore complex plays a dual role in the immune regulation network: on the one hand, it negatively regulates the ETI signaling pathway through the binding of RNA recognition motif (RRM) to inhibit programmed cell death (PCD); on the other hand, it is involved in pre-mRNA splicing, cell cycle regulation and endoplasmic reticulum stress response, and its loss-of-function mutant can activate the expression of immune-related genes. Studies have shown that CPR5 forms a complex with cell cycle protein-dependent kinase inhibitors (CKIs), splicing activator NTC and polyadenylation factor CPSF, dynamically regulates the balance between immunity and development, but the specific molecular mechanism remains to be analyzed.

[0004] MicroRNA (miRNA) as a highly conserved non-coding regulatory factor in eukaryotes can precisely regulate immune-related genes by targeted degradation of mRNA or inhibition of translation. In virus-host interaction, host miRNA can enhance resistance by silencing viral genes or host negative regulatory factors; and viruses can also encode miRNA to hijack host regulatory networks, for example, EB virus miR-BART2 maintains latent infection by targeting viral genes.

[0005] At present, the traditional chemical prevention and control method has low prevention and control efficiency on plant virus disease and easily causes environmental problems; development of a miRNA related to plant antiviral is not only beneficial to deepening the understanding of the host-virus game mechanism, but also provides a new tool for the research and development of a breakthrough plant antiviral strategy, and has important value for guaranteeing food safety and sustainable development of agriculture. SUMMARY

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

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] The miR159a related to plant antiviral is nbe-miR159a, and the nucleotide sequence thereof is shown in SEQ ID NO: 1.

[0009] The application of the above-mentioned miR159a related to plant antiviral in improving plant resistance to viruses is to improve the resistance of plants to viruses by negatively regulating the expression of the CPR5 gene.

[0010] On the basis of the above-mentioned scheme, the expression amount of nbe-miR159a in the plant body is increased, and the expression of the CPR5 gene is negatively regulated.

[0011] On the basis of the above-mentioned scheme, an overexpression vector of nbe-miR159a is constructed, and the plant body is transformed, so as to increase the expression amount thereof in the plant body.

[0012] On the basis of the above-mentioned scheme, the method for transforming the plant body is one of Agrobacterium-mediated method, gene gun method, electric shock method, PEG method and liposome method.

[0013] On the basis of the above-mentioned scheme, the Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

[0014] On the basis of the above-mentioned scheme, the plant is tobacco.

[0015] On the basis of the above-mentioned scheme, the virus is potato Y virus.

[0016] Advantages of the technical scheme of the present application

[0017] The application provides a plant virus resistance related miR159a, and the nucleotide sequence of the miR159a is shown as SEQ ID NO:1. The CPR5 gene is an immune negative regulator of a plant, and by inhibiting the expression of the CPR5 gene, the resistance of tobacco to potato virus Y can be improved. However, the immune negative regulation mechanism of the CPR5 gene is complex, and directly editing the gene will affect the growth and development of the plant. The target gene of the nbe-miR159a is the CPR5 gene, an overexpression vector of the nbe-miR159a is constructed, the nbe-miR159a is transformed into tobacco plants, the expression amount of the nbe-miR159a in the tobacco is improved, the relative expression amount of the CPR5 is reduced, and therefore the resistance of the tobacco to the potato virus Y is improved. Therefore, the nbe-miR159a has important application prospects in the prevention and treatment of plant viral diseases and the improvement of the resistance of plants to viruses. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a nucleic acid mutation site map of three cpr5 mutant homozygous lines;

[0019] Figure 2 It is a phenotype map of cpr5-KO plants and wild type WT plants after PVY infection for 7 days;

[0020] Figure 3 It is a qRT-PCR detection of the relative PVY RNA accumulation amount in cpr5-KO plants and wild type WT plants;

[0021] Figure 4 It is a WB detection of the PVY coat protein expression in cpr5-KO plants and wild type WT plants;

[0022] Figure 5 It is a phenotype map of CPR5-OE plants and wild type WT plants after PVY infection for 7 days;

[0023] Figure 6 It is a qRT-PCR detection of the relative CPR5 expression amount in CPR5-OE plants and wild type WT plants;

[0024] Figure 7 It is a qRT-PCR detection of the relative PVY RNA accumulation amount in CPR5-OE plants and wild type WT plants;

[0025] Figure 8 It is a WB detection of the PVY coat protein expression in CPR5-OE plants and wild type WT plants;

[0026] Figure 9 Comparison of the growth of two CPR5-OE lines and two cpr5-KO lines of Nicotiana benthamiana (the scale is 5 cm).

[0027] Figure 10 For qRT-PCR detection of predicted target gene expression;

[0028] Figure 11 Fluorescent in vitro activity imaging test chart;

[0029] Figure 12 Dual luciferase reporter gene test to verify the target gene of nbe-miR159a;

[0030] Figure 13 5' RLM-RACE test to verify the target gene binding site;

[0031] Figure 14 Electrophoresis chart for 5' RLM-RACE test to verify the target gene binding site (where A is the full length of CPR5 CDS; B is the length after cutting);

[0032] Figure 15 p35S:nbe-miR159a plants and control group NC plants after PVY infection for 7d phenotype chart;

[0033] Figure 16 qRT-PCR detection of the relative level of nbe-miR159a in p35S:nbe-miR159a plants and control group NC plants;

[0034] Figure 17 qRT-PCR detection of the relative PVY RNA accumulation in p35S:nbe-miR159a plants and control group NC plants;

[0035] Figure 18 WB detection of PVY coat protein expression in p35S:nbe-miR159a plants and control group NC plants;

[0036] Figure 19 TRV-miR159a plants and TRV:00 control group plants after PVY infection for 7d phenotype chart (scale is 3cm);

[0037] Figure 20 qRT-PCR detection of the relative level of nbe-miR159a in TRV-miR159a plants and TRV:00 control group plants;

[0038] Figure 21 qRT-PCR detection of the relative PVY RNA accumulation in TRV-miR159a plants and TRV:00 control group plants;

[0039] Figure 22To detect the expression of PVY coat protein in TRV-miR159a plants and TRV:00 control plants by WB.

[0040] 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

[0041] The terms used in the present application have the meanings generally understood by those of ordinary skill in the art, unless otherwise specified. The present application is described in further detail below in conjunction with specific examples and with reference to the data. The following examples are merely intended to illustrate the present application and in no way limit the scope of the present application.

[0042] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. In the following examples, the test materials, reagents, and drugs used, unless otherwise specified, can be purchased through general channels.

[0043] The materials such as N. benthamiana and plant virus sources in the following examples are preserved and cultured by the Plant Protection Research Group of Tobacco. N. benthamiana is cultured in a greenhouse under the conditions of humidity 55%±5%, temperature 25℃±1℃, and light cycle 16 h\8 h.

[0044] The plasmid pRS300 is provided by Addgene Beijing Zhongyuan Company;

[0045] pC1300s-GFP, E. coli with pGWC entry vector, and Gateway 100 expression vector are preserved by the Tobacco Research Institute of Chinese Academy of Agricultural Sciences;

[0046] pTRV1, pTRV2, and pTRV2-PDS are provided by the laboratory of Professor Liu Yule of Tsinghua University.

[0047] Example 1

[0048] A single-stranded small molecule RNA, which is nbe-miR159a, has a nucleic acid sequence as shown in SEQ ID NO: 1.

[0049] nbe-miR159a: 5'-AAGCTGCCGACCTATGGATTCC-3' (SEQ ID NO: 1).

[0050] Example 2

[0051] By regulating the expression amount of the CPR5 gene to improve the resistance of the plant to PVY infection

[0052] 1. Effect of targeted knockout of CPR5 gene on plant resistance to PVY infection

[0053] The CRISPR-Cas9 gene editing technology was used to target knockout of the N. benthamiana CPR5 gene, and a gene knockout mutant (cpr5-KO) was successfully created. The nucleic acid sequence of the N. benthamiana CPR5 gene is shown in SEQ ID NO: 2. Three homozygous mutant lines (4-3, 6-7, and 19-15) were obtained by T2 generation genetic segregation screening, and the genomic target site sequencing results showed frame shift mutation characteristics after deletion of 4 bases, deletion of 7 bases, and insertion of 1 base, respectively. Figure 1 ).

[0054] PVY infection test: PVY-infected tobacco leaves (systemic infection stage) were homogenized in pre-cooled 0.03 M potassium phosphate buffer (pH 7.2) or PBS buffer containing 1% (w / v) sodium sulfite (1:30, w / v), and the tissue debris was removed by centrifugation at 12,000 x g for 10 min at 4°C. The supernatant was the crude virus extract. The OD 260 value of the crude virus extract was determined by ultraviolet spectrophotometry, and the final concentration of the inoculum was adjusted to 0.8-1.2 OD 260 / mL. The dilution ratio was usually 1:30 (leaf weight / volume of buffer), ensuring an inoculum of about 10 μL per unit area (cm 2 ). The mechanical rubbing inoculation method was used to inoculate PVY virus solution to healthy N. benthamiana leaves of wild type (WT) and cpr5-KO plants, with 3 replicates for each test; the disease incidence was observed.

[0055] Compared with the wild type (WT), the cpr5-KO plants had a milder disease after 7 days of PVY infection, and no obvious new leaf deformity was observed. Figure 2 The qRT-PCR detection of the 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 after 3 days, 5 days, and 7 days of PVY infection. Figure 3 The WB further analyzed and detected the expression of PVY coat protein in cpr5-KO plants and wild type plants WT, with β-Actin as the internal reference protein, and the results are shown in Figure 4 Table 1. The PVY / β-Actin ratio was used for data normalization, and the results showed that the accumulation of PVY virion protein in cpr5-KO plants was significantly reduced compared with the wild type plants WT after 7 days of PVY infection.

[0056] In summary, the results showed that the loss of CPR5 function can effectively enhance the host's ability to resist viruses.

[0057] Table 1 PVY / β-Actin ratio to data normalization

[0058]

[0059] SEQ ID NO: 2 (5'→ 3')

[0060]

[0061] 2. Effect of overexpression of CPR5 gene on plant resistance to PVY infection

[0062] The cDNA of N. benthamiana was used as a template to amplify the CPR5 gene sequence using primers (Super-1300-F and Super-1300-R), and the target fragment was obtained. The pC1300s-GFP overexpression vector was treated with KpnI and BamHI for double enzyme digestion and the linear fragment was recovered, and then recombined with the CPR5 amplification product. The recombination product was transformed into E. coli. Single colony strains were shaken, and colony PCR was performed using primers to verify positive clones, and sequencing was performed for verification. The correct one was the successfully constructed CPR5-GFP fusion overexpression vector. The CPR5-GFP fusion overexpression vector was sent to the "Wuhan Tianwen Biological Company" to complete the genetic transformation test of tissue culture, and after obtaining the T0 generation, the stable genetic expression was verified. Three stable genetic transformation strains of N. benthamiana (CPR5-OE) were obtained. 1 -OE, CPR5 2 -OE, CPR5 3 -OE).

[0063] The primer sequences used are as follows:

[0064] Super-1300-F: 5'-CTGCAGGGGCCCGGGGTCGACATGCTTGGTGTACCACAAACTCC-3' (SEQ ID NO: 3);

[0065] Super-1300-R: 5'-GCCCTTGCTCACCATGGTACCGTATTCAACTGGAGTTATAAAAGCG TC-3' (SEQ ID NO: 4);

[0066] The CPR5 gene overexpression plants (CPR5-OE) and wild type WT plants were infected with PVY, and the method was the same as above. After 7 days of PVY infection, the phenotypes of the CPR5-OE plants and the wild type WT plants were observed as shown in Figure 5 The CPR5-OE plants showed typical diseased phenotypes: the top new leaves showed obvious curling.

[0067] qRT-PCR was used to detect the relative expression amount of CPR5 in the three CPR5-OE strains and the wild type WT plants, and the results are shown in Figure 6 The relative expression amount of CPR5 in the three CPR5-OE strains was significantly higher than that in the WT control group.

[0068] qRT-PCR was used to detect the relative PVY RNA accumulation amount in the CPR5-OE strains and the wild type WT plants after PVY infection, and the results are shown inFigure 7 As shown in Figure 6, the relative PVY RNA accumulation in CPR5-OE plants was significantly higher than that in wild type WT plants at 3d, 5d and 7d after PVY infection.

[0069] The PVY protein expression in CPR5-OE lines and wild type plants WT was further analyzed by WB, and β-Actin was used as the internal reference protein, and the results are shown in Figure 7. Figure 8 As shown in Figure 7, the data was normalized using the PVY / β-Actin ratio, and the results are shown in Table 2. The PVY virion protein accumulation in CPR5-OE plants was significantly increased compared with wild type plants WT at 5d and 7d after PVY infection.

[0070] Table 2 Normalization of data using PVY / β-Actin ratio

[0071]

[0072] The above results show that CPR5 overexpression significantly weakens the host defense response, and CPR5 as a host factor negatively regulates the plant antiviral immune response. CPR5 affects RNA alternative splicing, and coordinates between the immune response and normal growth and development of plants. 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 N. benthamiana. Figure 9

[0073] Example 3

[0074] Construction method of nbe-miR159a transient overexpression vector (p35S:nbe-miR159a) based on p35S promoter

[0075] (1) Using plasmid pRS300 as a template, the nbe-miR159a precursor backbone sequence was used to replace the ath-MIR319a sequence in the pBluescript SK backbone by overlapping PCR technology, and the oligonucleotide sequence protruding from the stem loop structure in the MIR319a precursor was retained. In the first group 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 the amiR319a. Then the three fragments were fused by PCR reaction (reaction d). The reaction program is as follows Table 3:

[0076] Table 3 Overlapping PCR reaction program

[0077]

[0078] ​The primer sequences used are as follows:

[0079] Table 4 Primer sequences used in the overlap PCR reaction

[0080]

[0081] (2) The product (fragment d) after the reaction of step (1) above was subjected to agarose gel electrophoresis and gel recovery to obtain anbe-miR159a. The gel recovery method is described in the EasyPure® Quick Gel Extraction Kit DNA Gel Rapid Purification Kit Instructions.

[0082] (3) The pGWC entry vector was extracted from E. coli with the pGWC entry vector, and the plasmid extraction method is described in the EasyPure® Plasmid MiniPrep Kit Instructions.

[0083] (4) The pGWC entry vector plasmid was digested with restriction enzyme AhdI (BioLabs) to obtain linearized pGWC entry vector. The linear fragment of the pGWC entry vector was recombined with the anbe-miR159a fragment using one-step cloning method to obtain the pGWC-anbe-miR159a recombinant vector. E. coli transformation was performed and sequencing was performed to obtain the correct vector. The one-step cloning method is described in the ClonExpress II One Step Cloning Kit Instructions.

[0084] (5) The pGWC-anbe-miR159a vector with correct sequencing results was subjected to LR reaction with Gateway 100 expression vector to construct p35S:nbe-miR159a expression vector.

[0085] Method for obtaining p35S:nbe-miR159a-LBA4404 recombinant bacteria:

[0086] The p35S:nbe-miR159a expression vector with correct sequencing results was subjected to plasmid extraction and Agrobacterium transformation. The Agrobacterium transformation method is described in the LBA4404 Agrobacterium competent cell instructions (Bomeda Biological). The p35S:nbe-miR159a-LBA4404 recombinant bacteria were obtained.

[0087] Example 4

[0088] An infection solution for improving the resistance of plants to PVY virus:

[0089] (1) The p35S:nbe-miR159a-LBA4404 recombinant bacteria liquid was centrifuged in a centrifuge at 8000 rpm for 4 min, and the bacterial bodies were collected;

[0090] (2) About 5 mL of resuspension was added to the collected bacterial bodies to wash away the antibiotics, and the precipitate was dissolved by resuspension. The bacterial bodies were centrifuged at 8000 rpm for 4 min, and the supernatant was discarded;

[0091] Resuspension preparation (1 L): 100 mL, 0.1 mol / L MES; 10 mL, 1 mol / L MgCl2; 1 mL Acetosyringone (As).

[0092] (3) The bacterial bodies in step (2) were resuspended by adding resuspension again, and the OD 600 value of the bacterial bodies was about 0.8-1.0. The obtained infection liquid was stored in the dark.

[0093] Example 5

[0094] Target genes of nbe-miR159a and their molecular interaction mechanism

[0095] Based on the molecular mechanism of miRNAs mediating the degradation or translation inhibition of target mRNA through sequence complementarity, the function verification and target gene identification of nbe-miR159a were focused. The psRNATarget software was used for nbe-miR159a target gene prediction and screening (Score>160), and 5 candidate target genes (respectively: Niben101Scf00319g00006.1, Niben101Scf10749g00007.1, Niben101Scf01182g01020.1, Niben101Scf07296g01002.1, Niben101Scf01269g05002.1) were obtained, and their binding sites were located in the coding region (CDS).

[0096] The infection liquid of p35S:nbe-miR159a-LBA4404 recombinant bacteria and the LBA4404 (control NC) bacteria liquid of the empty vector were injected (transiently infiltrated) into N. benthamiana, and the relative expression amounts of the 5 candidate target genes in different treatment groups were detected after 48 hours of treatment. The relative expression amounts of the 5 candidate target genes in the p35S:nbe-miR159a overexpression plants and the control plants NC were detected by qRT-PCR, and the results are shown in Table 1. Figure 10As shown, differential expression analysis revealed that only the CPR5 gene (Niben101Scf00319g00006.1) showed a significant downregulation trend in p35S:nbe-miR159a overexpressing plants (p<0.001), thus suggesting that this gene is a functional target of nbe-miR159a.

[0097] 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 tumefaciens carrying the reporter vector and Agrobacterium tumefaciens carrying the p35S:nbe-miR159a vector were co-infiltrated into Tobacco Bengal leaves. Empty vector was used as the control group (NC), and Renilla luciferase was used as an internal control. The in vivo imaging results of the dual-luciferase system are shown below. Figure 11 and Figure 12 As shown, the luciferase activity in the treatment group was significantly lower than that in the control group (p<0.001), indicating that nbe-miR159a can inhibit CPR5 expression. To confirm the binding site specificity, a CPR5 binding domain mutant (CPR5m) was designed based on the predicted binding domain, and a CPR5m-luciferase reporter system was constructed. This system was then co-infiltrated with *Agrobacterium* carrying the p35S:nbe-miR159a vector into *Tobacco Bengal* leaves. The results are as follows: Figure 11 and Figure 12 As shown, mutant detection revealed that the inhibitory effect of nbe-miR159a on the mutant reporter vector was completely eliminated (p>0.05), confirming that targeted binding depends on the integrity of the predicted site. Specific splicing sites were detected in p35S:nbe-miR159a overexpressing plants using 5' RLM-RACE technology. Figure 13 The electrophoresis results of the target gene binding site were verified using the 5'RLM-RACE assay, as shown in the figure below. Figure 14 As shown, its location largely matches the predicted binding domain. These results systematically confirm that nbe-miR159a mediates post-transcriptional silencing by precisely targeting the CDS region of CPR5 mRNA.

[0098] Example 6

[0099] Application of nbe-miR159a in enhancing plant resistance to PVY

[0100] PVY infection test: The infection solution of p35S:nbe-miR159a-LBA4404 recombinant bacteria (p35S:nbe-miR159a) prepared by the method in Example 4 and the LBA4404 (control NC) bacterial solution with empty vector were injected (instantly infiltrated) into Nigeriensis. After 24 h, PVY virus solution (prepared by the same method as above) was rubbed onto the two largest unfolded leaves of Nigeriensis. PVY infection was detected on the two upper systematic leaves of Nigeriensis at 3d, 5d and 7d respectively.

[0101] The p35S:nbe-miR159a-LBA4404 treated plants and the empty vector control plants were observed for disease symptoms 7 days after PVY infection, and the results are shown in Figure 15 Compared with the empty vector control (NC), the p35S:nbe-miR159a-LBA4404 plants showed a significant resistance phenotype: the newly grown leaves did not show typical crinkling symptoms, and the systemic infection process was significantly delayed.

[0102] The relative levels of nbe-miR159a and the relative PVY RNA accumulation in the p35S:nbe-miR159a-LBA4404 treated plants and the empty vector control plants (NC) after PVY infection were detected by qRT-PCR, and the results are shown in Figure 16 and Figure 17 The relative levels of nbe-miR159a in the p35S:nbe-miR159a-LBA4404 treated plants were significantly higher than those in the NC control group, and the relative expression of PVY in the p35S:nbe-miR159a-LBA4404 plants was significantly lower than that in the control group. The PVY protein expression in the p35S:nbe-miR159a-LBA4404 plants and the empty vector control plants (NC) was further analyzed and detected by WB, and β-Actin was used as the internal reference protein, and the results are shown in Figure 18 The data were normalized using the PVY / β-Actin ratio, and the results are shown in Table 5. After 3 days, 5 days and 7 days of PVY infection, the accumulation of PVY virion 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 ability to resist viruses.

[0103] Table 5. Normalization of data using the PVY / β-Actin ratio

[0104]

[0105] Example 7

[0106] The nbe-miR159a silencing system (TRV2-miR159a) was constructed using the short tandem target mimic (STTM) technology.

[0107] The STTM-nbe-miR159a sequence (SEQ ID NO:1) was designed using the PmiREN website (https: / / www.pmiren.com / tooloverexpress) and the whole genome was synthesized by Shanghai Sangon Biotech Co., Ltd. This sequence was homologously recombinated into the pTRV2 vector (with EcoRI and KpnI restriction sites) to obtain the nbe-miR159a silencing vector (TRV2-miR159a).

[0108] Plasmids were extracted from the TRV2-miR159a silencing vector with correct sequencing results, and then transformed into Agrobacterium. pTRV1, pTRV2, and pTRV2-PDS vectors were also transformed into Agrobacterium. The Agrobacterium transformation procedure was performed according to the LBA4404 Agrobacterium competent cell instruction manual (Bomaide Biotechnology).

[0109] Example 8

[0110] Application of nbe-miR159a in reducing plant resistance to PVY

[0111] PVY infection test:

[0112] Agrobacterium cells correctly sequenced and carrying pTRV2-miR159a, pTRV1, pTRV2, and pTRV2-PDS were incubated overnight in a shaker at 28°C. The cells 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 OD500 was reached. 600 The concentration reached 0.8. The Agrobacterium tumefaciens resuspension of pTRV1 was mixed with Agrobacterium tumefaciens resuspensions of pTRV2, pTRV2-PDS, and pTRV2-miR159a at a volume ratio of 1:1. After standing for 3 hours in the dark at room temperature, the mixture was applied to tobacco leaves using a needle-free syringe for the VIGS test. After approximately 13 days, when the new leaves of the TRV-PDS control (positive control, a mixture of Agrobacterium tumefaciens pTRV1 and pTRV2-PDS) showed complete whitening, PVY inoculation was performed on leaves at the same location in the TRV-miR159a (a mixture of Agrobacterium tumefaciens pTRV1 and pTRV2-miR159a) and TRV:00 (negative control, a mixture of Agrobacterium tumefaciens pTRV1 and pTRV2) treatment groups (PVY virus solution was prepared as above). Two upper leaves from the top of the system of the tobacco plant were collected for PVY infection at 3, 5, and 7 days after PVY infection for testing.

[0113] Seven days after PVY infection, the disease incidence was observed in TRV-miR159a-infected plants and TRV:00 control plants. The results are as follows: Figure 19As shown, compared with TRV:00, TRV-miR159a infected plants showed typical disease symptoms: the newly emerged leaves at the top were obviously curled.

[0114] The relative level of nbe-miR159a in TRV-miR159a infected plants and TRV:00 control plants was detected by qRT-PCR, and the results are shown in Figure 4. Figure 20 As shown, the relative level of nbe-miR159a in TRV-miR159a infected plants was significantly lower than that in TRV:00 control plants.

[0115] The relative PVY RNA accumulation in TRV-miR159a infected plants and TRV:00 control plants after PVY infection was detected by qRT-PCR, and the results are shown in Figure 5. Figure 21 As shown, the relative expression of PVY in TRV-miR159a infected plants was significantly higher than that in TRV:00 control plants at 5d and 7d after PVY infection. WB was further used to analyze and detect the PVY protein expression in TRV-miR159a infected plants and TRV:00 control plants, and β-Actin was used as the internal reference protein, and the results are shown in Figure 6. Figure 22 As shown, the data were normalized by using PVY / β-Actin ratio, and the results are shown in Table 6. After PVY infection for 5d and 7d, the accumulation of PVY virion protein in TRV-miR159a infected plants was significantly increased compared with TRV:00 control plants, which confirmed that the inhibition of nbe-miR159a function significantly enhanced the susceptibility of the host to PVY.

[0116] Table 6. Normalization of data by using PVY / β-Actin ratio

[0117]

[0118] The above description is only the preferred embodiments of the present application, and is not intended to limit the present application in other forms. Any skilled person in the art can modify or change the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent change and modification of the above embodiments without departing from the technical solution of the present application, and according to the technical essence of the present application, still belongs to the protection scope of the present application.

Claims

1. The use of miR159a associated with plant antiviral in improving the resistance of plants to viruses, characterized in that, The miR159a is nbe-miR159a, and its nucleotide sequence is shown as SEQ ID NO:1; The expression of the nbe-miR159a in the plant is improved, the expression of the CPR5 gene is negatively regulated, and the resistance of the plant to the virus is improved. The plant is tobacco, and the virus is a potato Y virus.

2. Use of the plant virus resistance-related miR159a according to claim 1 for increasing the resistance of a plant to a virus, characterized in that, The overexpression vector of the nbe-miR159a is constructed, and the plant is transformed, so that the expression of the nbe-miR159a in the plant is improved.

3. Use of the plant virus resistance-related miR159a according to claim 2 for increasing the resistance of a plant to a virus, characterized in that, The transformation method of the plant is one of an agrobacterium-mediated method, a gene gun method, an electric shock method, a PEG method and a liposome method.

4. The use of the plant virus resistance related miR159a according to claim 3 for improving the virus resistance of plants, characterized in that, The agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

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