CN116334023A - Application of Calcium-Dependent Protein Kinase Gene GhCDPK29 in Plant Resistance to Verticillium Wilt - Google Patents

Abstract

The invention discloses application of a calcium-dependent protein kinase gene GhCDPK29 in verticillium wilt resistance of plants. Belongs to the technical field of genetic engineering. The invention utilizes the sequence information of the gene GhCDPK29 to amplify the gene and constructs a VIGS plant expression vector to transform upland cotton TM-1, so as to obtain transgenic cotton, which shows the resistance to verticillium wilt after being inoculated with cotton verticillium wilt bacteria V991, and the high correlation between the gene GhCDK 29 and the resistance to cotton verticillium wilt is demonstrated. The invention lays an application foundation for molecular mechanism research of verticillium wilt resistance and cultivation of new varieties of new verticillium wilt-resistant plants, and provides new reference and reference for screening new disease-resistant genes and cultivating resistant plants.

Description

Application of Calcium-Dependent Protein Kinase Gene GhCDPK29 in Plant Resistance to Verticillium Wilt

technical field

The invention relates to the technical field of genetic engineering, and more specifically relates to the application of calcium-dependent protein kinase gene in plant resistance to verticillium wilt.

Background technique

Cotton occupies an important position in my country's economic development and is an important fiber crop in my country. Upland cotton is the main cotton cultivar due to its high yield and good fiber quality. Cotton verticillium wilt is caused by the soil-borne fungus Verticillium dahliae, which causes damage to the vascular bundles of cotton, leading to wilting, wilting, yellowing and shedding of leaves, which seriously restricts the growth and development of cotton. Although a number of Verticillium wilt-resistant cotton varieties have been screened through conventional breeding methods, due to the lack of germplasm resources, poor broad-spectrum, and long breeding time, no excellent Verticillium wilt-resistant varieties have been bred yet. In order to meet the needs of my country's breeding industry and break through resource constraints, it is very important to mine disease-resistant genes from upland cotton resources, analyze disease-resistant mechanisms, and use transgenes to innovate new disease-resistant germplasm.

Plant disease resistance mechanisms are complex and diverse. Under the stimulation of pathogenic bacteria, plants produce a series of disease resistance signal transduction and defense mechanisms, among which there are a large number of transcriptional regulatory factors and genes. Disease resistance gene (Disease Resistance, R) is a key factor in mediating plant defense response. It can directly or indirectly discover the avirulent gene product of pathogenic bacteria, and then trigger a series of downstream defense responses. The expression level of R gene was low under the condition of no pathogenic bacteria, and the expression of R gene was significantly activated under the induction of pathogen infection. R gene overexpression can even affect the normal growth of plants or cause plant death. In addition, reactive oxygen species (ROS) have also been found to play roles in calcium ion regulation signals and hormone regulation factors in plant disease resistance defense responses, thereby changing plant disease resistance.

CDPKs are a class of threonine/serine protein kinases widely distributed in plants. CDPKs transmit calcium signals downstream and cause plant physiological responses by phosphorylating various substrates, including transcription factors, metabolic substrates and metabolic enzymes (Chen et al., 2021). With the in-depth study of CDPKs, it was found that they perform various biological functions by participating in cellular Ca ²⁺ signal transduction, including plant pollen tube growth, hormone signal transduction, rhizome development, and stomatal movement (Atifetal., 2019). It also plays an important role in plant abiotic and biotic stress responses. Studies have shown that overexpression of CDPK7 improves the cold resistance, drought resistance and salt tolerance of rice (Szczegielniak et al., 2012). In tobacco, gibberellin or salt treatment can significantly induce the expression of CDPK4 (Zhang et al., 2005). CDPK10 is involved in the regulation of abscisic acid and stomatal regulation in Arabidopsis under drought conditions (Zuo et al., 2010). Overexpression of CDPK6 improves the salt tolerance of Arabidopsis (Xu et al., 2010). Cotton CDPK5 gene can respond to the induction of salt stress (Zhen Junbo et al., 2011).

In summary, it is an urgent problem for those skilled in the art to find a calcium-dependent protein kinase gene that regulates cotton Verticillium wilt resistance.

Contents of the invention

In view of this, the present invention provides the application of calcium-dependent protein kinase gene GhCDPK29 in plant resistance to verticillium wilt.

In order to achieve the above object, the present invention adopts the following technical solutions:

The application of the calcium-dependent protein kinase gene GhCDPK29 in regulating plant resistance to Verticillium wilt, the nucleotide sequence of the gene GhCDPK29 is shown in SEQ ID NO:1.

ATGGGACTCTGCCAATCTCTGGGATTTTGCTTGAGAAGATCTCACTCTCATGAAATCCCAATCTCTTCCTCATCTGAGTCTCTCTCTCGTCCATCTCATCTATTCCCAAAAACCACCCCGCAACACTTTTAACCCTTCTTCTTCTAAAGCTACTTCATCCGGTATTGGAACCATTTTGCTCAAACCATACGTTGATGTCACCACCATTTATGATCTTGACAAAGA GTTGGGGAGAGGTCAGTTTGGCATCACTTATCTTGCACCGAAAAGGCAACGGGGCGAAAATACGCCTGCAAATCTATTTCAAGGCGTAAACTGCGCACTGACAGAGACATAGAGGATGTTAGAAGAGAGACTTCGATAATGCAGCACCTAACAGGGCAACCCAATATTGTGGAATTCAAAGGTGCTTATGAAGATAGGCAGAATGTGCATTTGGTGATGGA ACTGTGCTCAGGTGGCGAGCTTTTTGATCGGATCATAGCTAAAGGGAGTTATTCAGAACGTCAAGCGGCTTCTATTTGTCGACAGGTTGTGAATGTGGTGAATGCCTGTCATTTTTATGGGGGTTATGCATAGGGACTTGAAACCTGAAAACTTTTTGCTGGTCAGCAAGGATGAGATTTCTCCAATAAAAGCCCAGATTTTGGACTCTCCGTCTTCATT GAGGAAGGCAGAATGTATAAGGACCTTGTTGGAAGTGCATACTATGTTGCACCAGAGGTGTTAAACCGGAAATATGGCAAGGAGATAGATGTGTGGAGTGCTGGTGTCATTTTATACATTCTTCTTAGTGGAGTGCCTCCATTTTGGGGTGAGACTGAGAAAGAAATATTTAAAGCGGTTTTGAAGGTAATCTTGACTTGAAAAGCTTGCCATGGCC ATCTATAACTGAAGGTGCAAAGGACCTCATCAGAAAGATGCTAACAAGGGATCCTACGAAACGGATCACTGCTGCCCAAGCTCTAGAACATCCATGGCTGAAGGAGGGTGGTGATGCTTCCGACAAACCTATTGATAGTGCTGTCCTTAGCAGGTTGAAGCAGTTCAGAGTAATGAACAAGCTCAAAAACTAGCATTGAAGGTGATAGCAGAAAGCCTATCAACGGA AGAAGAGATCAAGGGCTTGCAACAGATGTTCAAAAACATCGACACTGACGGAAGTGGTACAATAACACTAGGAGAACTCAGGGATGGATTAGCTCGCTTGGGATCTAAGTTAACTGAAACTGAAATAAAGCAGCTTATGGATGCTGCTGATGTTGACAATAGTGGAACCATTGATTACATTGAATTCATAACTGCAACAATGCATCGACATAGGCTTGA GAGGGAAGAAAATATAGTCAAGGCTTTTCAGTTCTTTGACAAGGATAACAGTGGGTTTTATTACAAGAGATGAACTAAGACAAGCTATGACGCAGTACGGAATGGGAGATGAGGCTACAATAGATGAAGTCATTGAGGATGTTGATACTGATAAGGATGGGAGGATCAACTACGAGGAATTTGTAGCCATGATGAAAAGGGGAACTCATGATGGGG ATGGCAATTGGCAAAGACATATGAACAGCTAA; SEQ ID NO:1.

The application of the protein or the substance regulating the expression of the protein-coding gene in plant resistance to Verticillium wilt, the amino acid sequence of the protein is shown in SEQ ID NO:2.

MGLCQSLGFCLRRSHHEIPISSSSSESSSPRPSHLFPKTTPQHFNPSSSKATSSSGIGTILLKPYVDVTTIYDLDKELGRGQFGITYLCTEKATGRKYACKSISRRKLRTDRDIEDVRRETSIMQHLTGQPNIVEFKGAYEDRQNVHLVMELCSGGELFDRIIAKGSYSERQAASICRQVVNV VNACHFMGVMHRDLKPENFLLVSKDEISPIKATDFGLSVFIEEGRMYKDLVGSAYYVAPEVLNRKYGKEIDVWSAGVILYILLSGVPPFWGETEKEIFKAVLEGNLDLKSLPWPSITEGAKDLIRKMLTRDPTKRITAAQALEHPWLKEGGDASDKPIDSAVLSRLKQFRVMNKLKKLALKVIAES LSTEEEIKGLQQMFKNIDTDGSGTITLGELRDGLARLGSKLTETEIKQLMDAADDVDNSGTIDYIEFITATMHRHRLEREENIVKAFQFFDKDNSGFITRDELRQAMTQYGMGDEATIDEVIEDVDTDKDGRINYEEFVAMMKRGTHDGDGNWQRHMNS*; SEQ ID NO: 2.

The application of the above-mentioned gene GhCDPK29 or the above-mentioned protein-related biological material in plant resistance to Verticillium wilt, the biological material is any of the following:

A: Nucleotide sequence such as the above-mentioned nucleic acid molecules or nucleic acid molecules encoding the above-mentioned proteins;

B: an expression cassette containing the silence of the nucleic acid molecule described in A;

C: an expression vector containing the nucleic acid molecule described in A, or a recombinant vector containing the expression cassette described in B;

D: A recombinant microorganism containing the nucleic acid molecule described in A, or a recombinant microorganism containing the expression cassette described in B, or a recombinant microorganism containing the recombinant vector described in C.

The term "expression cassette" refers to a DNA capable of expressing the protein described in the above applications in a host cell, and the DNA may include not only a promoter for initiating the transcription of the protein-encoding gene, but also a terminator for terminating the transcription of the protein-encoding gene. Further, the expression cassette may also include an enhancer sequence.

Further, the application of the above-mentioned gene GhCDPK29 or the above-mentioned protein-related biological materials in the breeding of plant varieties with enhanced or weakened resistance to Verticillium wilt.

Further, the application of the above-mentioned gene GhCDPK29 or the above-mentioned protein-related biological materials in plant breeding.

Further, the plant is a cotton plant.

Further, the plant is cotton.

Further, the plant is Upland cotton TM-1.

A method for improving plant resistance to Verticillium wilt, the ability of plants to resist Verticillium wilt is improved by silencing or inhibiting the expression of the gene GhCDPK29 in plants or the activity of the above-mentioned protein.

The above-mentioned silencing or suppression of the expression of the protein-coding gene in the plant can be achieved by any method in the prior art, so that the gene produces a deletion mutation, an insertion mutation or a base conversion mutation, thereby reducing or losing the function of the gene.

It can be seen from the above-mentioned technical scheme that compared with the prior art, the beneficial effects obtained by the present invention are as follows: the present invention utilizes the sequence information of the gene GhCDPK29 to amplify the gene, and constructs a VIGS plant expression vector to transform Upland Cotton TM-1, and the obtained transgene Cotton showed resistance to Verticillium dahliat after inoculation with cotton Verticillium dahliae V991, indicating that the gene GhCDPK29 is highly related to the resistance of cotton Verticillium dahliat. The invention lays an application foundation for the research on the molecular mechanism of Verticillium wilt resistance and the cultivation of new varieties of Verticillium wilt-resistant plants, and also provides new references and references for screening new disease-resistant genes and cultivating resistant plants.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only It is an embodiment of the present invention, and those skilled in the art can also obtain other drawings according to the provided drawings without creative work.

Fig. 1 accompanying drawing is the electrophoresis figure of the PCR product of gene GhCDPK29 in the embodiment 1 of the present invention, wherein Marker is 2000bp LadderMarker;

Fig. 2 accompanying drawing is the qRT-PCR result of the expression level of gene GhCDPK29 induced by signal molecules (jasmonic acid JA, salicylic acid SA, H ₂ O ₂ infiltration) in Example 2 of the present invention;

Fig. 3 accompanying drawing is the qRT-PCR analysis result of the expression pattern of gene GhCDPK29 induced by Verticillium dahliae V991 infection in Example 2 of the present invention;

Fig. 4 accompanying drawing is the plant phenotype diagram of upland cotton TM-1 after injecting VIGS vector in the embodiment 3 of the present invention, wherein: A is the plant phenotype after the VIGS vector of GhCLA1 gene injection 2 weeks; B is the plant phenotype of upland cotton TM-1 TRV:00 (control) and TRV:GhCDPK29 were inoculated with Verticillium dahliae V991 spore liquid (10 ⁷ conidia/mL) for 21 days by dipping the root method;

Fig. 5 accompanying drawing is the qRT-PCR result of gene GhCDPK29 in TRV: 00 and TRV: GhCDPK29 plant three-leaf stage leaves of upland cotton TM-1 in Example 3 of the present invention;

Fig. 6 accompanying drawing is the statistical result of disease index after transgenic upland cotton TM-1 is inoculated with Verticillium dahliae in Example 3 of the present invention;

Fig. 7 accompanying drawing is the silencing plant and control plant cotton stalk longitudinal cut diagram of VIGS interfering upland cotton TM-1 plant resistance analysis result to Verticillium dahliae in embodiment 3 of the present invention;

Figure 8 is a comparison of the results of the fungal recovery experiment between the silent plant and the stem section of the control plant surface disinfection of the results of the VIGS interference with the resistance analysis of the upland cotton TM-1 plant to Verticillium dahliae in Example 3 of the present invention;

Fig. 9 accompanying drawing is the statistical result of the recovery rate of Verticillium dahliae of cotton of silent plant and control plant in VIGS interference upland cotton TM-1 plant resistance analysis result to Verticillium dahliae in embodiment 3 of the present invention;

Fig. 10 accompanying drawing is the relative abundance of DNA of Verticillium dahliae in the cotton stem section of the silenced plant and the control plant in VIGS interference analysis result of the resistance of upland cotton TM-1 plant to Verticillium dahliae in Example 3 of the present invention;

Fig. 11 is a drawing showing DAB staining diagrams of leaves of silenced plants and control plants in Example 3 of the present invention in which VIGS interferes with the resistance analysis of upland cotton TM-1 plants to Verticillium dahliae.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Cotton materials include:

Upland cotton TM-1, provided by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences;

Plant VIGS silencing expression vector:

Verticillium dahliae V991: preserved in this laboratory, the public can obtain it from the Institute of Nuclear Technology and Biotechnology of Xinjiang Academy of Agricultural Sciences;

Primer sequences: all provided by Shanghai Sangong Synthetic;

Other biological materials include: Escherichia coli strain DH5α, Agrobacterium strain GV3101, BP reaction entry vector, VIGS interference technology vector pTRV1 and pTRV2, etc. are all commercial strains or vectors, and will not be described again;

Experimental reagents: plasmid extraction kit, oligo(dT)18, RNAase inhibitor, dNTP, pMD18-T Vector, T4-DNA ligase, endonuclease EcoRI and KpnI, ExTaq enzyme, PCR product recovery kit, etc. are TaKaRa The company's products, fluorescent quantitative PCR kits are products of TOYOBO;

The fluorescent quantitative PCR special board is the product of Labwares;

RNA extraction kit was purchased from TIANGEN (Beijing, China) company;

The medium and solutions used are: LB liquid (solid) medium, YEP liquid (solid) medium, Czapek (Czapek) medium, PDA medium, 50×TAE Buffer (Na ₂ EDTA·2H ₂ O 37.2g, Glacial acetic acid 57.1mL, NaOH to adjust pH=8.3, add water to make up to 1L), etc., all can be prepared according to the conventional preparation methods in this field;

Other reagents such as antibiotics and hormones not described are commonly used reagents in this field and will not be repeated here.

Example 1

Acquisition of Gene GhCDPK29 in Upland Cotton

Plant upland cotton TM-1, extract RNA from cotton seedling leaves, and perform reverse transcription to obtain cDNA;

For the RNA extraction steps, refer to the TIANGEN Plant RNA Rapid Extraction Kit. The reverse transcription system is as follows:

Execute the program: 30min at 42°C, 5s at 85°C.

With the ID number in the CottonFGD website (https://cottonfgd.org) being the cDNA of the gene Gh_A06G0013.1 (the nucleotide sequence is shown in SEQ ID NO: 1, the amino acid sequence is shown in SEQ ID NO: 2) as a template, Design primers and carry out gene amplification, the primer sequence is:

GhCDPK29-F:5'-ATGGGACTCTGCCAATCTCTG-3', SEQ ID NO: 3;

GhCDPK29-R: 5'-TTAGCTGTTCATATGTCTTTGCC-3', SEQ ID NO:4.

The PCR amplification system is as follows:

PCR amplification program: 95°C for 2min; 95°C for 20s, 55°C for 30s, 72°C for 30s, 35 cycles; 72°C for 5min.

The PCR electrophoresis products were detected by 1% agarose gel electrophoresis, and the results are shown in Figure 1. It can be seen from Figure 1 that the size of the gene GhCDPK29 is 1581bp.

Example 2

1. The expression pattern of the gene GhCDPK29 under the stress of signal molecules (jasmonic acid JA, salicylic acid SA, H ₂ O ₂ dipping)

TM-1 cotton seeds were sterilized with 75% alcohol for 30 seconds, soaked in sterile water for 24 hours, sown in nutrient soil, cultivated under 12 hours of light/12 hours of darkness at a temperature of 26-28°C. Keep the humidity at 60% or above, water once every 4-5 days, select cotton seedlings with the same growth and size when the seedlings have two leaves, and soak the roots of cotton seedlings with 200μM jasmonic acid, 2mM salicylic acid, and 1mM H ₂ O ₂ respectively After 10 minutes, the cotton seedlings were continued to be planted in the nutrient pot. Cotton roots were taken at 0.5h, 1h, and 3h after treatment, and RNA was extracted, and the following quantitative PCR primers were used for qPCR amplification:

QRT-PCR-GhCDPK29-F:5'-TTGGCATCACTTATCTTTG-3', SEQ ID NO: 5;

QRT-PCR-GhCDPK29-R:5'-CAGGTTTCAAAGTCCCTATGCA-3', SEQ ID NO:6.

Ubiquitin 7 (UB7) was selected as an internal reference gene, and the primer sequences were designed as follows:

UBQ7-F:5'-GAAGGCATTCCACCTGACCAAC-3', SEQ ID NO: 7;

UBQ7-R:5'-CTTGACCTTTCTTCTTCTTGTGCTTG-3', SEQ ID NO:8.

qRT-PCR was carried out on the ABI 7500 Real-time PCR sequence detection system and software (AppliedBiosystems, USA);

The 20μL reaction system was designed as follows:

The reaction system was 10 μL SYBR Green Realtime PCR Master Mix, 1 μL cDNA product, 0.4 μL (10 μM) of upstream and downstream primers, and Nuclease-free Water supplemented to a total reaction volume of 20 μL. 4 technical replicates for each gene.

The corresponding program is that the quantitative program is 94°C for 30s; 95°C for 5s, 57°C for 15s, 72°C for 31s, 40 cycles.

The specificity of the amplified product after 40 cycles was checked by melting curve analysis. Each reaction included at least three replicates. A single template was diluted to different concentrations, and the amplification efficiency of the primers was detected by plotting the log value of the template dilution factor against the Ct value of each diluted sample.

The detection results of qRT-PCR are shown in Figure 2. The expression level of gene GhCDPK29 was the highest when JA was treated for 1 hour, and then decreased; the expression level of gene GhCDPK29 showed an upward trend after both SA and H ₂ O ₂ treatments. It shows that the expression level of the gene GhCDPK29 is affected by the stress of signal molecules.

2. The expression pattern of the gene GhCDPK29 under the infection of Verticillium dahliae

The upland cotton TM-1 with two leaves and one heart was selected for root injury, and the conidia liquid concentration of Verticillium dahliae V991 used was 1×10 ⁷ spores/mL, and water treatment was used as a negative control. Soak the roots of cotton seedlings in the bacterial solution for 10 minutes, and then plant them in a nutrient pot. Take the cotton roots at 0h, 0.5h, 2h, 6h, 12h, 24h, and 48h after inoculation, extract RNA, and invert The cDNA was recorded for expression pattern analysis.

The relevant primers and PCR reaction procedures for qRT-PCR analysis are the same as those in Part 1 of this example "Expression pattern of gene GhCDPK29 under stress of signal molecules (jasmonic acid JA, salicylic acid SA, H ₂ O ₂ infiltration)".

The detection results of qRT-PCR are shown in Figure 3. After the conidia liquid of Verticillium dahliae V991 and water were treated, the expression of the gene GhCDPK29 showed a trend of first increasing and then decreasing. volume tends to be at a minimum. The results indicated that the expression level of the gene GhCDPK29 changed significantly after the roots of TM-1 were infiltrated by Verticillium dahliae.

Example 3

Using VIGS technology to construct a VIGS interference vector to silence the gene GhCDPK29. Observing the phenotypic changes of cotton when the gene GhCDPK29 was silenced when it was infiltrated by Verticillium dahliae further proved that the gene GhCDPK29 is highly related to the resistance to Verticillium dahliae.

1. Construction of recombinant expression vector

(1) Primer design and PCR amplification

Design primers in the non-conserved segment of the gene GhCDPK29, the primer design principle is: add the restriction enzyme EcoRI restriction enzyme cutting site and protective base to the upstream primer, add the restriction enzyme KpnI restriction enzyme cutting site and the downstream primer protected bases;

GhCDPK29-1F:5'-GGAATTCTTGGCATCACTTATCTTTG-3', SEQ ID NO: 9;

GhCDPK29-1R: 5'-GGGGTACCCAGGTTTCAAAGTCCCTATGCA-3' SEQ ID NO: 10.

The total RNA of upland cotton TM-1 was extracted and reverse-transcribed into cDNA. Using the cDNA as a template, PCR amplification was performed with a primer pair consisting of primer GhCDPK29-1F and primer GhCDPK29-1R to obtain PCR amplification products.

The PCR amplification system is as follows:

PCR amplification program: 95°C for 2min; 95°C for 20s, 57°C for 30s, 72°C for 30s, 35 cycles; 72°C for 5min.

The target sequence (about 351 bp) was amplified by PCR; the amplified product was subjected to 1% agarose gel electrophoresis.

(2) Obtain recombinant expression vector after sequencing

The PCR amplification product was recovered by gel, ligated with pEASY-Blunt Zero Cloning Kit (cloning vector), and the positive transformants identified by PCR with GhCDPK29-1F/R primers were sent for sequencing.

Select the positive transformant with successful sequencing to extract the plasmid, and use the enzyme EcoRI and the enzyme KpnI to perform double enzyme digestion on the plasmid and the empty vector pTRV2 respectively in a water bath at 37°C. The enzyme digestion system is as follows:

Enzyme digestion program: 37°C for 1h; 80°C for 10min.

The digested products were subjected to 1% agarose gel electrophoresis and ligated with T4 DNA ligase. The recombinant plasmid TRV2-GhCDPK29 of the plant expression vector was transformed into Agrobacterium GV3101. After PCR screening and identification with GhCDPK29-1F/R primers, VIGS interference Vector TRV2:GhCDPK29.

2. Obtaining genetically modified cotton

(1) Bacteria culture

pTRV1 and TRV2-GhCDPK29 were transformed into Agrobacterium, respectively, and the cells containing resistance to kanamycin (50 μg·mL ^-1 ), gentamicin (50 μg·mL ^-1 ) and rifampicin (25 μg·mL ^-1 ) LB culture flasks were cultured at 28°C until the OD ₆₀₀ reached 0.6-0.8.

(2) Resuspension obtained

The bacterial cells were collected by centrifugation at 4000rpm for 5min, and resuspended with an appropriate volume of resuspension solution (recipe: 10mmol·L ^-1 MgCl ₂ , 10mmol·L ^-1 MES and 200μmol·L ^-1 acetosyringone) to a final concentration of OD ₆₀₀ is 1.5. The resuspension was kept at room temperature in the dark for more than 3 h. A resuspension containing the recombinant plasmid TRV2-GhCDPK29 was obtained. A resuspension containing the pTRV1 vector, a resuspension of the TRV2-GhCLA1 vector containing the GhCLA1 gene fragment, and a resuspension containing the pTRV2 empty vector were prepared in a similar manner.

The resuspension containing the pTRV1 vector and the resuspension of the recombinant plasmid TRV2-GhCDPK29 containing the target gene fragment were mixed at a volume ratio of 1:1 to inject a TRV:GhCDPK29 solution into cotton cotyledons for obtaining gene silencing transformants; containing the pTRV1 vector The resuspension of the TRV2-GhCLA1 vector containing the GhCLA1 gene fragment was mixed at a volume ratio of 1:1, and the TRV:GhCLA1 solution was injected into the cotton cotyledons to detect whether the gene silencing system was correct; the resuspension containing the pTRV1 vector solution and the resuspension solution containing the pTRV2 empty vector were mixed at a volume ratio of 1:1 to inject a TRV:00 solution into cotton cotyledon, which was used as a genetic transformation control strain.

(3) Receptor culture

TM-1 cotton seeds were sterilized with 75% alcohol for 30 seconds, soaked in sterile water for 24 hours, then sowed in nutrient soil, cultivated under 12 hours of light/12 hours of darkness at a temperature of 26-28°C. Keep the humidity at 60% and above, water once every 4-5 days, and use the VIGS operation when the two cotyledons are spread flat and the true leaves have not yet developed.

(4) Obtaining of GhCDPK29 gene silencing transformant

Gently puncture the back of the cotyledon with a syringe needle to create a micro-injury, and inject the resuspension prepared in the above step (2) according to the volume ratio of 1:1 from the wound to obtain the cotton GhCDPK29 gene silencing transformant. Protected from light for 24 hours, 12 hours of light/12 hours of darkness, and cultured at 26-28°C under light.

The GhCLA1 gene silencing test strain and pTRV2 empty vector genetically transformed control strain were obtained in a similar manner.

Specific methods of Agrobacterium VIGS reference: Gao, X., Shan, L. Functional genomic anal ysis of cotton genes with agrobacterium-mediated virus-induced genesis. Methods Mol Biol, 2013, 975: 157-165.

3. Detection of VIGS genetic transformation system

(1) After 2 weeks, observe the phenotypes of cotton with different treatments, and use the upland cotton GhCLA1 gene (cloroplastosalterados 1 gene) as a marker gene for VIGS detection. This gene is involved in the development of chloroplasts and encodes 1-deoxyxylulose 5-phosphate synthase protein, which is highly conserved in evolution. After the GhCLA1 gene is silenced, cotton plants have obvious albino phenotype, which is an easy-to-identify marker trait. As shown in Figure 4, after 2 weeks of VIGS infection, the true leaves of the plants injected with TRV1 and TRV2-GhCLA1 were almost completely albino (Figure 4A), while the leaves of the plants injected with empty vectors pTRV1 and pTRV2 as controls (TRV:00) had no change (FIG. 4B TRV:00). It indicated that TRV-mediated VIGS system was successfully established in upland cotton TM-1.

(2) Fluorescence quantitative Real time-PCR detection

Take treated cotton leaves to extract RNA (preferably new long true leaves), and then perform qRT-PCR to detect whether the expression of the target gene is reduced, and detect the expression of the target gene. 30 individual plants were treated for each material.

Total RNA was extracted from the leaves of VIGS-infiltrated cotton plants using RNA extraction kits. Using cotton UBQ7 as an internal reference gene, the expression of the silenced gene GhCDPK29 was detected by fluorescence quantitative Real time-PCR.

For qRT-PCR analysis related primers and PCR reaction procedures, refer to Part 1 of Example 2 "Expression pattern of gene GhCDPK29 under stress of signal molecules (jasmonic acid JA, salicylic acid SA, H ₂ O ₂ infiltration)".

The qRT-PCR results are shown in Figure 5. Compared with the empty vector (TRV:00) control, the expression level of the gene GhCDPK29 was significantly decreased in the randomly selected VIGS-infected plants of the GhCDPK29 gene, and the silencing effect was obvious.

4. Cotton Verticillium wilt resistance inoculation and resistance identification

(1) Inoculation of cotton Verticillium dahliae

The preserved Verticillium dahliae strain V991, which causes Verticillium dahliae, was activated on PDA medium. The picked bacteria were cultured in Czapek _'s culture medium at 25°C and 200 rpm for 3-5 days. Filter the pathogen culture solution with 4 layers of gauze, use a hemocytometer to count the concentration of pathogens, adjust the final concentration to 1.0× ¹⁰⁷ spores/mL with sterilized double distilled water, and add Tween-20 to a final concentration of 0.001% (vol. content). Inoculate the spore liquid of Verticillium dahliae V991 into the transformed strain obtained after silencing the VIGS gene for 2 weeks by root injury method.

(2) Statistics on the incidence of Verticillium wilt

When the true leaves began to turn yellow and wilt, the incidence of Verticillium wilt was investigated and counted, and the disease grade was counted using the method of 0-4. 30 individual plants were treated for each material. Three biological replicates were set up.

References for disease index statistics: Xu Li, Zhu Longfu, Zhang Xianlong. Research progress on the mechanism of cotton resistance to Verticillium wilt. Acta Crops, 2012,38:1553–1560; Xu L, Zhu L F, Zhang X L. Research on resistance mechanism of cotton to Verticillium wilt . Acta Agron Sin, 2012, 38:1553–1560.

Disease index = [(number of diseased plants at all levels × corresponding disease level)/total number of investigated plants × highest disease level (4)] × 100

Statistics of test results: Compared with the empty vector control (TRV:00) plants of TRV:GhCDPK29 silenced plants 2 weeks after VIGS infection, Verticillium dahliae strain V991 was inoculated, and the incidence of Verticillium wilt after 21 days. It can be seen from Figure 4B that compared with the GhCDPK29 gene silencing transformant, the leaves of the control (TRV:00) plants have more light yellow patches and larger areas, and the leaf margins curl downward more obviously. Through 3 times of biological repeated observation and statistical analysis, the disease index results are shown in Figure 6, the average disease index of the control plants injected with empty vectors reached 52%, and the disease resistance of GhCDPK29 gene silenced transformants was significantly enhanced, and the average disease index was 52%. The index is 37%. It indicated that the gene GhCDPK29 was involved in the stress response induced by Verticillium dahliae. After silencing the gene GhCDPK29 in upland cotton TM-1, the incidence and disease index of cotton were significantly reduced when infected by Verticillium dahliae, indicating that the silencing of gene GhCDPK29 improved the resistance of cotton to Verticillium wilt.

(3) Verification that the gene GhCDPK29 is involved in Verticillium wilt-induced allergic response

In order to verify the accuracy of the above phenotypic results, this study continued to do the recovery culture experiment of Verticillium dahliae, the treatment of diseased plants and the detection of the relative abundance of DNA of Verticillium dahliae.

Verticillium dahliae recovery culture experiment method is as follows: use scissors to cut the stems of cotton seedlings 6cm away from the cotyledons after treatment with Verticillium dahliae into 1cm-long fragments, soak them in 70% alcohol for 1min, and then soak them in 30% hydrogen peroxide for 30mins. Rinse 4-5 times with sterile water, and finally place the treated cotton stem fragments on PDA medium and culture them at 25°C for 4 days to observe the growth status of Verticillium dahliae recovery culture.

The method of dissection of diseased plants is as follows: take a number of cotton stalks 21 days after inoculation with Verticillium dahliae, cut them longitudinally with a sharp blade, expose the longitudinal section, and observe the phenotype of the vascular tissue of the stalks. If the vascular bundle tissue of the stalk is brown, it means that it has been infected with Verticillium dahliae and has the disease. If the vascular bundle tissue of the stalk is not brown and the phenotype is normal, it means that it has not developed the disease.

The method for detecting the relative abundance of plant Verticillium dahliae DNA relative abundance is as follows: extract RNA from the stems of cotton seedlings treated with Verticillium dahliae, reverse to cDNA, and perform qPCR amplification.

The primers used are:

ITS1-F: 5'-AAAGTTTTAATGGTTCGCTAAGA-3', SEQ ID NO: 11;

ST-VE1-R: 5'-CTTGGTCATTTAGAGGAAGTAA-3', SEQ ID NO:12.

The corresponding program is that the quantitative program is 94°C for 30s; 95°C for 5s, 52°C for 15s, 72°C for 31s, 40 cycles.

For conditions of reverse transcription and qPCR reaction procedures, refer to Part 1 of Example 2 "Expression pattern of gene GhCDPK29 under stress of signal molecules (jasmonic acid JA, salicylic acid SA, H ₂ O ₂ infiltration)".

The experimental results are shown in Figures 7-10. After the stem was cut longitudinally, it was found that the stem vascular bundles of the control plants injected with the empty vector turned light brown, while the stem vascular bundles of the GhCDPK29 gene silenced transformants were normal in color, and the browning degree of the dissected stem material of TRV:GhCDPK29 plant material lower than TRV:00 control plant material injected with empty vector (Figure 7). TRV:GhCDPK29 plant material had fewer Verticillium dahlia colonies cultured in PDA medium than TRV:00 plant material (Figure 8). The recovery rate of Verticillium dahliae in TRV:00 control plants was 72%, while the recovery rate of Verticillium dahliae in plants after TRV:GhCDPK29 gene silencing was 18% ( FIG. 9 ). The relative abundance of Verticillium dahliae DNA in TRV:00 plants was twice that of plants after TRV:GhCDPK29 gene silencing (Fig. 10).

These results also showed that after silencing the gene GhCDPK29, the resistance of cotton to Verticillium dahliae was improved, indicating that the gene GhCDPK29 was involved in regulating the resistance of Verticillium dahliae.

5. Verification that the gene GhCDPK29 is involved in the accumulation of H ₂ O ₂ in leaves

Plant susceptibility often causes altered _{H2O2 accumulation} in leaves. We demonstrated H ₂ O ₂ accumulation by DAB staining of leaves of control plants and GhCDPK29 gene silenced transformants.

DAB dye preparation: Weigh 500mg of DAB powder, dissolve it in 500mL of distilled water to make the concentration 1mg/mL, use 0.2M hydrochloric acid to adjust the pH=3.0, store in the dark at 4°C for later use, add 20ul Tween20 and 200mM Na ₂ before use HPO ₄ adjusted the pH of the DAB stain to 5.8.

Decolorization solution preparation: ethanol: acetic acid: glycerol = 3:1:1.

Take the leaves of the control plant and the GhCDPK29 gene silencing transformant in the same part of the fully expanded leaves and place them in a 15mL tube containing DAB staining solution, vacuumize the leaves to sink to the bottom of the tube, and stain in the dark at 28°C for more than 12h. The leaves were taken out and placed in a centrifuge tube containing decolorization solution, boiled in a water bath until the leaves were green, and finally stored in 80% ethanol at 4°C and photographed.

The experimental results are shown in FIG. 11 , and the results showed that more reddish-brown spots were formed on the leaves of the GhCDPK29 gene-silencing transformed plants, indicating that the accumulation of H ₂ O ₂ was higher than that of the control plants.

Each embodiment in this specification is described in a progressive manner, each embodiment focuses on the difference from other embodiments, and the same and similar parts of each embodiment can be referred to each other.

The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (5)

1. The application of the gene GhCDPK29 of the calpain in regulating and controlling verticillium wilt resistance of plants is characterized in that the nucleotide sequence of the gene GhCDPK29 is shown in SEQ ID NO: 1.

2. The application of protein or a substance for regulating the expression of a protein coding gene in plant verticillium wilt resistance is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO: 2.

3. Use of the gene GhCDPK29 according to claim 1 or the protein related biomaterial according to claim 2 for plant verticillium resistance, characterized in that the biomaterial is any of the following:

a: a nucleic acid molecule according to claim 1 or a nucleic acid molecule encoding a protein according to claim 2;

b: an expression cassette comprising silencing of the nucleic acid molecule of a;

c: an expression vector comprising the nucleic acid molecule of A, or a recombinant vector comprising the expression cassette of B;

d: a recombinant microorganism comprising the nucleic acid molecule of A, a recombinant microorganism comprising the expression cassette of B, or a recombinant microorganism comprising the recombinant vector of C.

4. Use according to any one of claims 1 to 3, wherein the plant is a cotton plant.

5. A method for increasing verticillium wilt resistance in a plant, comprising increasing verticillium wilt resistance in a plant by silencing or inhibiting expression of the gene GhCDPK29 of claim 1 or activity of the protein of claim 2 in the plant.

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