Application of calcium-dependent protein kinase gene GhCDPK29 in plant resistance to verticillium wilt

Abstract

The application discloses application of a calcium-dependent protein kinase gene GhCDPK29 in plant resistance to verticillium wilt, and belongs to the technical field of genetic engineering. The gene GhCDPK29 is amplified by using sequence information of the gene, a VIGS (Virus Induced Gene Silencing) plant expression vector is constructed, the VIGS plant expression vector is transformed into Gossypium hirsutum TM-1, and transgenic cotton is obtained; after inoculation of cotton verticillium V991, the transgenic cotton shows resistance to verticillium wilt, which indicates that the gene GhCDPK29 is highly related to the resistance of cotton to verticillium wilt. The application lays an application foundation for research on a molecular mechanism of verticillium wilt resistance, cultivation of new plant varieties with verticillium wilt resistance, and provides a new reference for screening of new disease-resistant genes and cultivation of disease-resistant plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, and more particularly to the application of calcium-dependent protein kinase gene in plant resistance to Verticillium wilt. BACKGROUND

[0002] Cotton plays an important role in the economic development of our country and is an important fiber crop in China. 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 vascular bundle lesions in cotton, leading to leaf wilting, yellowing and shedding, and severely restricting the growth and development of cotton. Although a number of Verticillium wilt-resistant cotton varieties have been selected through conventional breeding methods, due to the lack of germplasm resources, poor broad-spectrum resistance, and long breeding time, there is still no excellent Verticillium wilt-resistant variety. To meet the needs of our country's breeding industry, it is very important to break through resource constraints, mine disease-resistant genes from upland cotton resources, analyze disease resistance mechanisms, and use transgenic innovation to develop new disease-resistant germplasm.

[0003] 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, which involve a large number of transcriptional regulatory factors and genes. Disease resistance (R) genes are key factors that mediate plant defense responses, which can directly or indirectly detect avirulence gene products of pathogenic bacteria, and then trigger a series of downstream defense responses. The expression level of R genes is low under the condition of no pathogenic bacteria, but is significantly activated under the induction of pathogenic bacteria. Overexpression of R genes can even affect the normal growth of plants or cause plant death. In addition, reactive oxygen species (ROS) also play a role in calcium ion regulation signals and hormone regulatory factors in plant disease defense response, thereby changing the disease resistance of plants.

[0004] CDPKs are a class of threonine / serine protein kinases widely distributed in plants. CDPKs transmit calcium signals to downstream by phosphorylating various substrates, including transcription factors, metabolic substrates and metabolic enzymes, and cause physiological responses in plants (Chen et al., 2021). With the in-depth study of CDPKs, it was found that they participate in cell Ca 2+Signal transduction performs a variety of biological functions, including plant pollen tube growth, hormone signal transduction, root stem development, stomatal movement (Atif et al., 2019). At the same time, it plays an important role in the response of plants to abiotic and biotic stress. Studies have shown that overexpression of CDPK7 improves the cold, drought 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 salt stress induction (Jinbo et al., 2011).

[0005] In summary, it is found that a calcium-dependent protein kinase gene regulating cotton Verticillium wilt resistance is a problem that those skilled in the art urgently need to solve. SUMMARY

[0006] Therefore, the application provides the application of calcium-dependent protein kinase gene GhCDPK29 in plant resistance to Verticillium wilt.

[0007] In order to achieve the above purpose, the application adopts the following technical scheme:

[0008] The application of calcium-dependent protein kinase gene GhCDPK29 in regulating plant resistance to Verticillium wilt, the nucleotide sequence of the gene GhCDPK29 is shown as SEQ ID NO: 1.

[0009]

[0010] The protein or a substance regulating expression of a gene encoding the protein, wherein the amino acid sequence of the protein is shown as SEQ ID NO: 2, is used in plant resistance to Verticillium wilt.

[0011] MGLCQSLGFCLRRSHSHEIPISSSSESSPRPSHLFPKTTPQHFNPSSSKATSSSGIGTILLKPYVDVTTIYDLDKELGRGQFGITYLCTEKATGRKYACKSISRRKLRTDRDIEDVRRETSIMQHLTGQPNIVEFKGAYEDRQNVHLVMELCSGGELFDRIIAKGSYSERQAASICRQVVNVVNACHFMGVMHRDLKPENFLLVSKDEISPIKATDFGLSVFIEEGRMYKDLVGSAYYVAPEVLNRKYGKEIDVWSAGVILYILLSGVPPFWGETEKEIFKAVLEGNLDLKSLPWPSITEGAKDLIRKMLTRDPTKRITAAQALEHPWLKEGGDASDKPIDSAVLSRLKQFRVMNKLKKLALKVIAESLSTEEEIKGLQQMFKNIDTDGSGTITLGELRDGLARLGSKLTETEIKQLMDAADVDNSGTIDYIEFITATMHRHRLEREENIVKAFQFFDKDNSGFITRDELRQAMTQYGMGDEATIDEVIEDVDTDKDGRINYEEFVAMMKRGTHDGDGNWQRHMNS*; SEQ ID NO: 2.

[0012] The above-mentioned gene GhCDPK29 or the above-mentioned protein related biological material is used in plant resistance to Verticillium wilt, wherein the biological material is any one of the following:

[0013] A: a nucleic acid molecule or a nucleic acid molecule encoding the above-mentioned protein;

[0014] B: an expression cassette containing the nucleic acid molecule of A;

[0015] C: an expression vector containing the nucleic acid molecule of A, or a recombinant vector containing the expression cassette of B;

[0016] D: a recombinant microorganism containing the nucleic acid molecule of A, or a recombinant microorganism containing the expression cassette of B, or a recombinant microorganism containing the recombinant vector of C.

[0017] The term "expression cassette" refers to DNA capable of expressing the protein described in the above applications in a host cell, which can include not only a promoter that initiates transcription of the protein coding gene, but also a terminator that terminates transcription of the protein coding gene. Further, the expression cassette can also include an enhancer sequence.

[0018] Further, the above-mentioned gene GhCDPK29 or the above-mentioned protein related biological material is applied in breeding of plant varieties with enhanced or weakened Verticillium wilt resistance.

[0019] Further, the above-mentioned gene GhCDPK29 or the above-mentioned protein related biological material is applied in plant breeding.

[0020] Further, the plant is a Gossypium plant.

[0021] Further, the plant is cotton.

[0022] Further, the plant is Gossypium hirsutum TM-1.

[0023] A method for improving the Verticillium wilt resistance of a plant by silencing or inhibiting the expression of the gene GhCDPK29 or the activity of the above-mentioned protein in the plant to improve the Verticillium wilt resistance of the plant.

[0024] The above-mentioned silencing or inhibition of the expression of the protein coding gene in the plant can be achieved by any means in the prior art to cause deletion mutation, insertion mutation or base substitution mutation of the gene, and thus reduce or lose the function of the gene.

[0025] According to the technical solutions described above, compared with the prior art, the present application has the following beneficial effects: the present application uses the sequence information of the gene GhCDPK29 to amplify the gene, constructs a VIGS plant expression vector to transform Gossypium hirsutum TM-1, and obtains transgenic cotton that shows resistance to Verticillium wilt after being inoculated with the cotton Verticillium wilt fungus V991, which indicates that the gene GhCDPK29 is highly related to the resistance of cotton to Verticillium wilt. The present application lays a foundation for the research on the molecular mechanism of Verticillium wilt resistance and the cultivation of new plant varieties with Verticillium wilt resistance, and also provides a new reference for the screening of new disease-resistant genes and the cultivation of disease-resistant plants. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without any creative effort.

[0027] Figure 1Figure is the electrophoresis map of PCR product of gene GhCDPK29 in embodiment 1 of the present application, wherein Marker is 2000bp LadderMarker;

[0028] Figure 2 Figure is the qRT-PCR result of expression amount of gene GhCDPK29 induced by signal molecules (jasmonic acid JA, salicylic acid SA, H2O2) in embodiment 2 of the present application;

[0029] Figure 3 Figure is the qRT-PCR analysis result of expression pattern of gene GhCDPK29 induced by V991 of Verticillium wilt in embodiment 2 of the present application;

[0030] Figure 4 Figure is the phenotype map of Gossypium hirsutum TM-1 plant after injection of VIGS carrier in embodiment 3 of the present application, wherein: A is the plant phenotype after injection of VIGS carrier of GhCLA1 gene for 2 weeks; B is the disease incidence of Gossypium hirsutum TM-1 TRV:00 (control) and TRV:GhCDPK29 inoculated with spore solution (10 7 conidia / mL) of Verticillium wilt V991 for 21 days by root dipping method;

[0031] Figure 5 Figure is the qRT-PCR result of expression amount of gene GhCDPK29 in the third leaf stage leaf of Gossypium hirsutum TM-1 TRV:00 and TRV:GhCDPK29 plants in embodiment 3 of the present application;

[0032] Figure 6 Figure is the statistical result of disease index of transgenic Gossypium hirsutum TM-1 inoculated with Verticillium wilt in embodiment 3 of the present application;

[0033] Figure 7 Figure is the longitudinal cutting map of cotton stem of silenced plants and control plants in the resistance analysis result of VIGS interference Gossypium hirsutum TM-1 plants to Verticillium wilt in embodiment 3 of the present application;

[0034] Figure 8 Figure is the comparison of fungus recovery experiment result of surface sterilized stem segments of silenced plants and control plants in the resistance analysis result of VIGS interference Gossypium hirsutum TM-1 plants to Verticillium wilt in embodiment 3 of the present application;

[0035] Figure 9 Figure is the statistical result of recovery rate of cotton Verticillium wilt of silenced plants and control plants in the resistance analysis result of VIGS interference Gossypium hirsutum TM-1 plants to Verticillium wilt in embodiment 3 of the present application;

[0036] Figure 10Figure 1 is a diagram of the relative abundance of Verticillium dahliae DNA in cotton stem segments of silenced plants and control plants in the analysis of the resistance of VIGS-silenced Gossypium hirsutum TM-1 plants to Verticillium dahliae in Example 3 of the present application;

[0037] Figure 11 Figure 2 is a diagram of the DAB staining of leaves of silenced plants and control plants in the analysis of the resistance of VIGS-silenced Gossypium hirsutum TM-1 plants to Verticillium dahliae in Example 3 of the present application. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0039] The cotton material includes:

[0040] Gossypium hirsutum TM-1 was provided by the Cotton Research Institute of Chinese Academy of Agricultural Sciences;

[0041] The plant VIGS silencing expression vector includes:

[0042] Verticillium dahliae V991: preserved in the laboratory and available from the Institute of Nuclear and Biological Technology, Xinjiang Academy of Agricultural Sciences;

[0043] The primer sequences were synthesized by Shanghai Shengong;

[0044] Other biological materials include: Escherichia coli strain DH5α, Agrobacterium strain GV3101, BP reaction primer vector, VIGS interference technology vector pTRV1 and pTRV2, etc. are all commercial strains or vectors, and will not be described again;

[0045] The experimental reagents include 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. which are all products of TaKaRa Company, and the fluorescent quantitative PCR reagent kit is a product of TOYOBO Company;

[0046] The fluorescent quantitative PCR special plate is a product of Labwares Company;

[0047] The RNA extraction kit was purchased from TIANGEN (Beijing, China) Company;

[0048] The used culture medium and solution are: LB liquid (solid) culture medium, YEP liquid (solid) culture medium, Czapek culture medium, PDA culture medium, 50x TAE Buffer (Na2EDTA·2H2O 37.2g, glacial acetic acid 57.1mL, NaOH to pH = 8.3, add water to 1L), etc., which are prepared according to the conventional preparation method in the art.

[0049] Other un-described antibiotics, hormones and other reagents are commonly used in the art, and will not be described.

[0050] Example 1

[0051] Obtaining of land cotton gene GhCDPK29

[0052] Land cotton TM-1 was planted, and the RNA of cotton seedling leaves was extracted, and cDNA was obtained by reverse transcription;

[0053] The RNA extraction step refers to TIANGEN plant RNA rapid extraction kit, and the reverse transcription system is as follows:

[0054]

[0055] Execution program: 42℃ 30min, 85℃ 5s.

[0056] The cDNA of the gene with ID number Gh_A06G0013.1 in CottonFGD website (https: / / cottonfgd.org) (nucleotide sequence as shown in SEQ ID NO: 1, amino acid sequence as shown in SEQ ID NO: 2) was used as a template, primers were designed and the gene was amplified, and the primer sequences were as follows:

[0057] GhCDPK29-F: 5'-ATGGGACTCTGCCAATCTCTG-3', SEQ ID NO: 3;

[0058] GhCDPK29-R: 5'-TTAGCTGTTCATATGTCTTTGCC-3', SEQ ID NO: 4.

[0059] The PCR amplification system is as follows:

[0060]

[0061] PCR amplification program: 95℃ 2min; 95℃ 20s, 55℃ 30s, 72℃ 30s, 35 cycles; 72℃ 5min.

[0062] The PCR electrophoresis product was detected by 1% agarose gel electrophoresis, and the results were as follows Figure 1As shown in Fig. 1, the size of the gene GhCDPK29 is 1581 bp. Figure 1 As can be seen from Fig. 1, the size of the gene GhCDPK29 is 1581 bp.

[0063] Example 2

[0064] 1. Expression pattern of the gene GhCDPK29 under the stress of signal molecules (jasmonic acid JA, salicylic acid SA, H2O2 immersion)

[0065] TM-1 cotton seeds were soaked in sterile water for 24 h after being sterilized with 75% alcohol for 30 s, and then were sowed in nutrient soil, and were cultured under 12 h light / 12 h darkness, temperature of 26-28°C, and humidity of 60% or above. The seedlings were watered once every 4-5 d, and when the seedlings had two leaves and one heart, the seedlings with uniform growth and size were selected, and the roots of the seedlings were immersed with 200 μM jasmonic acid, 2 mM salicylic acid, and 1 mM H2O2 for 10 min, respectively. Then the seedlings were planted in nutrient pots. The roots of the seedlings were collected at 0.5 h, 1 h, and 3 h after the treatment, respectively, and RNA was extracted, and qPCR amplification was performed using the following quantitative PCR primers:

[0066] QRT-PCR-GhCDPK29-F: 5'-TTGGCATCACTTATCTTTG-3', SEQ ID NO: 5;

[0067] QRT-PCR-GhCDPK29-R: 5'-CAGGTTTCAAGTCCCTATGCA-3', SEQ ID NO: 6.

[0068] The reference gene was ubiquitin 7 (UB7), and the primer sequences were designed as follows:

[0069] UBQ7-F: 5'-GAAGGCATTCCACCTGACCAAC-3', SEQ ID NO: 7;

[0070] UBQ7-R: 5'-CTTGACCTTCTTCTTCTTGTGCTTG-3', SEQ ID NO: 8.

[0071] qRT-PCR was performed on an ABI 7500 Real-time PCR sequence detection system and software (Applied Biosystems, USA);

[0072] The 20 μL reaction system was designed as follows:

[0073] The reaction system was 10 μL SYBR Green Realtime PCR Master Mix, 1 μL cDNA product, 0.4 μL (10 μM) of each upstream and downstream primer, and Nuclease-free Water to make up the total reaction volume of 20 μL. There were 4 technical replicates for each gene.

[0074] The corresponding procedure was as follows: the quantitative procedure was 94°C for 30 s; 95°C for 5 s, 57°C for 15 s, 72°C for 31 s, and 40 cycles.

[0075] The specificity of the amplified products after 40 cycles was detected by using the melting curve analysis. Each reaction included at least three replicates. The amplification efficiency of the primers was detected by plotting the log value of the template dilution factor against the Ct value of each dilution sample using a single template diluted to different concentrations.

[0076] The detection results of qRT-PCR are shown in Table 1. Figure 2 As shown in Table 1, the expression level of gene GhCDPK29 was the highest at 1 h after JA treatment, and then decreased; the expression level of gene GhCDPK29 showed an upward trend after SA and H2O2 treatment. It is indicated that the expression level of gene GhCDPK29 is affected by signal molecule stress.

[0077] 2. Expression pattern of gene GhCDPK29 under Verticillium dahliae infection

[0078] The two-leaf one-heart upland cotton TM-1 was selected for root wounding, and the concentration of Verticillium dahliae V991 conidial suspension used was 1×10 7 spores / mL, and the water treatment was used as a negative control. After the cotton seedling roots were soaked in the conidial suspension for 10 min, the cotton seedlings were planted in a nutrient pot, and the roots were taken at 0 h, 0.5 h, 2 h, 6 h, 12 h, 24 h and 48 h after inoculation, respectively, and then the RNA was extracted and the cDNA was reverse transcribed for expression pattern analysis.

[0079] The related primers and PCR reaction procedure for qRT-PCR analysis were the same as those in the first part of this embodiment “Expression pattern of gene GhCDPK29 under signal molecule stress (jasmonic acid JA, salicylic acid SA, H2O2 treatment)”.

[0080] The detection results of qRT-PCR are shown in Table 2. Figure 3 As shown in Table 2, after the Verticillium dahliae V991 conidial suspension and water treatment, the expression level of gene GhCDPK29 showed a trend of first increasing and then decreasing, and the expression level was the highest at 2 h after inoculation with the Verticillium dahliae pathogen, and the expression level tended to be the lowest at 48 h. This result shows that the expression level of gene GhCDPK29 changes significantly after the roots of TM-1 are infected with Verticillium dahliae.

[0081] Example 3

[0082] The VIGS interference vector is constructed by using the VIGS technology to silence the gene GhCDPK29. The phenotype change of the cotton after the gene GhCDPK29 is silenced and the cotton is infiltrated by Verticillium dahliae is observed, and it is further proved that the gene GhCDPK29 is highly related to the Verticillium wilt resistance.

[0083] 1. Construction of the recombinant expression vector

[0084] (1) Primer design and PCR amplification

[0085] The primer is designed in the non-conserved segment of the gene GhCDPK29, and the principle of the primer design is that the restriction enzyme EcoRI enzyme cutting site and the protection base are added to the upstream primer, and the restriction enzyme KpnI enzyme cutting site and the protection base are added to the downstream primer.

[0086] GhCDPK29-1F: 5'-GGAATTCTTGGCATCACTTATCTTTG-3', SEQ ID NO: 9;

[0087] GhCDPK29-1R: 5'-GGGGTACCCAGGTTTCAAGTCCCTATGCA-3' SEQ ID NO: 10.

[0088] The total RNA of Gossypium hirsutum TM-1 is extracted and is reversely transcribed into cDNA, and the cDNA is used as a template, and the primer pair composed of the primer GhCDPK29-1F and the primer GhCDPK29-1R is used for PCR amplification to obtain a PCR amplification product.

[0089] The PCR amplification system is as follows:

[0090]

[0091]

[0092] The PCR amplification program is as follows: 95℃ for 2min; 95℃ for 20s, 57℃ for 30s, 72℃ for 30s, 35 cycles; 72℃ for 5min.

[0093] The target sequence (about 351bp) of the PCR amplification is amplified, and the amplification product is subjected to 1% agarose gel electrophoresis.

[0094] (2) Obtaining the recombinant expression vector after sequencing

[0095] The PCR amplification product is recovered by gel recovery, and is connected with pEASY-Blunt Zero Cloning Kit (cloning vector), and the positive transformant after the primer GhCDPK29-1F / R is identified by PCR is sent for sequencing.

[0096] The plasmid of the positive transformant with successful sequencing was extracted, and the plasmid and empty vector pTRV2 were respectively subjected to double enzyme digestion with enzyme EcoRI and enzyme KpnI under the condition of 37℃ water bath, and the enzyme digestion system was as follows:

[0097]

[0098] The enzyme digestion procedure was 37℃ for 1h and 80℃ for 10min.

[0099] The enzyme digestion product was subjected to 1% agarose gel electrophoresis, and was subjected to ligation by using T4 DNA ligase, the plant expression vector recombinant plasmid TRV2-GhCDPK29 was transformed into Agrobacterium GV3101, and after screening and identification by PCR with GhCDPK29-1F / R primers, the VIGS interference vector TRV2:GhCDPK29 was obtained.

[0100] 2. Obtaining of transgenic cotton

[0101] (1) Bacterial liquid culture

[0102] pTRV1 and TRV2-GhCDPK29 were respectively transformed into Agrobacterium, and were cultured in LB culture bottles containing kanamycin (50μg·mL -1 ), gentamicin (50μg·mL -1 ) and rifampicin (25μg·mL -1 ) resistance at 28℃ until OD 600 reached 0.6-0.8.

[0103] (2) Obtaining of resuspension

[0104] The bacterial cells were collected by centrifugation at 4000rpm for 5min, and were resuspended to a final concentration of OD 600 1.5 by using a proper volume of resuspension (formula: 10mmol·L -1 MgCl2, 10mmol·L -1 MES and 200μmol·L -1 acetyl vanillyl ketone). The resuspension was placed at room temperature for more than 3h in dark. The resuspension containing the recombinant plasmid TRV2-GhCDPK29 was obtained. The resuspension containing pTRV1 vector, the resuspension containing TRV2-GhCLA1 vector with GhCLA1 gene fragment and the resuspension containing pTRV2 empty vector were obtained by using similar method.

[0105] The resuspension containing pTRV1 vector and the resuspension containing recombinant plasmid TRV2-GhCDPK29 with the gene fragment of interest are mixed at a volume ratio of 1:1 to obtain a TRV:GhCDPK29 solution for injecting cotton cotyledons to obtain a gene silencing transformation strain; the resuspension containing pTRV1 vector and the resuspension containing TRV2-GhCLA1 vector with the GhCLA1 gene fragment are mixed at a volume ratio of 1:1 to obtain a TRV:GhCLA1 solution for injecting cotton cotyledons to detect whether the gene silencing system is correct; the resuspension containing pTRV1 vector and the resuspension containing pTRV2 empty vector are mixed at a volume ratio of 1:1 to obtain a TRV:00 solution for injecting cotton cotyledons to obtain a genetic transformation control strain.

[0106] (3) Receptor culture

[0107] TM-1 cotton seeds are soaked in sterile water for 24 hours after being sterilized with 75% alcohol for 30 seconds, and then sown in nutrient soil, and cultured under 12h light / 12h dark at a temperature of 26-28°C. The humidity is maintained at 60% or above, and watered once every 4-5 days. When two cotyledons are flat and true leaves have not yet developed, VIGS operation can be performed.

[0108] (4) Obtaining of GhCDPK29 gene silencing transformation strain

[0109] First, a syringe needle is used to lightly pierce the back of the cotyledon to create a small wound, and then the resuspension prepared in step (2) above is injected into the wound using a syringe without a needle, to obtain a cotton GhCDPK29 gene silencing transformation strain. Avoid light for 24 hours, and culture under 12h light / 12h dark at a temperature of 26-28°C.

[0110] GhCLA1 gene silencing detection strain and pTRV2 empty vector genetic transformation control strain are obtained by a similar method.

[0111] For specific methods of Agrobacterium VIGS, refer to Gao, X., Shan, L. Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing. Methods Mol Biol, 2013, 975: 157-165.

[0112] 3. Detection of VIGS genetic transformation system

[0113] (1) 2 weeks after observation of the phenotype of different treatments of cotton, using the GhCLA1 gene (cloroplastosalterados 1 gene) as a marker gene for VIGS system detection. The gene is involved in the process of chloroplast development, encoding 1-deoxyxylulose 5-phosphate synthase protein, which is highly conserved in evolution. After silencing of the GhCLA1 gene, the cotton plants have a significant white phenotype, which is an easily recognizable marker trait. As shown in Figure 2, after 2 weeks of VIGS infection, the injected TRV1 and TRV2-GhCLA1 plants have almost completely white leaves (Figure 2A), while the leaves of the injected empty vector pTRV1 and pTRV2 control (TRV:00) have no changes (Figure 2B, TRV:00). Figure 4 Figure 4 Figure 4 A), while the leaves of the injected empty vector pTRV1 and pTRV2 control (TRV:00) have no changes (Figure 2B, TRV:00). This indicates that the TRV-mediated VIGS system has been successfully established in TM-1 upland cotton.

[0114] (2) Fluorescent quantitative Real time-PCR detection

[0115] Take the treated cotton leaves to extract RNA (preferably newly grown true leaves), and then perform qRT-PCR detection to determine whether the target gene is reduced in expression, and to detect the expression of the target gene. 30 single plants are treated for each material.

[0116] Total RNA is extracted from the leaves of VIGS-infected cotton plants using an RNA extraction kit. With cotton UBQ7 as the internal reference gene, the expression of the silenced gene GhCDPK29 is detected by fluorescent quantitative Real time-PCR.

[0117] The qRT-PCR analysis of related primers and PCR reaction procedures is as described in Example 2, Part 1 “Expression pattern of gene GhCDPK29 under signal molecule stress (jasmonic acid JA, salicylic acid SA, H2O2 immersion)”.

[0118] The qRT-PCR results are shown in Figure 3. Compared with the empty vector (TRV:00) control, the expression of the GhCDPK29 gene in randomly selected GhCDPK29 VIGS-infected plants was significantly reduced, and the silencing effect was obvious. Figure 5

[0119] 4. Cotton Verticillium wilt inoculation and resistance identification

[0120] (1) Inoculation of cotton Verticillium wilt

[0121] The stored Verticillium dahliae pathogen strain V991 was activated on PDA medium. The picked mycelium was inoculated on Czapek agar medium and incubated at 25°C for 2 weeks. , ​​​s culture solution, 25℃, 200rpm, 3-5d. The pathogen culture solution was filtered with 4 layers of gauze, and the concentration of pathogen was counted with a hemocytometer. The final concentration was adjusted to 1.0x10 7

[0122] (2) Statistics of Verticillium wilt incidence

[0123] When the first true leaves began to appear yellow and wilt, the Verticillium wilt incidence was investigated and counted by using the 0-4 grade method. 30 single plants were counted for each material treatment. Three biological replicates were set.

[0124] The reference for disease index statistics: Xu L, Zhu L F, Zhang X L. Research on resistance mechanism of cotton to Verticillium wilt. Acta Agron Sin, 2012, 38: 1553-1560.

[0125] Disease index = [(number of plants at each grade x corresponding grade) / total number of plants surveyed x highest disease grade (4)] x 100

[0126] Test results statistics: the Verticillium wilt incidence of TRV:GhCDPK29 silenced plants compared with the empty vector control (TRV:00) plants after 21d of inoculation with V991 strain of L. theochromae. As shown in Figure 2B, the control (TRV:00) plants had more and larger yellow patches on the leaves and more obvious downward curling of the leaf edges than the GhCDPK29 gene silenced plants. Through 3 biological replicate observations and statistical analysis, the disease index results are shown in Figure 2C. The average disease index of the control plants injected with the empty vector was 52%, while the average disease index of the GhCDPK29 gene silenced plants was 37%. This indicates that the gene GhCDPK29 is involved in the stress response induced by Verticillium wilt. After silencing the gene GhCDPK29 in Gossypium hirsutum TM-1, the incidence and disease index of cotton were significantly reduced when infected with Verticillium wilt, indicating that silencing of the gene GhCDPK29 improved the resistance of cotton to Verticillium wilt. Figure 4 B can be seen, the control (TRV:00) plants had more and larger yellow patches on the leaves and more obvious downward curling of the leaf edges than the GhCDPK29 gene silenced plants. Through 3 biological replicate observations and statistical analysis, the disease index results are shown in Figure 2C. The average disease index of the control plants injected with the empty vector was 52%, while the average disease index of the GhCDPK29 gene silenced plants was 37%. This indicates that the gene GhCDPK29 is involved in the stress response induced by Verticillium wilt. After silencing the gene GhCDPK29 in Gossypium hirsutum TM-1, the incidence and disease index of cotton were significantly reduced when infected with Verticillium wilt, indicating that silencing of the gene GhCDPK29 improved the resistance of cotton to Verticillium wilt. Figure 6 B can be seen, the control (TRV:00) plants had more and larger yellow patches on the leaves and more obvious downward curling of the leaf edges than the GhCDPK29 gene silenced plants. Through 3 biological replicate observations and statistical analysis, the disease index results are shown in Figure 2C. The average disease index of the control plants injected with the empty vector was 52%, while the average disease index of the GhCDPK29 gene silenced plants was 37%. This indicates that the gene GhCDPK29 is involved in the stress response induced by Verticillium wilt. After silencing the gene GhCDPK29 in Gossypium hirsutum TM-1, the incidence and disease index of cotton were significantly reduced when infected with Verticillium wilt, indicating that silencing of the gene GhCDPK29 improved the resistance of cotton to Verticillium wilt.

[0127] (3) Verification of the involvement of gene GhCDPK29 in the hypersensitive response induced by Verticillium wilt ​

[0128] In order to verify the accuracy of the above phenotype results, the present study further carried out Verticillium dahliae recovery culture experiment, stem sectioning treatment of diseased plants and detection of DNA relative abundance of Verticillium dahliae in plants.

[0129] The method of Verticillium dahliae recovery culture experiment is as follows: the stems of cotton seedlings treated by Verticillium dahliae for 21 days are cut into 1 cm long segments at 6 cm from the cotyledon with scissors, then the segments are soaked in 70% alcohol for 1 min, 30% hydrogen peroxide for 30 min, and then washed with sterile water for 4-5 times, finally the treated cotton stem segments are placed on PDA medium and cultured at 25°C for 4 days, and then the growth condition of Verticillium dahliae recovery culture can be observed.

[0130] The method of stem sectioning treatment of diseased plants is as follows: several cotton stems treated by Verticillium dahliae for 21 days are taken and longitudinally cut with a sharp blade to expose the longitudinal section, and then the phenotype of vascular bundle tissue of the stem is observed, if the vascular bundle tissue of the stem is brown, it indicates that the stem has been infected by Verticillium dahliae and has developed disease, and if the vascular bundle tissue of the stem is not brown and the phenotype is normal, it indicates that the stem has not developed disease.

[0131] The method of detection of DNA relative abundance of Verticillium dahliae in plants is as follows: the RNA of the stem of cotton seedling treated by Verticillium dahliae is extracted, and then the cDNA is obtained and subjected to qPCR amplification.

[0132] The primers used are as follows:

[0133] ITS1-F: 5'-AAAGTTTTAATGGTTCGCTAAGA-3', SEQ ID NO: 11;

[0134] ST-VE1-R: 5'-CTTGGTCATTTAGAGGAAGTAA-3', SEQ ID NO: 12.

[0135] The corresponding program is as follows: the quantitative program is 94°C for 30 s; 95°C for 5 s, 52°C for 15 s, 72°C for 31 s, 40 cycles.

[0136] The reverse transcription and qPCR reaction program conditions are as described in Example 2, Part 1 "Expression pattern of gene GhCDPK29 under stress of signal molecules (jasmonic acid JA, salicylic acid SA, H2O2 immersion)".

[0137] The experimental results are shown in Table 1. Figures 7-10 After longitudinal sectioning of the stem, it was found that the vascular bundle of the stem of the control plant injected with empty vector turned light brown, while the vascular bundle of the stem of the GhCDPK29 gene silenced transformation plant was normal in color, and the browning degree of the stem sectioning material of the TRV:GhCDPK29 plant material was lower than that of the TRV:00 control plant material injected with empty vector. Figure 7). The TRV: GhCDPK29 plant material produced less Verticillium dahliae colonies than the TRV: 00 plant material in PDA medium Figure 8 ). The Verticillium dahliae recovery rate of the TRV: 00 control plant was 72%, while the Verticillium dahliae recovery rate of the TRV: GhCDPK29 gene-silenced plant was 18% Figure 9 ). The Verticillium dahliae DNA relative abundance of the TRV: 00 plant was 2 times that of the TRV: GhCDPK29 gene-silenced plant Figure 10

[0138] These results also indicate that the resistance of cotton to Verticillium dahliae is improved after silencing of the GhCDPK29 gene, indicating that the GhCDPK29 gene is involved in the regulation of Verticillium wilt resistance.

[0139] 5. Verification of the involvement of the GhCDPK29 gene in the accumulation of H202 in leaves

[0140] Plant disease usually causes changes in the accumulation of H202 in leaves. We proved the H202 accumulation by DAB staining of the leaves of the control plant and the GhCDPK29 gene-silenced transformation plant.

[0141] DAB dye preparation: 500 mg of DAB powder was dissolved in 500 mL of distilled water to make a concentration of 1 mg / mL, 0.2 M hydrochloric acid was used to adjust the pH to 3.0, and it was stored at 4°C in the dark for standby, and 20 ul Tween20 and 200 mM Na2HPO4 were added before use to adjust the pH of the DAB dye solution to 5.8.

[0142] Decolorizing solution preparation: ethanol: acetic acid: glycerol = 3: 1: 1.

[0143] The same part of the fully expanded leaves of the control plant and the GhCDPK29 gene-silenced transformation plant was taken and placed in a 15 mL tube containing DAB dye, vacuumed to sink the leaves to the bottom of the tube, and stained in the dark at 28°C for 12 hours or more. The leaves were taken out and placed in a centrifuge tube containing a decolorizing solution, boiled in a water bath until the green color of the leaves was removed, and finally stored in 80% ethanol at 4°C and photographed.

[0144] The experimental results are shown in Figure 11 The results show that more red-brown spots are formed in the leaves of the GhCDPK29 gene-silenced transformation plant, indicating that the accumulation of H202 is higher than that of the control plant.

[0145] Each embodiment in the specification is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between each embodiment can be referred to each other.

[0146] ​The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Silencing calcium-dependent protein kinase genes GhCDPK 29. Application in improving the resistance of upland cotton to Verticillium wilt, characterized in that, The gene GhCDPK The nucleotide sequence of 29 is shown in SEQ ID NO:

1.

2. Silencing calcium-dependent protein kinase genes GhCDPK The application of substance 29 in improving the resistance of upland cotton to Verticillium wilt is characterized by, Gene encoding calcium-dependent protein kinase GhCDPK The amino acid sequence of protein 29 is shown in SEQ ID NO:

2.

3. A method for improving plant resistance to Verticillium wilt, characterized in that, By silencing the gene described in claim 1 in upland cotton GhCDPK The expression or inhibition of the activity of the protein described in claim 29 can enhance the resistance of upland cotton to Verticillium wilt.

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