Application of plant 14-3-3h protein and its encoding gene in resisting potato virus Y

Abstract

The present invention discloses the application of plant 14-3-3h protein and its coding gene in resisting Potato virus Y, belonging to the field of biotechnology. The present invention for the first time studies and proves that plant 14-3-3h protein can interact with 6K1 protein participating in the replication process in Potato virus Y; silencing the 14-3-3h gene in plants will promote the infection of PVY; while overexpressing the 14-3-3h gene will inhibit the infection of PVY. Therefore, plant 14-3-3h protein can be used as a new target for resisting Potato virus Y, providing a new idea for the prevention and control of Potato virus Y disease.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a plant 14-3-3h protein and a coding gene thereof in resistance to potato virus Y. Background Art

[0002] Potyviruses are numerous and cause serious damage, causing significant economic losses to my country's grain production every year. The genome of Potyvirus Y is +ssRNA, with a total length of about 9700nt and two open reading frames (ORFs). One ORF encodes a large polyprotein that is cleaved into 10 mature proteins by its own protease; the other small ORF encodes the P3N-PIPO protein. Viruses need the help of host proteins to complete replication, movement and other processes, and then successfully infect. Studying the interaction between viruses and host factors and clarifying their molecular mechanisms, as well as screening new antiviral targets, can provide a reference for a deeper understanding of the pathogenicity and prevention of Potatovirus Y.

[0003] 14-3-3 proteins are widely distributed in eukaryotes, and there are many 14-3-3 genes in plants. Among them, the model plant Arabidopsis has a total of 13 14-3-3 protein subtypes; tomato has 12 subtypes; 8 subtypes were found in rice; tobacco has 17 subtypes, which is the species with the most 14-3-3 genes identified. 14-3-3 is a protein with signal transduction function unique to eukaryotes. It plays a role in regulating metabolism, hormone signaling, cell division, cell cycle regulation, light signal response, stress resistance, growth and development, stomatal movement, oil synthesis, and responding to biotic and abiotic stresses. 14-3-3 proteins themselves have no enzymatic activity and rely on protein-protein interactions to exert regulatory effects. The functional mechanism of 14-3-3 protein can be roughly divided into five modes of action: (1) interfering with the binding of interacting proteins to other interacting factors; (2) changing the localization of interacting proteins; (3) connecting two interacting proteins together; (4) modifying the catalytic activity of interacting proteins; (5) protecting interacting proteins from post-translational modification and proteolysis. Different 14-3-3 proteins will change their transcription levels or protein abundance and properties when plants are infected by pathogens. However, the interaction between plant 14-3-3h protein and potato virus Y has not been reported. Summary of the invention

[0004] In view of the above prior art, the object of the present invention is to provide a plant 14-3-3h protein and its encoding gene for use in resistance to potato virus Y.

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

[0006] In the first aspect of the present invention, there is provided the use of plant 14-3-3h protein in regulating plant resistance to Potato virus Y;

[0007] The amino acid sequence of the plant 14-3-3h protein is as shown in SEQ ID NO.1 or SEQ ID NO.2. Among them:

[0008] The amino acid sequence of Nicotiana benthamiana 14-3-3h protein is as shown in SEQ ID NO.1, specifically as follows:

[0009] MASPREENVYMAKLAEQAERYEEMVEFMEKVIAAADGAEELTVEERNLLSVAYKNVIGARRAS

[0010] WRIISSIEQKEESRGNEDHVAYIKEYRSKIETELTSICNGILKLLDSKLIGAAATGDSKVFYLKMKGDYH

[0011] RYLAEFKTGAERKEAAENTLSAYKSAQDIANTELAPTHPIRLGLALNFSVFYYEILNSPDRACNLAKQAFDEAIAELDTLGEESYKDSTLIMQLLRDNLTLWTSDMQDDGTDEIKEAAKPDNEQQ.

[0012] The amino acid sequence of Solanum tuberosum 14-3-3h protein is as shown in SEQ ID NO.2, specifically as follows:

[0013] MASPREENVYMAKLAEQAERYEEMVEFMEKVVAAADGAEELTVEERNLLSVAYKNVIGARRAS

[0014] WRIISSIEQKEESRGNEDHVASIKEYRSKIESELTSICNGILKLLDSKLIGSAATGDSKVFYLKMKGDYH

[0015] RYLAEFKTGAERKEAAENTLSAYKAAQDIANADLAPTHPIRLGLALNFSVFYYEILNSPDRACNLAKQAFDEAIAELDTLGEESYKDSTLIMQLLRDNLTLWTSDMQDDGTDEIKEAAPKPDNNE.

[0016] In the above application, the plant 14-3-3h protein inhibits the infection of Potato virus Y by interacting with the 6K1 protein in Potato virus Y.

[0017] The second aspect of the present invention provides the use of the coding gene of plant 14-3-3h protein in the following (1) or (2):

[0018] (1) Regulating the resistance of plants to Potato virus Y;

[0019] (2) Cultivating plant varieties resistant to Potato virus Y.

[0020] In the above application, the coding gene of the plant 14-3-3h protein is a nucleic acid molecule shown in any one of the following (i)-(iv):

[0021] (i) A nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3;

[0022] (ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.1 other than (i);

[0023] (iii) A nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.4;

[0024] (iv) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than (iii).

[0025] Among them, the CDS sequence of the coding gene of Nicotiana benthamiana 14-3-3h protein is as shown in SEQ ID NO.3, specifically as follows:

[0026] ATGGCGTCGCCACGCGAGGAGAACGTGTACATGGCAAAGCTTGCCGAGCAAGCCGAGCGTTACGAGGAGATGGTTGAATTCATGGAGAAAGTCATCGCCGCCGCCGACGGCGCCGAGGAACTTACCGTCGAAGAACGGAACCTCCTCTCCGTCGCATACAAAAATGTTATCGGAGCACGGCGAGCCTCGTGGCGTATCATCTCCTCCATTGAGCAAAAAGAGGAGAGCCGCGGTAACGAAGATCACGTTGCCTACATCAAGGAGTACAGATCTAAGATCGAGACCGAACTCACCTCGATCTGTAACGGCATTCTCAAGCTCCTCGATTCTAAGCTCATTGGTGCCGCTGCTACCGGTGATTCTAAGGTGTTTTACTTGAAAATGAAAGGAGATTATCATCGTTATTTGGCTGAGTTTAAGACTGGTGCCGAGCGAAAGGAAGCCGCCGAAAATACTCTCTCGGCTTACAAATCCGCTCAGGATATTGCAAATACCGAGCTTGCTCCTACGCATCCAATCCGATTGGGACTTGCTCTCAATTTCTCTGTATTTTACTACGAGATTTTGAATTCTCCTGATCGTGCTTGTAATCTCGCCAAACAGGCTTTTGATGAGGCAATTGCCGAGCTGGACACATTGGGCGAAGAGTCATACAAGGATAGCACTCTGATCATGCAGCTTCTTCGCGATAACCTCACTTTATGGACTTCGGATATGCAGGATGATGGAACTGATGAGATCAAAGAAGCTGCAAAACCAGATAATGAGCAGCAGTAA。

[0027] The CDS sequence of the coding gene of potato 14-3-3h protein is shown in SEQ ID NO.4, as follows:

[0028] ATGGCGTCGCCACGCGAGGAAAACGTGTACATGGCGAAGCTCGCCGAGCAAGCTGAGCGTTA

[0029] CGAGGAGATGGTAGAGTTCATGGAGAAAGTTGTCGCTGCCGCCGATGGTGCCGAGGAGTTAACC

[0030] GTTGAAGAACGAAACCTCCTCTCCGTTGCGTATAAGAATGTGATCGGAGCACGGAGAGCTTCATG

[0031] GAGGATCATTTCCTCCATTGAGCAGAAAGAGGAGAGCCGTGGTAACGAAGATCATGTTGCTTCCA

[0032] TTAAGGAATACAGATCTAAGATCGAGTCTGAACTTACCTCGATCTGTAATGGAATTCTTAAGCTGCT

[0033] TGATTCTAAGCTCATTGGCTCTGCTGCTACCGGTGACTCTAAGGTGTTTTACTTGAAAATGAAGGG

[0034] AGATTATCATCGTTATTTGGCTGAGTTTAAGACTGGTGCTGAGCGAAAAGAAGCTGCTGAGAATAC

[0035] TCTCTCGGCTTACAAAGCTGCTCAGGATATTGCTAATGCCGACCTTGCTCCTACACATCCAATCCGA

[0036] TTGGGACTTGCTCTCAATTTCTCTGTGTTTTACTACGAGATTTTGAATTCTCCTGATCGTGCCTGTA

[0037] ATCTCGCCAAACAGGCCTTTGATGAGGCAATTGCGGAGTTGGACACATTGGGCGAAGAGTCCTAC

[0038] AAGGATAGCACTCTGATCATGCAGCTTCTTCGTGATAACCTCACTTTATGGACCTCTGATATGCAGGATGATGGAACTGATGAGATCAAAGAAGCTGCACCTAAACCAGATAATAATGAATGA。

[0039] The third aspect of the present invention provides the use of a recombinant expression vector or an engineered bacterium containing the coding gene of plant 14-3-3h protein in the following (1) or (2):

[0040] (1) Regulating the resistance of plants to Potato virus Y;

[0041] (2) Cultivating plant varieties resistant to Potato virus Y.

[0042] In the above applications, the recombinant expression vector can be constructed using existing plant expression vectors. The plant expression vectors include pCAMBIA3300, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19 or other derivative plant expression vectors.

[0043] The fourth aspect of the present invention provides a method for improving the resistance of plants to Potato virus Y, including the step of overexpressing the coding gene of 14-3-3h protein in plants.

[0044] In the above method, the overexpression of the coding gene of 14-3-3h protein can be achieved by introducing the coding gene of 14-3-3h protein exogenously; or by upregulating the expression of the coding gene of 14-3-3h protein in the plant genome.

[0045] The fifth aspect of the present invention provides a method for cultivating transgenic plants resistant to Potato virus Y, including the following steps:

[0046] Introducing the 14-3-3h gene into the target plant to obtain a plant with high expression of the 14-3-3h gene; the plant with high expression of the 14-3-3h gene obtained has higher resistance to Potato virus Y than the target plant.

[0047] In the above method, the 14-3-3h gene is a nucleic acid molecule shown in any one of the following (i)-(iv):

[0048] (i) A nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3;

[0049] (ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.1 other than (i);

[0050] (iii) A nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.4;

[0051] (iv) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than (iii).

[0052] The sixth aspect of the present invention provides an agent for preventing and treating Potato virus Y disease, and the agent uses plant 14-3-3h protein as the active ingredient.

[0053] Advantages of the present invention:

[0054] The present invention firstly studies and confirms that the plant 14-3-3h protein can interact with the 6K1 protein involved in the replication process in Potato virus Y (PVY); silencing the 14-3-3h gene in plants will promote the infection of PVY; overexpressing the 14-3-3h gene will inhibit the infection of PVY. Therefore, the plant 14-3-3h protein can be used as a new target against PVY, providing a new idea for the prevention and control of PVY disease. Description of the drawings

[0055] Figure 1 : Phylogenetic analysis of 14-3-3 in Nicotiana benthamiana.

[0056] Figure 2 : Interaction study between the 6K1 protein of Potato virus Y and the Nb14-3-3 protein.

[0057] Figure 3 : The Nb14-3-3h protein inhibits the infection of PVY.

[0058] Figure 4 : Symptoms and detection of PVY-GFP inoculated on potato with silenced 14-3-3h. Detailed implementation manners

[0059] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0060] As mentioned above, the interaction relationship between PVY and its host is a current research hotspot. Plant 14-3-3 proteins have various physiological functions and are highly valuable for research. However, there are many subtypes of plant 14-3-3 proteins, and different 14-3-3 protein subtypes have different cell specificities and functional specificities. Therefore, the difficulty and workload in studying the functions of plant 14-3-3 proteins are extremely large.

[0061] In view of this, the present invention first studied the 14-3-3 proteins of Nicotiana benthamiana and found that nine 14-3-3 protein subtypes of Nicotiana benthamiana could interact with the 6K1 protein of Potato virus Y. To further clarify the biological functions of the 14-3-3 proteins of Nicotiana benthamiana during the infection process of Potato virus Y, nine 14-3-3 proteins of Nicotiana benthamiana were transiently overexpressed in tobacco respectively, and then inoculated with PVY replicons. The results showed that there was no significant difference in virus fluorescence and accumulation between the overexpression of Nb14-3-3a, b, e, f, i and the control, while the virus fluorescence was significantly weaker than that of the control after the overexpression of Nb14-3-3c, d, g, h, and the virus accumulation was significantly reduced, indicating that the overexpression of Nb14-3-3c, d, g, h could inhibit PVY infection. Since the virus accumulation was the lowest after the overexpression of Nb14-3-3h, the present invention selected Nb14-3-3h for the next step of research.

[0062] The present invention further constructed a gene silencing vector and an overexpression vector of Nb14-3-3h, and the results showed that silencing the Nb14-3-3h gene could promote PVY infection; overexpressing the Nb14-3-3h gene could inhibit PVY infection.

[0063] The present invention also silenced the St14-3-3h gene in potato and found that silencing the St14-3-3h gene could also promote PVY infection.

[0064] This proves that: the 14-3-3h gene can be used as a target for plants to resist Potato virus Y and can provide a new way for the prevention and control of Potato virus Y disease.

[0065] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific embodiments.

[0066] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. The experimental methods without specific conditions are carried out according to conventional test methods or the operation manuals recommended by the suppliers. Among them:

[0067] The plant total RNA extraction reagent Trizol was purchased from Beijing TransGen Biotech Co., Ltd.; DNA polymerase, high-fidelity enzyme, reverse transcriptase and fluorescence quantitative reagents were all purchased from Nanjing Novozymes Biotech Co., Ltd.; homologous recombination enzyme was purchased from Applied Biological Materials (abm) Company; restriction endonucleases were purchased from Thermo Fisher SCIENTIFIC Company; T4 DNA ligase was purchased from Takara Company; plasmid extraction and gel recovery kits were purchased from Omega Bio-Tek Company; nucleic acid molecular weight and protein molecular weight standards were all purchased from Suzhou New Semi Technology Co., Ltd., and GFP-trap, HA-trap, and MYC-trap agarose beads were purchased from Shenzhen Kangti Life Technology Co., Ltd.; other commonly used reagents were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0068] Nicotiana benthamiana, N. tabacum cv. Xanthi, and Solanum tuberosum cv. Desiree used in the present invention were preserved in the Plant Virology Laboratory of Shandong Agricultural University. Unless otherwise specified, the plants were grown in an artificial climate chamber or incubator at a temperature of 22 °C, with a 16 h light / 8 h dark cycle and a relative humidity of 65%.

[0069] The potato virus Y used in the present invention was the PVY N605 isolate (GenBank: X97895.1).

[0070] The full-length cDNA infectious clone of PVY with GFP was constructed by the following method:

[0071] The full-length sequence of the PVY N605 (GenBank: X97895.1) isolate was cloned from the cDNA of tobacco infected with PVY. It was ligated into the basic vector pCAM0390 using homologous recombination technology, and the green fluorescent protein GFP was inserted between NIb and CP. After inoculating Nicotiana benthamiana, systemic disease occurred, and the full-length cDNA infectious clone of PVY was obtained.

[0072] Example 1: Study on the interaction between the 6K1 protein of potato virus Y and Nb14-3-3

[0073] The 6K1 protein is involved in the replication process of potyviruses. To identify the role of host proteins interacting with 6K1 during virus infection, we fused 6K1 of PVY with the eGFP tag and inserted it between the NIb and CP of the high-expression vector TVBMV to obtain the virus vector pTVBMV-6K1-eGFP, which was then transformed into Agrobacterium for infiltration inoculation of Nicotiana benthamiana. After 12 days, the systemically diseased leaves were collected, and the protein complex bound to 6K1-eGFP was purified using GFP magnetic beads. Samples infected with TVBMV-eGFP were used as negative controls. The purified samples were analyzed by SDS-PAGE electrophoresis, and gel bands of the correct size were excised for analysis by liquid chromatography tandem mass spectrometry (LC-MS / MS). Through data analysis, 14-3-3g-1, 14-3-3h-1, and 14-3-3i-2 were found to be present in the 6K1-eGFP protein complex.

[0074] 1. In vivo co-immunoprecipitation to verify protein interaction:

[0075] There are 17 homologous genes of 14-3-3 proteins in tobacco (Konagaya et al., 2004). Since only 9 homologous genes were retrieved due to the incomplete genome of Nicotiana benthamiana ( Figure 1 ), we constructed transient expression vectors of Nb14-3-3a, Nb14-3-3b, Nb14-3-3c, Nb14-3-3d, Nb14-3-3e, Nb14-3-3f, Nb14-3-3g, Nb14-3-3h, and Nb14-3-3i with Myc tags and verified their interaction with 6K1-eGFP through in vivo co-immunoprecipitation experiments. The specific method is as follows:

[0076] (1) Construction of transient expression vectors:

[0077] Using a high-fidelity enzyme, the Nb14-3-3 homologous gene was amplified with Nicotiana benthamiana cDNA as a template, and the Rluc gene was artificially synthesized (reference: "Saha S, Hafren A, Makinen K (2019) Dynamics of Protein Accumulation from the 3' End of Viral RNA Are Different from Those in the Rest of the Genome in Potato Virus A Infection. J Virol 93"). Fragments containing the coding region sequences of Nb14-3-3s and Rluc genes were cloned into the pUTR-Myc vector (constructed according to the reference "Cheng DJ, Xu XJ, Yan ZY, Tettey CK, Fang L, Yang GL, Geng C, Tian YP, Li XD (2021) The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection. Plant Physiol 187:174-186") by restriction digestion ligation or homologous recombination. This vector contains the 35S promoter, the 5' UTR of TVBMV, and the 3Myc tag sequence), and plasmids pNb14-3-3a-Myc, pNb14-3-3b-Myc, pNb14-3-3c-Myc, pNb14-3-3d-Myc, pNb14-3-3e-Myc, pNb14-3-3f-Myc, pNb14-3-3g-Myc, pNb14-3-3h-Myc, pNb14-3-3i-Myc, and pRluc-Myc were obtained.

[0078] (2) Co-immunoprecipitation:

[0079] The transient expression vectors constructed above were separately transferred into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method. Single colonies were picked and cultured overnight in liquid LB containing 50 μg / mL kanamycin and 100 μg / mL rifampicin in a 28°C horizontal shaker. The overnight culture was centrifuged at 6000 rpm for 3 min to enrich the bacteria, and then resuspended with a resuspension solution (10 mM MES [pH 5.6], 10 mM MgCl2, and 200 mM AS), and left standing at 28°C for 3 h. The concentrations of the above bacterial solution and Agrobacterium tumefaciens containing the RNA silencing suppressor pBinP19 were adjusted to OD 600= 1.0, and then mix them evenly in equal volumes and co-infiltrate and inoculate Nicotiana benthamiana. Three days after inoculation, collect the inoculated leaves, add liquid nitrogen and grind them. Add protein extraction buffer (25 mM Tris-HCl [pH 7.5], 1 mM EDTA, 150 mM NaCl, 10 mM DTT, 10% glycerol, 0.15% Nonidet P-40 and 1× protease inhibitor cocktail) in a ratio of 1:1 (W / V). Vortex thoroughly to mix evenly and incubate on ice for 10 min. Centrifuge at 4°C and 12,000 rpm for 15 min, transfer the supernatant to a new pre-chilled centrifuge tube, repeat centrifugation, filter the supernatant through a 0.22 μm aqueous filter for later use. Balance the beads during centrifugation. Taking GFP-Trap magnetic beads as an example, vortex the magnetic beads to mix evenly, pipette 25 μL and transfer it to a 1.5 mL centrifuge tube, add 1 mL of pre-chilled 1× PBS solution, invert to mix evenly, centrifuge at 4°C and 2,500 rpm for 3 min, aspirate and discard the supernatant with a pipette, repeat three times. Incubate the filtered supernatant and the balanced magnetic beads on ice for 1 h. Centrifuge at 4°C and 5,000 rpm for 10 min, carefully discard the supernatant, transfer the magnetic beads to a 1.5 mL centrifuge tube, rinse the magnetic beads 4 - 6 times according to the method of balancing the magnetic beads, add 40 μL of 2× SDS, boil the sample for 10 min, and then perform SDS-PAGE electrophoresis using a 12% protein gel. After electrophoresis, transfer the protein to a nitrocellulose membrane using the wet transfer method. After the transfer is completed, block the membrane with 0.5% skim milk powder (add 0.5 g of skim milk powder to 10 mL of TBST buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl and 0.05% Tween-20)) at room temperature for 1 h or at 4°C overnight. Add the corresponding antibody according to the antibody titer ratio, incubate at room temperature for 1 h, then wash the membrane with 1× TBST, 10 min each time, wash three times. Add the secondary antibody according to the source of the primary antibody ratio, incubate at room temperature for 1 h, then wash the membrane with 1× TBST, 10 min each time, wash twice, and wash the membrane with 1× TBS (20 mM Tris-HCl [pH 7.5], 150 mM NaCl and 0.05% Tween-20), 10 min each time, wash once. After washing the membrane, add ECL western blotting substrate and detect the protein signal using a chemiluminescence detector.

[0080] The results showed that 6K1 could co-precipitate with Nb14-3-3 family proteins, but not with Rluc-Myc ( Figure 2 A).

[0081] 2. In vitro verification of protein-protein interaction:

[0082] To verify whether 6K1 interacts with 14-3-3 in vitro, 6K1 was fused with the GST tag, and eGFP was fused with 14-3-3 to construct expression vectors. The vectors were transformed into the Escherichia coli expression strain Rosetta(DE3), and the proteins were expressed and purified prokaryotically. The in vitro interaction of the proteins was verified by a Pulldown experiment. The specific method is as follows:

[0083] (1) Construction of prokaryotic expression vectors:

[0084] The 6K1-GST fragment was obtained by fusion PCR amplification and ligated to the prokaryotic expression vector pEHISTEV vector by homologous recombination to obtain the plasmid pE6K1-GST.

[0085] The fragment encoding the Nb14-3-3 homologous gene was cloned into the pEeGFP vector by restriction enzyme digestion and ligation (constructed according to the reference "Cheng DJ, Xu XJ, Yan ZY, Tettey CK, Fang L, Yang GL, Geng C, Tian YP, Li XD (2021) The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection. Plant Physiol 187: 174-186") to obtain the plasmids pEeGFP-Nb14-3-3a, pEeGFP-Nb14-3-3b, pEeGFP-Nb14-3-3c, pEeGFP-Nb14-3-3d, pEeGFP-Nb14-3-3e, pEeGFP-Nb14-3-3f, pEeGFP-Nb14-3-3g, pEeGFP-Nb14-3-3h and pEeGFP-Nb14-3-3i.

[0086] (2) Pull down experiment:

[0087] The successfully sequenced plasmids pEeGFP-Nb14-3-3a, pEeGFP-Nb14-3-3b, pEeGFP-Nb14-3-3c, pEeGFP-Nb14-3-3d, pEeGFP-Nb14-3-3e, pEeGFP-Nb14-3-3f, pEeGFP-Nb14-3-3g, pEeGFP-Nb14-3-3h, pEeGFP-Nb14-3-3i and pE6K1-GST were transferred into the expression strain Rosetta(DE3). Single colonies were picked and cultured in 300 mL of LB containing antibiotics at 37 °C and 200 rpm until the OD 600= 0.5. After cooling to room temperature, IPTG (final concentration 0.2 mM) was added, and induction was carried out at 16 °C and 150 rpm for 12 h. The induced bacterial solution was aliquoted and centrifuged at 4000 rpm for 10 min at 4 °C. The cells were resuspended in 30 mL of lysis buffer (containing 0.3 M NaCl, 30 mM imidazole, 1% protease inhibitor, 0.5 mg / ml lysozyme, 0.5% NP40 in 1× PBS [pH 7.4]), allowed to stand at 37 °C for 10 min, and then sonicated on ice using a tissue cell disruptor. Cell debris was removed by centrifugation at 12000 rpm for 20 min at 4 °C, and after filtration through a 0.22 μm aqueous filter, it was incubated with equilibrated nickel beads (GenScript) on ice for 30 min. Subsequently, the nickel beads were washed with wash buffer (containing 0.3 M NaCl, 45 mM imidazole in 1× PBS [pH 7.4]), and finally the protein was eluted with elution buffer (containing 0.3 M NaCl, 250 mM imidazole in 1× PBS [pH 7.4]). The eluted protein was dialyzed overnight at 4 °C against dialysis buffer (containing 0.3 M NaCl, 1 M Tris-HCl [pH 7.5], 1 mM DTT in 1× PBS [pH 7.4]) to remove the high-concentration imidazole for later use. Taking the verification of the interaction between 6K1 and Nb14-3-3h as an example, 20 μg of eGFP-Nb14-3-3h protein and 20 μg of 6K1-GST protein were respectively pipetted into a 1.5 mL centrifuge tube, pre-cooled 1× PBS [pH 7.4] solution and 20 μL of equilibrated GFP-Trap magnetic beads were added to make up to 1 mL, incubated at 4 °C for 1 h, then the magnetic beads were rinsed 6 - 10 times with pre-cooled 1× PBS [pH 7.4] solution, 20 μL of 2× SDS was added and the sample was boiled for 10 min to elute and denature the protein, and the target protein was separated by SDS-PAGE using a 12% protein gel, and detected by Western blot with specific antibodies.

[0088] The results showed that 6K1 interacted with the 14-3-3 protein in vitro, but did not interact with the control ( Figure 2 B).

[0089] 3. Verification of protein interaction under virus infection conditions:

[0090] To verify whether the interaction between 6K1 and 14-3-3 occurred under virus infection conditions, the bacterial solution of PVY-mCherry with the red fluorescent tag mCherry was co-infiltrated and inoculated into Nicotiana benthamiana together with the agrobacteria of 6K1-YN and 14-3-3-YC. Three days after inoculation, it was observed under a confocal microscope, and the cells infected with the virus were indicated by red fluorescence. The specific method was as follows:

[0091] The fragment encoding the Nb14-3-3 homologous gene was ligated into pCamYN (constructed according to the reference "Cheng DJ, Xu XJ, Yan ZY, Tettey CK, Fang L, Yang GL, Geng C, Tian YP, Li XD (2021) The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection. Plant Physiol 187: 174-186", and this vector contains the N-terminal sequence of YFP), and plasmids pNb14-3-3a-YN, pNb14-3-3b-YN, pNb14-3-3c-YN, pNb14-3-3d-YN, pNb14-3-3e-YN, pNb14-3-3f-YN, pNb14-3-3g-YN, pNb14-3-3h-YN, pNb14-3-3i-YN were obtained. The GUS gene was cloned from the bacterial genome and ligated into the pYN vector to obtain the plasmid GUS-YN.

[0092] The 6K1 gene was cloned from the PVY infectious clone and ligated into pCamYC (constructed according to the reference "Cheng DJ, Xu XJ, Yan ZY, Tettey CK, Fang L, Yang GL, Geng C, Tian YP, Li XD (2021) The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection. Plant Physiol 187: 174-186", and this vector contains the C-terminal sequence of YFP) to obtain the plasmid p6K1-YC.

[0093] Taking the verification of the interaction between 6K1 and Nb14-3-3h as an example, the Agrobacterium containing PVY-mCherry was adjusted to OD 600 = 0.5, and the bacterial solutions containing Nb14-3-3h-YN, 6K1-YC, and pBin19 were respectively adjusted to OD 600 = 0.3 and infiltrated into Nicotiana benthamiana for inoculation. After 2 days, the infiltrated area was cut and prepared for microscopy. Observation was carried out under a confocal microscope. The excitation wavelength of YFP was 514 nm, and the capture wavelength was 565 nm - 585 nm.

[0094] The results showed that YFP signals were detected for 6K1 and 14-3-3 in virus-infected cells, while no YFP signal was detected in the control, indicating that 6K1 interacts with 14-3-3 under PVY infection conditions ( Figure 2 C).

[0095] Example 2: Study on the inhibition of PVY infection by Nb14-3-3 protein

[0096] 1. Effect of overexpressing Nb14-3-3 protein on PVY infection:

[0097] To clarify the biological function of 14-3-3 during virus infection, Nb14-3-3-Myc was transiently overexpressed in tobacco, and PVY replicons were inoculated 3 days later. The specific method is as follows:

[0098] (1) Construction of transient expression vectors:

[0099] Using a high-fidelity enzyme, the Nb14-3-3 homologous gene was amplified with Nicotiana benthamiana cDNA as a template. The fragment containing the coding region sequence of the Nb14-3-3 homologous gene was cloned into the pUTR-Myc vector by digestion-ligation or homologous recombination to obtain plasmids pNb14-3-3a-Myc, pNb14-3-3b-Myc, pNb14-3-3c-Myc, pNb14-3-3d-Myc, pNb14-3-3e-Myc, pNb14-3-3f-Myc, pNb14-3-3g-Myc, pNb14-3-3h-Myc, pNb14-3-3i-Myc.

[0100] (2) Construction of PVY replicons:

[0101] Specific primers were designed according to the PVY N605 isolate sequence. After amplifying the full-length PVY-GFP sequence, it was ligated into the binary vector pCB301 to obtain pCB301-PVY-GFP. On this basis, the 35S-mCherry-HDEL expression cassette was ligated behind Nos of the pCB301-PVY-GFP vector. Mutations were introduced into P3N-PIPO of PVY (changing lysine at position 177 and glutamine at position 200 to stop codons) to obtain the pCB301-PVY-GFP replicon (pCB301-PVY-GFPrep) that cannot move between cells but can replicate normally.

[0102] (3) Transient overexpression of Nb14-3-3-Myc in tobacco:

[0103] The transient expression vectors constructed above were separately transferred into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method, and the OD of each bacterial solution and the bacterial solution of the RNA silencing suppressor pBin-P19 were adjusted to600 = 0.35, and infiltrate-inoculate tobacco.

[0104] (4) Inoculate with PVY replicons:

[0105] Three days later, inoculate the PVY replicons constructed above into Agrobacterium tumefaciens strain GV3101, and adjust the bacterial liquid concentration to OD 600 = 0.5, and inoculate tobacco.

[0106] Under ultraviolet light, it was observed that there was no significant difference in virus fluorescence between overexpressing Nb14-3-3a, b, e, f, i and the control, while the virus fluorescence was significantly weaker than that of the control after overexpressing Nb14-3-3c, d, g, h ( Figure 3 A), and the Western blot detection results showed that the virus accumulation level was significantly reduced after overexpressing Nb14-3-3c, d, g, h compared with the control ( Figure 3 B), indicating that overexpressing Nb14-3-3c, d, g, h could inhibit PVY infection. Since the accumulation level of overexpressing Nb14-3-3h was the lowest, we selected Nb14-3-3h for the next step of research.

[0107] 2. Study on the interaction between Nb14-3-3h and 6K1 under virus infection conditions:

[0108] To further verify whether Nb14-3-3h and 6K1 interact under virus infection conditions, infiltrate and inoculate Nb14-3-3h-YN and 6K1-YC into the leaves of the Nicotiana benthamiana system infected with PVY-mCherry. Observe under a confocal microscope 2 days after inoculation. The specific method is the same as that in Example 1.

[0109] The results showed that YFP fluorescence was observed in the co-infiltrated cells of Nb14-3-3h-YN and 6K1-YC, further indicating that Nb14-3-3h and 6K1 interact under the condition of PVY infection ( Figure 3 C); Nb14-3-3h-YN + GUS-YC and GUS-YN + 6K1-YC were used as negative controls.

[0110] 3. Study on the role of Nb14-3-3h in the process of PVY infection:

[0111] To study the role of Nb14-3-3h during PVY infection, we ligated the sequence of the 3'UTR of the Nb14-3-3h gene into a silencing vector based on tobacco rattle mosaic virus (TRV), and obtained the viral vector pTRV2-Nb14-3-3h for silencing Nb14-3-3h. Agrobacterium containing this plasmid was infiltrated and inoculated into Nicotiana benthamiana, and plants inoculated with TRV2-GUS were used as negative controls. The specific method is as follows:

[0112] Nb14-3-3h was amplified from Nicotiana benthamiana cDNA using rTaq DNA polymerase, and the gene fragment was cloned into the pMD18-T vector. Its 3’UTR sequence was obtained by sequencing and ligated into the TRV silencing vector to obtain the plasmid pTRV2-Nb14-3-3h.

[0113] The results showed that 12 days after inoculation, there was no obvious abnormal phenotype in the plants inoculated with pTRV2-Nb14-3-3h( Figure 3 D).

[0114] To determine the silencing efficiency of the Nb14-3-3h gene, we collected the systemic leaves of Nb14-3-3h-silenced and control plants on the 12th day after inoculation to extract total RNA for qPCR detection. The specific method is as follows:

[0115] Total plant RNA was extracted according to the instructions of TransZol (product number ET101-01) from TransGen Biotech. Weigh 50-100 mg and grind it thoroughly in a pre-cooled mortar. Transfer the sample powder to a 2.0 mL RNase-free centrifuge tube, add 1 mL TransZol, and shake vigorously in a tissue grinder for 1 min. Let it stand at room temperature for 3 min, then add 300 μL chloroform to each tube, shake well for 15 s, and let it stand for 5 min. Centrifuge at 12,000 rpm at 4℃ for 15 min, transfer the colorless supernatant to a 1.5 mL RNase-free centrifuge tube, add an equal volume of isopropanol, invert and mix well, and let it stand at room temperature for 10 min. Centrifuge at 12,000 rpm at 4℃ for 10 min and discard the supernatant. At this time, a white or gelatinous precipitate can be observed at the bottom or on the side wall of the tube. Add 1 mL of 75% ethanol solution to each tube, invert and mix well to wash the precipitate, centrifuge at 9,000 rpm at 4℃ for 5 min, and discard the supernatant. Let it stand at room temperature for 5 min to volatilize the residual liquid at the bottom of the tube. Add 50-100 μL of ultrapure water to each tube to dissolve the RNA sufficiently for standby.

[0116] Design qPCR primers according to the virus CP gene or plant gene sequence using Primer 5 software. In the experiment of detecting virus accumulation or gene expression level, the total RNA extracted is treated with gDNA eraser to remove the plant genome, and 1 - 2 μg of total RNA is used as a template for reverse transcription with 5×HiScriptⅡqRT Super MixⅡ. Using cDNA as a template, detect with 2×ChamQ SYBR qPCR Master Mix and specific primers. In the experiment of detecting virus replication level, taking the detection of PVY replication level as an example, the total RNA extracted is treated with gDNA eraser to remove the plant genome, 1 - 2 μg of total RNA is used as a template, and qPVY-CP-F + EF1α-R or qPVY-CP-R + EF1α-R is used as primers, and reverse transcription is carried out using 5×HiScript II RT select SuperMix. Using the obtained cDNA as a template, detect with 2×ChamQ SYBR qPCR Master Mix and CP specific primers.

[0117] The results showed that the relative mRNA accumulation level of the silenced plant Nb14-3-3h was about 15% of that of the control plants ( Figure 3 E).

[0118] Subsequently, both the silenced and control plants were inoculated with the full-length cDNA infectious clone of PVY carrying GFP, and green fluorescence can be used to indicate the virus. Figure 3 G statistically represents the proportion of plants in which the fluorescence reaches the systemic leaves after the silenced plants and the control are inoculated with PVY-GFP among the total number of experimental plants. By observing the fluorescence under ultraviolet light, count the number of plants in which the fluorescence reaches the systemic leaves. The analysis showed that the speed at which the virus GFP fluorescence reached the systemic leaves of the plants with silenced Nb14-3-3h was faster than that of the control ( Figure 3 G). Five days after inoculation, it was observed under ultraviolet light that the virus fluorescence in the systemic leaves of the plants with silenced Nb14-3-3h was stronger than that of the control ( Figure 3 F). The RT-qPCR detection results showed that on the 5th day after inoculation, the PVY RNA accumulation level in the Nb14-3-3h-silenced plants was significantly increased compared with that of the control plants ( Figure 3 H-3I), among which, Figure 3 H is to detect the virus genomic RNA, and the positive-strand RNA is detected; Figure 3 I is to detect the negative-strand RNA produced during the virus replication process. The Western blot detection results showed that the accumulation level of PVYCP in the Nb14-3-3h-silenced plants was significantly higher than that of the control plants ( Figure 3 J), indicating that silencing Nb14-3-3h would promote PVY infection.

[0119] To further verify the role of Nb14-3-3h in the process of PVY infection, the full-length cDNA infectious clone of PVY with GFP was inoculated onto transgenic Nicotiana benthamiana overexpressing Nb14-3-3h-3HA. The specific method is as follows:

[0120] The coding region sequence of the Nb14-3-3h gene was ligated into the pCam-UTR-HA vector (constructed according to the reference "Cheng DJ, Xu XJ, Yan ZY, Tettey CK, Fang L, Yang GL, Geng C, Tian YP, Li XD (2021) The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection. Plant Physiol 187: 174-186". This vector contains the 35S promoter, the 5’UTR of TVBMV, and the 3HA tag sequence) to obtain the plasmid pNb14-3-3h-HA. Agrobacterium tumefaciens containing this plasmid was infiltrated and inoculated onto Nicotiana benthamiana to obtain transgenic Nicotiana benthamiana overexpressing Nb14-3-3h-3HA. PVY-GFP was inoculated onto the transgenic Nicotiana benthamiana overexpressing Nb14-3-3h-3HA.

[0121] Figure 3 K shows the fluorescence of the systemic leaves of transgenic plants overexpressing Nb14-3-3h inoculated with PVY-GFP. Five days after inoculation, the fluorescence of the plants overexpressing Nb14-3-3h-3HA (Nb14-3-3h-OE) was weak, indicating less virus accumulation in the systemic leaves. The Western blot detection results showed that on the 5th day after inoculation, the accumulation level of PVY CP in the Nb14-3-3h overexpressing plants was significantly lower than that of the control ( Figure 3 L).

[0122] To further verify the effect of Nb14-3-3h on PVY replication, PVY replicons were inoculated onto Nicotiana benthamiana overexpressing Nb14-3-3h-3HA (Nb14-3-3h-OE), with wild-type Nicotiana benthamiana (WT) as the control. Samples were collected 60 hours after inoculation for RT-qPCR detection.

[0123] The results showed that: the accumulation level and replication level of PVY RNA in the Nb14-3-3h transgenic plants were significantly lower than those of the control plants ( Figure 3 M and 3N). The above results all indicate that overexpression of Nb14-3-3h inhibits PVY infection.

[0124] Example 3: Study on the effect of silencing potato 14-3-3h gene on PVY infection

[0125] To verify the role of 14-3-3h in PVY infection in potatoes, the St14-3-3h gene fragment was ligated into a PVX silencing vector (constructed according to the reference "Wang Y, Cong QQ, Lan YF, Geng C, Li XD, Liang YC, Yang ZY, Zhu XP, Li XD (2014) Development of new potato virus X-based vectors for gene over-expression and gene silencing assay. Virus Res 191:62-69"). The leaves of the Nicotiana benthamiana system infected with PVX-St14-3-3h were collected to prepare diseased sap, which was then rubbed onto potatoes. After 15 days, PVY-GFP was rubbed onto the silenced and control plants. Compared with the control, the fluorescence in the plants with silenced St14-33h reached the systemic leaves slightly faster ( Figure 4 A), and the fluorescence intensity was stronger than that of the control. The systemic leaves were collected for detection, and the results showed that the accumulation levels of viral proteins and RNAs in the silenced plants were higher than those in the control ( Figure 4 B and 4C), preliminarily indicating that silencing St14-3-3h promotes PVY infection.

[0126] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various modifications and changes can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. Use of overexpressed Nb14-3-3h protein in enhancing the resistance of tobacco to Potato virus Y; The amino acid sequence of the Nb14-3-3h protein is shown as SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The Nb14-3-3h protein inhibits the infection of Potato virus Y by interacting with the 6K1 protein in Potato virus Y.

3. Use of the coding gene of overexpressed Nb14-3-3h protein in the following (1) or (2): (1) Enhancing the resistance of tobacco to Potato virus Y; (2) Cultivating tobacco varieties resistant to Potato virus Y; The coding gene of the Nb14-3-3h protein is a nucleic acid molecule shown as the following (i) or (ii): (i) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.3; (ii) A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID NO.1 other than (i).

4. Use of a recombinant expression vector or engineered bacterium containing the coding gene of Nb14-3-3h in the following (1) or (2): (1) Enhancing the resistance of tobacco to Potato virus Y; (2) Cultivating tobacco varieties resistant to Potato virus Y; The coding gene of the Nb14-3-3h protein is a nucleic acid molecule shown as the following (i) or (ii): (i) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.3; (ii) A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID NO.1 other than (i).

5. A method for improving the resistance of tobacco to potato virus Y, characterized in that, Comprising: The step of overexpressing the coding gene of 14-3-3h protein in tobacco; the coding gene of the 14-3-3h protein is a nucleic acid molecule shown as the following (i) or (ii): (i) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.3; (ii) A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID NO.1 other than (i).

6. A method for cultivating a transgenic plant resistant to potato virus Y, characterized in that, Comprising the following steps: Introducing the 14-3-3h gene into a target plant to obtain a plant with high expression of the 14-3-3h gene; the obtained plant with high expression of the 14-3-3h gene has higher resistance to Potato virus Y than the target plant; The target plant is tobacco; The 14-3-3h gene is a nucleic acid molecule shown as the following (i) or (ii): (i) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.3; (ii) A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID NO.1 other than (i).

7. A medicament for preventing and treating potato virus Y disease, characterized in that, The medicament uses the plant 14-3-3h protein as the active ingredient; the amino acid sequence of the plant 14-3-3h protein is shown as SEQ ID NO.1.

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