Method for preventing and controlling maize chlorotic mottle virus by using disease-resistant gene ZmCDPK7 and application of disease-resistant gene ZmCDPK7

Abstract

The invention discloses a method for preventing and controlling maize chlorotic mottle virus by using an anti-disease gene ZmCDPK7 and application of the anti-disease gene ZmCDPK7, and belongs to the technical field of plant protection. According to the application, through instantaneous silencing of the ZmCDPK7, an overexpression and knockout line of the ZmCDPK7 is constructed, the resistance reaction of the overexpression and knockout line to maize chlorotic mottle virus infection is analyzed, and it is proved that the ZmCDPK7 positively regulates the immunity of maize to the MCMV. In order to further clarify the molecular mechanism of the ZmCDPK7 for regulating and controlling immunity, interaction between the ZmCDPK7 and a key pathogenic factor P31 of the MCMV is discovered and proved through technologies such as yeast two-hybridization and bimolecular fluorescence complementation. ROS detection proves that the ROS content of the ZmCDPK7 knockout strain is higher than that of a wild type after MCMV infection. In biological and abiotic stress reactions, the resistance of the ZmCDPK7 knockout strain to biological and abiotic stress is proved to be reduced.

Description

Technical Field

[0001] This invention relates to the field of plant protection technology, and in particular to a method and application of the disease resistance gene ZmCDPK7 for controlling maize chlorotic mottle virus. Background Technology

[0002] Maize chlorotic mottle virus (MCMV) is a virus belonging to the Tomato Cluster Dwarf Virus family (Tomato Cluster Dwarf Virus family). Tombusviridae Maize chlorotic mottle virus ( ) Machlomovirus The only member of the family MCMV. MCMV can co-infect with other Potato Virus Y viruses (such as maize dwarf mosaic virus and wheat stripe mosaic virus), causing maize lethal necrosis (MLN), posing a serious threat to global maize production. The MCMV genome is approximately 4.4 kb and encodes 7 proteins, among which the P31 protein is a key factor in viral pathogenicity.

[0003] Calcium is a ubiquitous second messenger in plant cells, participating in the regulation of plant responses to biotic and abiotic stresses. In plants, calcium-dependent protein kinases (CDPKs) are the only class of calcium signal decoding proteins possessing both calcium sensor and kinase domains, enabling them to directly translate calcium signals into downstream biological responses. CDPKs play crucial roles in plant growth and development, and in responses to biotic stresses (such as pathogen infection) and abiotic stresses (such as drought and salt stress). For example, in maize, ZmCPK39 has been found to negatively regulate numerical resistance to gray leaf spot, large leaf spot, and small leaf spot. Furthermore, ZmCPK35 and ZmCPK37 enhance drought tolerance in maize by activating the ZmSLAC1 channel protein in guard cells. However, although the importance of CDPKs in plant immunity is gradually being revealed, their specific regulatory mechanisms in maize, especially the signaling pathways associated with virus infection, remain poorly understood.

[0004] Although the functions of CDPKs in plant immunity are gradually being revealed, research on their interactions with plant viruses remains limited. In Arabidopsis, AtCPK3 restricts intercellular transmission of potato virus X (PVX) by phosphorylating the S74, T86, and S91 sites of the StRem1.3 protein. In wheat, TaTHI2 interacts with TaCPK5 to inhibit the infection of Chinese wheat mosaic virus (CWMV) by regulating the accumulation of reactive oxygen species (ROS). These studies suggest that CDPKs may play an important role in plant antiviral immunity. However, the specific mechanisms by which CDPKs contribute to antiviral immunity in maize remain unclear. Summary of the Invention

[0005] The application aims to provide a method for preventing and controlling maize chlorotic mottle virus by using the disease-resistant gene ZmCDPK7 and application thereof, so as to solve the problems in the prior art.

[0006] To achieve the above-mentioned object, the application provides the following solutions. In one of the technical solutions of the application, the ZmCDPK7 gene or the protein coded by the ZmCDPK7 gene is applied to improving the resistance of plants to maize chlorotic mottle virus.

[0007] In another of the technical solutions of the application, the recombinant vector, expression cassette or transgenic cell line containing the ZmCDPK7 gene is applied to improving the resistance of plants to maize chlorotic mottle virus.

[0008] In a third of the technical solutions of the application, a method for improving the resistance of plants to maize chlorotic mottle virus is provided, in which the ZmCDPK7 gene is overexpressed or the level of the protein coded by the ZmCDPK7 gene is improved, so as to improve the resistance of plants to maize chlorotic mottle virus.

[0009] In a fourth of the technical solutions of the application, the ZmCDPK7 gene or the protein coded by the ZmCDPK7 gene is applied to cultivating a maize line with high resistance to maize chlorotic mottle virus, in which the ZmCDPK7 gene is overexpressed or the level of the protein coded by the ZmCDPK7 gene is improved, so as to improve the resistance of plants to maize chlorotic mottle virus.

[0010] In a fifth of the technical solutions of the application, a method for cultivating a maize line with high resistance to maize chlorotic mottle virus is provided, in which the ZmCDPK7 gene is overexpressed or the level of the protein coded by the ZmCDPK7 gene is improved, so as to improve the resistance of plants to maize chlorotic mottle virus.

[0011] Based on the above technical solutions, the application has the following technical effects: The application first finds that overexpression of the ZmCDPK7 gene can improve the resistance of maize to MCMV, and identifies and analyzes the molecular mechanism of ZmCDPK7 in regulating the plant immune resistance in the MCMV infection process, and the results show that the key pathogenic protein factor P31 of MCMV interacts with ZmCDPK7, the resistance of the plant with overexpressed ZmCDPK7 to MCMV is increased, the virus accumulation amount of MCMV is higher than that of the control plant after ZmCDPK7 is silenced or knocked out, and in summary, overexpression of the ZmCDPK7 gene can enhance the resistance immunity to MCMV. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1The accumulation of MCMV is promoted by silencing ZmCDPK7, wherein A: detection of ZmCDPK7 silencing efficiency; B: phenotype of ZmCDPK7 silencing group and control group after inoculation of MCMV; C: qRTPCR detection of CMV-GFP and CMV-ZmCDPK7 silencing, and MCMV CP accumulation in corn at 7 days after inoculation of MCMV; D: Western Blot detection of CMV-GFP and CMV-ZmCDPK7 silencing, and MCMV CP protein accumulation in corn at 7 days after inoculation of MCMV.

[0013] Figure 2 The accumulation of MCMV is promoted by knocking out ZmCDPK7, wherein A: ZmCDPK7 knockout strain; B: phenotype of control and ZmCDPK7-KO corn after inoculation of MCMV for 7 days; C: qRT-PCR detection of MCMV CP accumulation in control and ZmCDPK7-KO corn at 7 days after inoculation of MCMV.

[0014] Figure 3 The accumulation of MCMV is inhibited by overexpression of ZmCDPK7, wherein A: qRT-PCR analysis of ZmCDPK7 expression in control and ZmCDPK7-OE corn; B: phenotype of control and ZmCDPK7-KO corn after inoculation of MCMV for 7 days; C: qRT-PCR analysis of MCMV CP accumulation in control and ZmCDPK7-OE corn at 7 days after inoculation of MCMV.

[0015] Figure 4 The interaction of ZmCDPK7 and P31, wherein A: yeast two-hybrid verification of P31 and ZmCDPK7 interaction; B: verification of P31 and ZmCDPK7 interaction by bimolecular fluorescence complementation; C: verification of P31 and ZmCDPK7 interaction by immunoprecipitation.

[0016] Figure 5 ROS content detection of ZmCDPK7 knockout strain; A: DAB staining of knockout strain and B104 control group, B: H2O2 detection of knockout strain and B104 control group.

[0017] Figure 6 Response of ZmCDPK7 to biotic and abiotic stresses; wherein A, B: phenotype of ZmCDPK7 knockout strain and B104 after drought treatment; C, D, E: MDA, electrolyte permeability and H2O2 content, F, G: rhizome phenotype after inoculation of F. verticillioides. DETAILED DESCRIPTION

[0018] The technical solutions described in the present application are conventional solutions in the art if not specifically stated, and the reagents or raw materials used are purchased from commercial channels or publicly disclosed if not specifically stated.

[0019] The application provides application of the ZmCDPK7 gene or the protein coded by the ZmCDPK7 gene in improving the resistance of plants to maize chlorotic mottle virus.

[0020] In some specific embodiments, the nucleotide sequence of the ZmCDPK7 gene is shown as SEQ ID NO. 1.

[0021] In some specific embodiments, overexpression of the ZmCDPK7 gene or the protein coded by the ZmCDPK7 gene improves the resistance of plants to maize chlorotic mottle virus.

[0022] In some specific embodiments, the plants include maize.

[0023] The application also provides application of the recombinant vector, the expression cassette or the transgenic cell line containing the ZmCDPK7 gene in improving the resistance of plants to maize chlorotic mottle virus.

[0024] The application also provides a method for improving the resistance of plants to maize chlorotic mottle virus, overexpression of the ZmCDPK7 gene or the protein coded by the ZmCDPK7 gene improves the resistance of plants to maize chlorotic mottle virus.

[0025] The application also provides application of the ZmCDPK7 gene or the protein coded by the ZmCDPK7 gene in breeding maize lines with high resistance to maize chlorotic mottle virus, overexpression of the ZmCDPK7 gene or the protein coded by the ZmCDPK7 gene improves the resistance of plants to maize chlorotic mottle virus.

[0026] The application also provides a method for breeding maize lines with high resistance to maize chlorotic mottle virus, overexpression of the ZmCDPK7 gene or the protein coded by the ZmCDPK7 gene improves the resistance of plants to maize chlorotic mottle virus.

[0027] In some specific embodiments, the method comprises the following steps: introducing the ZmCDPK7 gene into target plants.

[0028] In some specific embodiments, the ZmCDPK7 gene is introduced into target plants by the recombinant vector, the expression cassette or the transgenic cell line containing the ZmCDPK7 gene.

[0029] The application proves that ZmCDPK7 positively regulates the immunity of corn to MCMV by constructing overexpression and knockout lines of ZmCDPK7 through transient silencing of ZmCDPK7 and analyzing the resistance response of the corn to MCMV infection. In order to further clarify the molecular mechanism of ZmCDPK7 regulating immunity, the application finds and proves the interaction between ZmCDPK7 and the key pathogenic factor P31 of MCMV through techniques such as yeast two-hybrid and bimolecular fluorescence complementation. And it is proved that the ROS content of the ZmCDPK7 knockout line is higher than that of the wild type after MCMV infection through ROS detection. In the stress response of biotic and abiotic stress, it is proved that the resistance of the ZmCDPK7 knockout line to biotic and abiotic stress is reduced.

[0030] Example 1 Cloning of genes I. Extraction of total RNA of corn B73 1. Put the plant sample into a 2 ml centrifuge tube, put two 4 mm steel balls, and freeze in liquid nitrogen.

[0031] 2. Put the 2 ml centrifuge tube into the shock mill at 22 Hz for 45 s, and shock mill 4-5 times until the sample is powdered.

[0032] 3. Put the 2 ml centrifuge tube on ice, add 1 mL Trizol (4℃), homogenize with a vortex, stand for 5 min, and centrifuge at 12000 g for 10 min in a 4℃ centrifuge. Precool chloroform in advance.

[0033] 4. Absorb the supernatant into a new 1.5 ml centrifuge tube, put it on ice, add 250 µL of chloroform, homogenize with a vortex, stand for 10 min, and centrifuge at 12000 g for 10 min in a 4℃ centrifuge. Precool isopropanol at -20℃.

[0034] 5. Absorb the supernatant into a new 1.5 ml centrifuge tube, put it on ice, add an equal volume of isopropanol, homogenize with a vortex, stand for 20 min, and centrifuge at 12000 g for 10 min in a 4℃ centrifuge.

[0035] 6. Pour out the supernatant, add 1 ml of 75 % anhydrous ethanol, shake with a vortex, and centrifuge at 7500 g for 5 min in a 4℃ centrifuge.

[0036] 7. Repeat the previous step.

[0037] 8. Pour out the supernatant, stand at room temperature for 3-5 min, dissolve with DEPC water, and mix well with a vortex.

[0038] II. Synthesis of cDNA by reverse transcription Genomic DNA removal Table 1

[0039] Put into PCR instrument, 42℃, 2 min.

[0040] Synthesis of cDNA Table 2

[0041] Put into PCR instrument 37℃ 15 min, 85℃ 5 s, store in -20℃ refrigerator.

[0042] III. RT-PCR amplification of ZmCDPK7, P31 genes Using the above cDNA template, and according to the ZmCDPK7 gene nucleic acid sequence to design primers, PCR amplification, reaction system as shown in Table 3.

[0043] Table 3

[0044] The primer sequence used is shown in Table 4.

[0045] Table 4

[0046] After mixing the reagents, PCR was performed according to the conditions in Table 5.

[0047] Table 5

[0048] According to the detection results, use the gel cutting kit for purification and recovery.

[0049] The nucleotide sequence of ZmCDPK7, P31 genes is as follows: Nucleotide sequence of P31 gene (SEQ ID NO. 2): ATGTCTTCTTCTCAAACACAATCCCCTCCCACCGACGTAGCCCGCAGACAACAGACCGGCACTGCCGTGCGATCTGAAGACAACCATCGCAATCAGTTGGAAAACATTGCTGTTGGAAAACTCACTAAATCTGAAGGAGCACCTGCCCAACAGAACGTGATCATTGCGAAAGAGGTGGTTATCAATAACCACTTCAATTTCAACTATGCTGGAGTGTGTGTGTGTAGATTCGTCCTGGCCTCAGTGGTTAAGGAATTTAATCTTGGGGATTTTGATCTCCTCGATTCTGTTCATCTTGACGAAGACACAGGACACCGTTGCCGTTTATCACGAGCCATCGGTGTACTCGATCGATCAGACACAAAAATTCCAAAAGATCGACATACACAATGGCGGCAAGTAGCCGGTCTACCCGAGGTAGAAAGCAGCGCGGACGTAGCGTGGAAGCAAAATCCAGAGCTATTCGAGCCAACCCGCCTGTCCCTCGACCCAACCCGCAGCGAAACCGTCCCCCCCCTGCGGGAACAACCTGCTCCATGTCCGAAATTCTGCTTGCAGTGTCAGCAACAACTGCTGACCAGATTCTCGAGATTCCAGTGTGTGCAGGGATTGACTTCCCGGCTGGAACGTCGCCCCGATACATTGGGGCGGCCAAATGGCTGGCAGCACAATCACAGATGTGGAATACAATTGTGTTCAACTCTGTGCGCATCACTTGGGAGACATTCACAGCAGACACCACTAGCGGATACATTTCCATGGCATTCCTCTCCGATTACATGTTATCAATACCCACTGGGGTGGAGGATGTTGCCAGGATCGTGCCCTCAGCTACAAtAG.

[0050] Example 2 Effect of ZmCDPK7 gene silencing on MCMV infection I. Construction of silencing vector CMV-ZmCDPK7 Systemic silencing of ZmCDPK7 gene was performed by CMV infectious clone mediated VIGS technology. A 300 bp fragment was predicted by SGN VIGS Tool (http: / / vigs.solgenomics.net) using the cDNA of Zea mays B73 as the template for amplification. The primer was designed and the target fragment was amplified by PCR: The primer sequence is shown in Table 6.

[0051] Table 6

[0052] The fragment was cloned into the VIGS vector by homologous recombination. First, the vector was double-digested, and the system is shown in Table 7.

[0053] Table 7 Enzyme digestion system of vector

[0054] The reaction system was placed in a water bath at 37℃ for 6 h. The gel cutting kit was used for purification and recovery.

[0055] The fusion cloning was performed using the homologous recombination ligation kit (C112), and the above ligation product was transformed into DH5a; 37℃ incubation overnight, and single colony PCR was performed. The plasmid was extracted and sent for testing.

[0056] II. Identification of silencing efficiency of ZmCDPK7 silenced plants The agrobacterium was transformed with the silencing vector and combined with CMV-RNA1 and CMV-RNA3 vectors to infect Nicotiana benthamiana for 3-5 days. The virus liquid was extracted and the corn seeds were pricked. The leaves were taken at the two-leaf stage and the silencing efficiency was detected by qRT-PCR.

[0057] The primer sequence is shown in Table 8.

[0058] Table 8

[0059] The reaction system is shown in Table 9.

[0060] Table 9

[0061] The reaction procedure is shown in Table 10.

[0062] Table 10

[0063] The real-time fluorescent quantitative PCR results were analyzed and the data was arranged. The results are shown in Figure 1From the results of Fig. 1A, the relative mRNA level of ZmAPX1 was significantly reduced, and the gene ZmCDPK7 was silenced.

[0064] III. The effect of ZmCDPK7 silencing on MCMV infection MCMV was inoculated into CMV-GFP and CMV-ZmCDPK7 infected maize seedlings, and CMV-GFP was used as a control group to verify the effect of ZmCDPK7 silencing on MCMV infection. Seven days after inoculation, the virus accumulation was detected by qRT-PCR and western blot (Note: CMV-GFP is a plasmid preserved in the laboratory).

[0065] 1. qRT-PCR detection of virus accumulation Table 11 Primers

[0066] 2. Western Blot detection 1) Different protein gel formulations are selected according to the size of the protein.

[0067] 2) Prepare the separation gel according to the formula, add the separation gel to the glass plate, then add ddH2O2 to flatten it. Let it stand for 30 min.

[0068] 3) After the separation gel solidifies, prepare the concentrated gel according to the formula, pour off the ddH2O2 and add the concentrated gel, and insert the comb corresponding to the gel plate. Let it stand for 30 min.

[0069] 4) Add the deformed protein to the gel well, and after the protein Maker band is separated, adjust the voltage to 120 V.

[0070] 5) Cut 8 pieces of filter paper slightly larger than the gel and 1 piece of NC membrane. Soak the NC membrane and filter paper in the transfer solution.

[0071] 6) Transfer according to the order of 4 layers of filter paper-NC membrane-gel-4 layers of filter paper.

[0072] 7) Constant voltage 20 V, 25 min.

[0073] 8) After the transfer is completed, place the NC membrane in the blocking solution and seal it in a horizontal shaker at room temperature for 1 h.

[0074] 9) Add the primary antibody to the blocking solution according to the corresponding proportion, incubate on a horizontal shaker at room temperature for 2 h or in a refrigerator at 4°C overnight. Recover the primary antibody diluent in the -20°C refrigerator.

[0075] 10) Wash the NC membrane with TBST solution 3 times, 10 min each time, and wash it on a horizontal shaker at room temperature.

[0076] 11) Add secondary antibody to blocking solution at the corresponding ratio, incubate on a horizontal shaker at room temperature for 1 h, pour off the secondary antibody dilution and wash the NC membrane with TBST for 3 times.

[0077] 12) Add ECL chemiluminescence developing solution to the front of the NC membrane, and develop with a super-sensitive multifunctional imager.

[0078] Results from Figure 1 C, D, the phenotype results from Figure 1 B can be known. Compared with the control, the accumulation of MCMV RNA and protein in the plants transiently silenced by ZmCDPK7 was significantly increased.

[0079] Example 3 Effect of ZmCDPK7 gene knockout on MCMV infection I. Gene editing Further, ZmCDPK7 knockout mutants (ZmCDPK7-KO1, ZmCDPK7-KO2) were obtained by CRISPR / Cas9-mediated gene editing (FIG. 1A). Figure 2 A).

[0080] II. Phenotype observation after MCMV inoculation MCMV was inoculated on ZmCDPK7-KO and control corn, and symptoms were observed 7 days after inoculation. ZmCDPK7-KO lines showed more obvious mosaic symptoms than control B104 (FIG. 1B). Figure 2 B).

[0081] III. qRT-PCR and analysis of virus accumulation Compared with B104, the accumulation of MCMV CP mRNA in ZmCDPK7-knockout plants was significantly increased (FIG. 1C). Figure 2 C).

[0082] Example 4 Effect of overexpression of ZmCDPK7 gene on MCMV infection I. Vector construction A transgenic vector for overexpression of ZmCDPK7 was constructed, and transgenic corn overexpressing ZmCDPK7 (ZmCDPK7-OE) was obtained by transformation (FIG. 2A). Figure 3 A) II. Phenotype after MCMV inoculation MCMV was inoculated on ZmCDPK7-OE and control corn, and symptoms were observed 7 days after inoculation. The systemic leaf mosaic symptoms of ZmCDPK7-OE were lighter than the control (FIG. 2B). Figure 3 B).

[0083] III. qRT-PCR analysis of virus accumulation Compared with B104, the accumulation level of MCMV mRNA in ZmCDPK7 overexpressed plants was significantly reduced Figure 3 C).

[0084] Example 5 Interaction of ZmCDPK7 and P31 I. Yeast two-hybrid analysis The primers for amplifying fragments were designed by using the software snapgene, and the C116 ligase was used to connect the P31 and ZmCDPK7 full-length fragments to the corresponding AD and BD vectors to obtain the corresponding vectors AD-P31 and BD-ZmCDPK7. AD-T+BD-53a was used as a positive control and AD-T+BD-Lam was used as a negative control. The constructed AD-P31 and BD-ZmCDPK7 recombinant vectors were co-transformed into the yeast strain Y2H Gold by using the yeast two-hybrid system, and were plated on the two-lacking medium (SD-TL) and the four-lacking medium (SD-TLHA), respectively. The yeasts with the above plasmid combinations could grow normally on the two-lacking medium, and only AD-P31+ZmCDPK7 and the AD-T+BD53a positive control could grow normally on the four-lacking medium. Figure 4 A). The above results show that AD-P31 and ZmCDPK7 can interact in vitro. (Appendix: AD-T, BD-Lam, BD-53a are plasmids preserved in our laboratory).

[0085] Table 12 Primers

[0086] II. Bi-molecular fluorescence complementation experiment The P31 full-length reading frame was constructed into the vector pLIC-nYFP, and the ZmCDPK7 full-length reading frame was constructed into the vector pLIC-cYFP to obtain the recombinant vectors P31-nYFP and ZmCDPK7-cYFP. The recombinant plasmids were transformed into the Agrobacterium strain GV3101, and the Agrobacterium liquid P31-nYFP was mixed with ZmCDPK7-cYFP and empty cYFP and empty nYFP at a ratio of 1:1, and was infiltrated into the four-leaf stage Nicotiana benthamiana leaves, which were cultured in the greenhouse for 48 h. The YFP fluorescence of the Nicotiana benthamiana leaves was observed by using the laser confocal microscope. The results showed that only the ZmCDPK7+P31 combination could observe YFP fluorescence, and the other combinations could not observe YFP fluorescence Figure 4 B), which shows that ZmCDPK7 and P31 interact in plants.

[0087] III. CO-IP P31 full length reading frame was constructed into MYC vector, ZmCDPK7 full length reading frame was constructed into GFP vector, and the recombinant vectors P31-Myc and ZmCDPK7-GFP were obtained. Agrobacterium containing P31-Myc and ZmCDPK7-GFP and GUS-Myc were infiltrated into N. benthamiana leaves at the four-leaf stage, and cultured in the greenhouse for 48 h. GUS-Myc and ZmCDPK7-GFP were used as the control group, and P31-Myc and ZmCDPK7-GFP were used as the treatment group. Western blot was used for detection, and P31-Myc, ZmCDPK7-GFP and GUS-Myc were normally expressed. Immunoprecipitation was performed on total protein using GFP magnetic beads, and the results showed that P31-Myc and ZmCDPK7-GFP combination could detect the band of P31-Myc, while GUS-Myc and ZmCDPK7-GFP combination could not detect the band of GUS-Myc (Fig. 6C). Figure 4 Further proved that P31 and ZmCDPK7 could interact in plants. Figure 4

[0088] Example 6 Regulation of ROS by ZmCDPK7 I. DAB staining Preparation of DAB (3,3'-diaminobenzidine) staining solution: solvent: 50 mm Tris.HCl, concentration: 2 mg / mL (need to prepare fresh).

[0089] 1. Take a picture of the leaves before staining.

[0090] 2. Take the same batch of intact leaves for DAB staining, and stain for more than 8 h or overnight (avoid light staining).

[0091] 3. Boil and decolorize with 95 %±5 % alcohol, and boil until the leaves turn white.

[0092] 4. After decolorization, store in 75 %±5 % alcohol.

[0093] 5. Take a picture after decolorization, and match it with the picture of the leaves before staining.

[0094] In order to study whether ZmCDPK7 mediates the regulation of ROS by virus infection, the level of ROS in ZmCDPK7 knockout line was observed using DAB staining method, and the accumulation of H2O2 was detected. High level of H2O2 accumulation was found in ZmCDPK7 knockout line (Fig. 6A-B). Figure 5

[0095] Example 7 ​​Effect of ZmCDPK7 on drought stress and Fusarium verticillioides infection Fusarium verticillioides ) infection I. Phenotype observation To study the role of ZmCDPK7 under drought stress, two independent transgenic knock-out lines of ZmCDPK7 were selected for stress tolerance assays. Under drought conditions, the zmcdpk7-ko lines showed significantly wilted and curled leaves, while wild type plants showed moderate wilting ( Figure 6 Fig. A-B).

[0096] II. MDA (malondialdehyde) and electrolyte leakage assays Both WT and zmcdpk7 mutants showed a significant increase in MDA (malondialdehyde) content and electrolyte leakage, and the levels were higher in the zmcdpk7 lines than in the WT ( Figure 6 Fig. C-D). In addition, the H2O2 content was higher in the zmcdpk7 lines than in the WT ( Figure 6 Fig. E).

[0097] These findings support the observed greater sensitivity of the zmcdpk7 mutants to drought stress.

[0098] III. Effect of ZmCDPK7 on Fusarium verticillioides infection In addition, the present invention evaluated the resistance of ZmCDPK7-KO lines to Fusarium verticillioides (Fv), the main pathogen causing corn stalk rot. Infection assays showed that both zmcdpk7 lines showed an increased sensitivity to F. verticillioides ( Figure 6 Fig. F-G).

[0099] In the present invention, it is proved that the ZmCDPK7 transiently silenced plants have a weakened immunity to MCMV compared with the control CMV-GFP; the ZmCDPK7 knock-out lines are more sensitive to MCMV than the wild type plant B104; overexpression of ZmCDPK7 can enhance the immunity to MCMV; ZmCDPK7 interacts with the key pathogenic factor P31 of MCMV in vitro and in vivo; the reactive oxygen species content of the ZmCDPK7 transiently silenced plants increases after MCMV infection compared with the control, and ZmCDPK7 also has a positive regulatory effect on drought stress and FV. In summary, ZmCDPK7 positively regulates the immune response to viral diseases in corn.

[0100] Obviously, the above embodiments of the present application are merely exemplary but not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, it is not necessary or possible to exhaust all the embodiments. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. Use of ZmCDPK7 gene or its encoded protein in improving plant resistance to maize chlorotic mottle virus.

2. Use according to claim 1, characterized in that, The nucleotide sequence of the ZmCDPK7 gene is shown as SEQ ID NO.

1.

3. Use according to claim 1, characterized in that, Overexpression of ZmCDPK7 gene or increasing the level of its encoded protein improves plant resistance to maize chlorotic mottle virus.

4. Use according to claim 1, characterized in that, The plant includes maize.

5. Use of recombinant vector, expression cassette or transgenic cell line containing ZmCDPK7 gene in improving plant resistance to maize chlorotic mottle virus.

6. A method of increasing the resistance of a plant to maize chlorotic mottle virus, characterized in that, Overexpression of ZmCDPK7 gene or increasing the level of its encoded protein improves plant resistance to maize chlorotic mottle virus.

7. Use of the ZmCDPK7 gene or the protein encoded by the same in the breeding of corn lines with high resistance to maize chlorotic mottle virus, characterized in that, Overexpression of ZmCDPK7 gene or increasing the level of its encoded protein improves plant resistance to maize chlorotic mottle virus.

8. A method of breeding a corn line having high resistance to corn chlorotic mottle virus, comprising the steps of, Overexpression of ZmCDPK7 gene or increasing the level of its encoded protein improves plant resistance to maize chlorotic mottle virus.

9. The method of claim 8, wherein, The method comprises the following steps: The ZmCDPK7 gene is introduced into the target plant.

10. The method of claim 9, wherein, The ZmCDPK7 gene is introduced into the target plant by the recombinant vector, expression cassette or transgenic cell line containing ZmCDPK7 gene as described in claim 5.

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