Application of CDPK8 protein coded by solanaceae plants in prevention and treatment of pepper mild mottle virus

Abstract

The invention relates to the technical field of biotechnology and crop disease control, in particular to application of CDPK8 protein coded by solanaceae plants in control of pepper mild mottle virus. The invention finds that the CDPK8 protein (taking NbCDPK8 as an example) coded by solanaceae plants and the coding gene thereof play an important role in infection and proliferation of PMMoV, are susceptible genes of PMMoV coded by host plants, can be used for preventing and treating pepper light mottle virus diseases caused by PMMoV, and can be used for molecular breeding for resisting / resisting PMMoV and the like. According to the invention, a basis can be provided for researching the effect of plant protein in virus infection and the fields of antiviral breeding and the like, the antiviral research level of solanaceous vegetables is improved, and the production safety of solanaceous vegetables is ensured and promoted.

Description

Technical Field

[0001] The present invention relates to the technical fields of biotechnology and crop disease control, and in particular to the application of the CDPK8 protein encoded by solanaceous plants in controlling pepper mild mottle virus. Background Art

[0002] Peppers, tomatoes, etc. are important solanaceous vegetable crops. In recent years, the occurrence of pepper mild mottle virus (PMMoV) has shown an increasing trend year by year. The chlorotic mottling of plant leaves caused by PMMoV infection and the slow growth pose a serious threat to the high and stable yields of crops such as peppers. Since there are no resistant / susceptible varieties and no effective control agents, it is urgent to identify disease-resistant genes and develop resistant materials in order to control PMMoV and its hazards sustainably and more effectively, and ensure the production safety of solanaceous vegetables such as peppers.

[0003] As an obligate intracellular parasite, plant viruses must rely on and utilize host factors to complete life processes such as virus uncoating, genome replication, protein expression, virus particle assembly, and movement to adjacent cells during the processes of infection and proliferation. Therefore, identifying host factors involved in the processes of plant virus infection and proliferation can help to deeply understand the infection and pathogenic mechanisms of viruses, and thus provide new materials and theoretical basis for the prevention and control of virus diseases. Summary of the Invention

[0004] The present invention provides the application of the CDPK8 protein encoded by solanaceous plants in controlling pepper mild mottle virus.

[0005] The present invention provides an NbCDPK8 protein for regulating the expression of pepper mild mottle virus, and its amino acid sequence includes any one of the following:

[0006] (1) The amino acid sequence shown in SEQ ID No.1;

[0007] (2) An amino acid sequence having at least 77.71% (preferably at least 99%) identity with the amino acid sequence shown in SEQ ID No.1, and the protein has the function of regulating the expression of pepper mild mottle virus;

[0008] (3) An amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1, and the protein has the function of regulating the expression of pepper mild mottle virus.

[0009] The present invention also provides an NbCDPK8 gene for regulating the expression of pepper mild mottle virus, and its nucleotide sequence includes any one of the following:

[0010] (1) The nucleotide sequence shown in SEQ ID No.2 or its complementary sequence;

[0011] (2) A sequence having at least 89.98% (preferably at least 99%) identity with the nucleotide sequence shown in SEQ ID No.2, and the polypeptide encoded by it has the function of regulating the expression of pepper mild mottle virus;

[0012] (3) A sequence that hybridizes with SEQ ID No.2 under stringent conditions;

[0013] (4) A sequence obtained by substituting and / or deleting and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID No.2, and the polypeptide encoded by it has the function of regulating the expression of pepper mild mottle virus.

[0014] The above gene is derived from Nicotiana benthamiana.

[0015] In view of the high conservation of the NbCDPK8 gene in Nicotiana benthamiana and CDPK8 in solanaceous economic crops such as pepper, the present invention can also be used for solanaceous dicotyledonous host plants such as pepper.

[0016] The present invention also provides a biological material, including the NbCDPK8 gene that regulates the expression of pepper mild mottle virus.

[0017] Preferably, the biological material is an expression cassette, a vector, a host cell, a transgenic cell line or a recombinant microorganism.

[0018] The present invention also provides the application of the NbCDPK8 protein, or its coding gene, or the biological material containing its coding gene in any one or more of the following:

[0019] (1) Application in regulating the expression of pepper mild mottle virus;

[0020] (2) Regulating the disease resistance of plants to pepper mild mottle virus;

[0021] (3) Preventing and treating pepper mild mottle virus disease;

[0022] (4) Regulating crop yield;

[0023] (5) Application in improving plant germplasm resources;

[0024] (6) Application in preparing products for preventing and treating pepper mild mottle virus disease;

[0025] The amino acid sequence of the NbCDPK8 protein includes any one of the following:

[0026] (1) The amino acid sequence shown in SEQ ID No.1;

[0027] (2) An amino acid sequence having at least 99% identity with the amino acid sequence shown in SEQ ID No.1, and the protein has the function of regulating the expression of pepper mild mottle virus;

[0028] (3) An amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1, and the protein has the function of regulating the expression of pepper mild mottle virus;

[0029] Preferably, the biological material is an expression cassette, a vector, a host cell, a transgenic cell line or a recombinant microorganism;

[0030] Preferably, the plant or crop is a solanaceous plant, more preferably a plant of the genus Nicotiana or Solanum, and even more preferably tobacco or pepper.

[0031] According to the said application, the disease resistance of plants to pepper mild mottle virus disease, or the regulation of crop yield, or the prevention and control of pepper mild mottle virus disease, or the improvement of germplasm resources are regulated by adjusting the content or activity of the NbCDPK8 protein.

[0032] According to the said application, the content or activity of the NbCDPK8 protein is regulated by genetic engineering techniques.

[0033] In the specific implementation process, the content or activity of the NbCDPK8 protein can be regulated by controlling substances such as cDNA and RNA. For example, it can be RNA encoded by the nucleotide sequence shown at positions 1414 - 1606 of SEQ ID NO.2 (silencing the NbCDPK8 gene).

[0034] In the specific implementation process, it includes but is not limited to regulating the content or activity of the NbCDPK8 protein in plant cells by biotechnological methods such as using Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc.

[0035] According to the said application, the replication and proliferation of pepper mild mottle virus are inhibited by reducing the content or activity of the NbCDPK8 protein, so as to improve the disease resistance of plants to pepper mild mottle virus disease, or to increase crop yield, or to improve germplasm resources.

[0036] According to the said application, the coding gene of the NbCDPK8 protein includes the sequence shown in SEQ ID No.2;

[0037] The present invention also provides a product, and the product is a pesticide, a fertilizer or a fertilizer additive; the product realizes at least one of the following purposes by regulating the content or activity of the NbCDPK8 protein:

[0038] (1) Regulating the disease resistance of plants to pepper mild mottle virus disease;

[0039] (2) Regulate crop yield;

[0040] (3) Control pepper mild mottle virus disease.

[0041] The present invention also provides a method for inhibiting the replication and proliferation of pepper mild mottle virus in plants, including: reducing the content or activity of NbCDPK8 protein by genetic engineering techniques.

[0042] Preferably, by introducing a substance for overexpressing the NbCDPK8 gene into plants, thereby promoting the replication and proliferation of pepper mild mottle virus in plants.

[0043] Preferably, by introducing a substance for inhibiting the expression of the NbCDPK8 gene into plants, thereby inhibiting the replication and proliferation of pepper mild mottle virus in plants.

[0044] The pepper mild mottle virus disease described in the present invention refers to a viral disease occurring in plants such as peppers and tobaccos caused by PMMoV infection.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] The present invention discovers that the NbCDPK8 protein and its coding gene play an important role in the infection and proliferation of PMMoV, and it is a susceptible gene of PMMoV encoded by the host plant. It can be used to control pepper mild mottle virus disease caused by PMMoV and can be used for molecular breeding work such as resistance / tolerance to PMMoV. The present invention can provide a basis for studying the role of plant proteins in virus infection and for fields such as antiviral breeding, improve the research level of antiviral in solanaceous vegetables, and ensure and promote the production safety of solanaceous vegetables. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0048] Figure 1 Results of inhibiting PMMoV infection by silencing NbCDPK8; among them, A is the infection photo; B is the statistical chart of the number of green fluorescent dots; C is the result chart of the expression level of the NbCDPK8 gene; D is the result chart of the accumulation amount of PMMoV RNA; E is the result chart of the accumulation amount of PMMoV CP protein. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0049] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0050] Unless otherwise specified, the embodiments are all carried out under conventional experimental conditions, such as those described in the Molecular Cloning experimental manual by Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended in the manufacturer's instructions.

[0051] In the following quantitative tests of the embodiments, three repeated experiments are set, and the results are averaged.

[0052] The biological materials used in the following embodiments are as follows:

[0053] Nicotiana benthamiana: See Goodin MM, Zaitlin D, Naidu RA, Lommel SA. 2008. Nicotiana benthamiana: its history and future as a model for plant - pathogen interactions. Molecular Plant - Microbe Interactions 21:1015 - 1026.

[0054] Vectors pJM23, pJG1024, pLoxP - Cre: See Yin, Y., Hua M., Zhao K., Wan Q., Bu S., Lu Y., Zhang, H., Rao, S., Yan F., Peng J., Chen H., and Chen, J. 2022. Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre / loxP system. Journal of Integrative Agriculture 21:2456 - 2463.

[0055] The vectors pTRV1 and pTRV2 are referred to: Liu, Y., Schiff M and Dinesh-Kumar SP. 2002. Virus-induced gene silencing in tomato. The Plant journal 32:777-786.

[0056] Pepper mild mottle virus (PMMoV) is referred to: Yin, Y., Hua M., Zhao K., Wan Q., Bu S., Lu Y., Zhang, H., Rao, S., Yan F., Peng J., Chen H., and Chen, J. 2022. Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre / loxP system. Journal of Integrative Agriculture 21:2456-2463.

[0057] The NbCDPK8 protein sequence in the following examples is shown in SEQ ID No. 1, and the gene encoding the NbCDPK8 protein is named the NbCDPK8 gene, and the sequence is shown in SEQ ID No. 2.

[0058] SEQ ID No. 1:

[0059] 1mgnccvtpgkssekkknkkk nkpnpfaidy gatqssaegd gnklvvlkdptghninekyd61lgrelgrgefgvtylctdvd tgekyacksi skkklrtavd iddvrrevqi mkhlpkhpni121vtlrdtyedd savhivmelc eggelfdriv arghyteraa agimrtivevvqmchrhgvm181hrdlkpenfl fgnkketapl kaidfglsvf fkpgehfneivgspyymapevlkrnygpev 241dvwsagvilyillcgvppfwaeteqgvaqaiirsvvdfkrdpwpkvsdnakdlvkkmldp 301dptrrltaqqvlehtwlqni kkapnvslge tvkarlkqfs vmnklkkraltviaeflsae 361evagmkeafdqmdtgkkgki nlgelkiglq klghqipdad lqilmeaadvdrdgclnyae 421fvavsvhlrk mandehlhkafsffdrnqsgyieieelrsalsdeddgnseevinaimhdv 481dtdkdgrisy eefaammkag tdwrkasrqysrerfnslslklmrdgslqlgneak。

[0060] SEQ ID No.2:

[0061]

[0062] Example 1 Construction of Recombinant Vector pTRV2-NbCDPK8

[0063] 1. Extract the total RNA from Nicotiana benthamiana and reverse transcribe it into cDNA.

[0064] 2. Using the cDNA obtained in step 1 as a template, perform PCR amplification with the primer pair consisting of pTRV2::NbCDPK8-F and pTRV2::NbCDPK8-R to obtain the PCR amplification product of the nucleotide sequence gene fragment shown at positions 1414-1606 of the NbCDPK8 (SEQ ID No.2) coding region; recover the PCR product of the gene fragment shown at positions 1414-1606 of the NbCDPK8 coding region.

[0065] pTRV2::NbCDPK8-F(SEQ ID No.3)

[0066] 5'-TAAGGTTACCGAATTCTCTAGAGTCATCAATGCCATTATGCAT GA-3'

[0067] pTRV2::NbCDPK8-R(SEQ ID No.4)

[0068] 5'-GAGACGCGTG AGCTCGGTAC CATTTAGCCT CGTTTCCAAG TTGTA-3'

[0069] 3. Digest the vector pTRV2 with the restriction endonucleases XbaI and KpnI, and recover the vector backbone.

[0070] 4. Ligate the recovery product of step 2 and the vector backbone of step 3 to obtain the recombinant vector pTRV2-NbCDPK8.

[0071] 6. According to the sequencing results, the structure of the recombinant vector pTRV2-NbCDPK8 is described as follows: A double-stranded DNA molecule shown in SEQ ID No.11 was inserted between the XbaI and KpnI restriction enzyme cleavage sites of the vector pTRV2.

[0072] SEQ ID No.11:

[0073] GTCATCAATGCCATTATGCATGATGTTGACACTGACAAGGATGG

[0074] TCGCATTAGTTACGAAGAATTTGCTGCAATGATGAAGGCTGGAA

[0075] CGGATTGGAGAAAAGCATCGAGACAGTATTCTCGTGAACGTTT

[0076] CAATAGTCTCAGCTTAAAATTGATGAGGGACGGCTCATTACAACTTGGAAACGAGGCTAAAT。

[0077] Example 2: Silencing experiment of NbCDPK8 gene

[0078] 1. Extraction of PMMoV-GFP crude extract

[0079] (1) Streak the Agrobacterium tumefaciens strains of pJM23, pJG1024, and pLoxP-Cre stored at -80°C on an LB resistant plate (containing 50 μg / ml Kan and 100 μg / ml Rif), and culture at 28°C for about 48 h; Inoculate the single colonies on the LB resistant plate into 3 - 4 ml of LB liquid resistant medium (containing 50 μg / ml Kan and 100 μg / ml Rif), and culture at 28°C and 180 rpm for 14 - 18 h.

[0080] (2) Inoculate 100 μL of the bacterial solution into 10 mL of LB liquid resistant medium (containing 50 μg / ml Kan and 100 μg / ml Rif) at a ratio of 1:100, and culture at 28°C and 180 rpm for 10 - 12 h to obtain the bacterial solutions of Agrobacterium tumefaciens pJM23, pJG1024, and pLoxP-Cre.

[0081] (3) At room temperature, centrifuge at 4000 rpm for 10 min to collect the bacterial cells, suspend the precipitate with 3 mL of infiltration buffer (10 mM MES, pH 5.7, 10 mM MgCl2, 200 μM acetosyringone), and measure the concentration of the bacterial suspension with a spectrophotometer.

[0082] (4) Mix 3 mL of the bacterial suspension of Agrobacterium tumefaciens pJM23 (OD 600nm concentration is 0.5), 3 mL of the bacterial solution of Agrobacterium tumefaciens pJG1024 (OD 600nm concentration is 0.5), and 3 mL of the bacterial solution of Agrobacterium tumefaciens pLoxP-Cre (OD 600nm concentration is 0.25), let it stand at room temperature for 2 - 3 h to obtain a mixed solution; Then use a syringe without a needle (specification 1 mL) to inject the mixed solution into the lower epidermis of Nicotiana benthamiana leaves, and culture at 22 - 24°C for 6 days.

[0083] (5) Take the Nicotiana benthamiana leaves, add 0.1M phosphate buffer for grinding, centrifuge at 4500g for 5 min, and collect the virus supernatant containing the crude extract of PMMoV.

[0084] 2. Use TRV VIGS to silence the NbCDPK8 gene in Nicotiana benthamiana

[0085] (1) Transform the recombinant plasmid pTRV2-NbCDPK8 into Agrobacterium tumefaciens GV1301 respectively to obtain the recombinant Agrobacterium tumefaciens TRV2-NbCDPK8 successively.

[0086] (2) Streak the Agrobacterium tumefaciens strains carrying pTRV1 and pTRV2-mcherry stored at -80°C on an LB resistant plate (containing 50 μg / ml Kan and 100 μg / ml Rif), and culture at 28°C for about 48 h; Inoculate the single colonies on the LB resistant plate into 3 - 4 ml of LB liquid resistant medium (containing 50 μg / ml Kan and 100 μg / ml Rif), and culture at 28°C and 180 rpm for 14 - 18 h.

[0087] (3) Inoculate 100 μL of the bacterial solution into 10 mL of LB liquid resistant medium (containing 50 μg / ml Kan and 100 μg / ml Rif) at a ratio of 1:100, and culture at 28°C and 180 rpm for 10 - 12 h to obtain the Agrobacterium tumefaciens solution carrying pTRV1.

[0088] (4) At room temperature, centrifuge at 4000 rpm for 10 min to collect the bacterial cells, suspend the precipitate with 3 mL of infiltration buffer (10 mM MES, pH 5.7, 10 mM MgCl2, 200 μM acetosyringone), and measure the concentration of the bacterial suspension with a spectrophotometer.

[0089] (5) Mix 3 mL of the Agrobacterium tumefaciens solution carrying pTRV1 (OD 600nm with a concentration of 1.0) with 3 mL of the bacterial suspension of the recombinant Agrobacterium tumefaciens pTRV2-NbCDPK8 (OD 600nm with a concentration of 1.0) and 3 mL of the Agrobacterium tumefaciens solution carrying pTRV2-mcherry (OD 600nm with a concentration of 1.0), place at room temperature for 2 - 3 h to obtain a mixed solution; Then use a needleless syringe (specification 1 mL) to inject the mixed solution into the lower epidermis of the 3rd - 4th true leaves of Nicotiana benthamiana, and culture at 22 - 24°C for 6 days.

[0090] 3. Challenge-inoculate Nicotiana benthamiana plants with PMMoV-GFP

[0091] (1) Take the Nicotiana benthamiana plants with silenced NbCDPK8 or the Nicotiana benthamiana plants with silenced mcherry (as a control) that have completed step (5) in step 2, and continue to culture them under alternating light and dark conditions (16 h at 20 °C light, 8 h at 18 °C dark) for 6 days. Sprinkle a little carborundum on the 5th - 6th true leaves of the Nicotiana benthamiana plants, and rub and inoculate with PMMoV - GFP (the inoculation amount is 50 μL, and the concentration is 0.3 g / mL). After that, continue to culture the inoculated Nicotiana benthamiana plants under alternating light and dark conditions (16 h at 20 °C light, 8 h at 18 °C dark).

[0092] 3. Detect the replication and proliferation levels of PMMoV in Nicotiana benthamiana plants with silenced NbCDPK8

[0093] Continuously observe the disease incidence of Nicotiana benthamiana leaves after inoculation with PMMoV. On the 4th day after PMMoV inoculation, take the inoculated leaves, extract total RNA and reverse - transcribe it into cDNA. Using cDNA as a template, adopt the fluorescence quantitative PCR kit of ComWin Biotech Co., Ltd., use the Actin gene as an internal reference gene, and identify the relative expression level of the NbCDPK8 gene and the content of PMMoV genomic RNA by RT - qPCR; at the same time, extract the total protein of Nicotiana benthamiana leaves and detect the accumulation amount of PMMoV CP protein.

[0094] The primer pairs for identifying the NbCDPK8 gene are as follows:

[0095] NbCDPK8 - qRT - F: 5'-CGGAGGTTGATGTGTGGAGT - 3'(SEQ ID No.5)

[0096] NbCDPK8 - qRT - R: 5'-GAGCCGTCGAGTTGGATCTG - 3'(SEQ ID No.6)

[0097] The primer pairs for identifying the Actin gene are as follows:

[0098] NbActin - qRT - F: 5'-TGCCATTCTCCGTCTTGACT - 3'(SEQ ID No.7)

[0099] NbActin - qRT - R: 5'-TGCAGTCTCGAGTTCCTGTT - 3'(SEQ ID No.8)

[0100] The primer pairs for identifying PMMoV CP are as follows:

[0101] PMMoV CP - qRT - F: 5'-TGGAAGACTATTCCGACCGC - 3'(SEQ ID No.9)

[0102] PMMoV CP - qRT - R: 5'-CCGTGCCACGAACTAACTCA-3' (SEQ ID No.10)

[0103] The data obtained were analyzed by the 2 -ΔΔCT method as follows:

[0104] First, for the experimental group samples (test) and the control group samples (calibrator), normalize the C T value of the target gene (target) with the C T value of the reference gene (ref):

[0105] ΔC T(test) = C T(target,test) - C T(ref,test)

[0106] ΔC T(calibrator) = C T(target,calibrator) - C T(ref,calibrator)

[0107] Second, normalize the ΔC T value of the experimental group samples with the ΔC T value of the control group samples:

[0108] ΔΔC T = ΔC T(test) - ΔC T(calibrator)

[0109] Finally, calculate the expression level ratio: 2 -ΔΔCT = ratio of expression levels

[0110] The results showed that: on the 4th day after PMMoV inoculation, compared with the control group (TRV - mcherry), the number of green fluorescent dots on the infected leaves of Nicotiana benthamiana in the experimental group (TRV - NbCDPK8) with silenced NbCDPK8 gene was less, as shown in Figure 1 (A, B); compared with the control group, in Nicotiana benthamiana plants with silenced NbCDPK8 gene, the expression level of NbCDPK8 was down - regulated by about 70%, as shown in Figure 1 (C); the accumulation level of PMMoV CP was down - regulated by about 75%, as shown in Figure 1 (D).

[0111] Using the anti - serum against TMV CP as the primary antibody and the HRP - labeled goat anti - rabbit antibody as the secondary antibody, western blot analysis was performed. The results showed that, compared with the control group, in Nicotiana benthamiana plants with silenced NbCDPK8 gene, the accumulation level of PMMoV CP protein was down - regulated by about 40%, as shown in Figure 1 (E, the quantitative value was calculated by ImageJ software. Anti - actin indicates using the expression level of actin as the internal reference).

[0112] The above research results indicate that by silencing the NbCDPK8 gene in Nicotiana benthamiana, the levels of PMMoV genomic RNA and CP protein can be significantly reduced, inhibiting the infection of PMMoV. These results suggest that the NbCDPK8 protein and its encoding gene play important roles in the replication and proliferation of PMMoV, and are host susceptibility genes that promote the infection of PMMoV. They can be used for controlling pepper mild mottle virus disease caused by PMMoV, as well as for molecular breeding against PMMoV and other work.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An NbCDPK8 protein for regulating the expression of pepper mild mottle virus, characterized in that, Its amino acid sequence includes any one of the following: (1) The amino acid sequence shown in SEQ ID No.1; (2) An amino acid sequence having at least 77.71% identity with the amino acid sequence shown in SEQ ID No.1, and the protein has the function of regulating the expression of pepper mild mottle virus; (3) An amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1, and the protein has the function of regulating the expression of pepper mild mottle virus.

2. An NbCDPK8 gene for regulating the expression of pepper mild mottle virus, characterized in that, Its nucleotide sequence includes any one of the following: (1) The nucleotide sequence shown in SEQ ID No.2 or its complementary sequence; (2) A sequence having at least 89.98% identity with the nucleotide sequence shown in SEQ ID No.2, and the polypeptide encoded by it has the function of regulating the expression of pepper mild mottle virus; (3) A sequence that hybridizes with SEQ ID No.2 under stringent conditions; (4) A sequence obtained by substituting and / or deleting and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID No.2, and the polypeptide encoded by it has the function of regulating the expression of pepper mild mottle virus.

3. A biological material, characterized in that, Including the NbCDPK8 gene for regulating the expression of pepper mild mottle virus described in claim 2; Preferably, the biological material is an expression cassette, a vector, a host cell, a transgenic cell line or a recombinant microorganism.

4. Use of the NbCDPK8 protein, or its coding gene, or a biological material containing its coding gene in any one or more of the following: (1) Use in regulating the expression of pepper mild mottle virus; (2) Regulating the disease resistance of plants to pepper mild mottle virus; (3) Controlling pepper mild mottle virus disease; (4) Regulating crop yield; (5) Use in improving plant germplasm resources; (6) Use in preparing products for controlling pepper mild mottle virus disease; The amino acid sequence of the NbCDPK8 protein includes any one of the following: (1) The amino acid sequence shown in SEQ ID No.1; (2) An amino acid sequence having at least 99% identity with the amino acid sequence shown in SEQ ID No.1, and the protein has the function of regulating the expression of pepper mild mottle virus; (3) An amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1, and the protein has the function of regulating the expression of pepper mild mottle virus; Preferably, the biological material is an expression cassette, a vector, a host cell, a transgenic cell line or a recombinant microorganism; Preferably, the plant or crop is a solanaceous plant, more preferably a plant of the genus Nicotiana or Solanum, and even more preferably tobacco or pepper.

5. The application according to claim 4, wherein the disease resistance of plants to pepper mild mottle virus, or the regulation of crop yield, or the prevention and control of pepper mild mottle virus, or the improvement of germplasm resources is regulated by adjusting the content or activity of NbCDPK8 protein.

6. The application according to claim 5, characterized in that Regulating the content or activity of NbCDPK8 protein by genetic engineering techniques.

7. The application according to claim 5 or 6, characterized in that Inhibiting the replication and proliferation of pepper mild mottle virus by reducing the content or activity of NbCDPK8 protein, so as to improve the disease resistance of plants to pepper mild mottle virus disease, or increase crop yield, or improve germplasm resources.

8. The application according to claim 4 or 5, characterized in that The coding gene of the NbCDPK8 protein includes the sequence shown in SEQID No.

2.

9. A product, characterized in that The product is a pesticide, a fertilizer or a fertilizer additive; the product achieves at least one of the following purposes by regulating the content or activity of NbCDPK8 protein: (1) Regulating the disease resistance of plants to pepper mild mottle virus disease; (2) Regulating crop yield; (3) Controlling pepper mild mottle virus disease.

10. A method for inhibiting the replication and proliferation of pepper mild mottle virus in plants, characterized in that Including: Reducing the content or activity of NbCDPK8 protein by genetic engineering techniques.

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