CN114790449A - Application of calcium-dependent protein kinase gene GhCPK4 in resisting verticillium wilt of plants - Google Patents

Abstract

The invention discloses an application of a calcium-dependent protein kinase gene GhCPK4 in plant verticillium wilt resistance, and relates to the technical field of genetic engineering. The invention utilizes GhCPK4 gene to construct VIGS plant expression vector to transform upland cotton TM-1, and the obtained transgenic cotton shows resistance to verticillium wilt after being inoculated with cotton verticillium wilt V991, which shows that GhCPK4 gene is highly related to resistance to cotton verticillium wilt. The invention lays an application foundation for the research of the molecular mechanism of verticillium wilt resistance and the cultivation of new varieties of new verticillium wilt resistant plants, and simultaneously provides new reference for the screening of new disease-resistant genes and the cultivation of resistant plants.

Description

Application of calcium-dependent protein kinase gene GhCPK4 in resisting verticillium wilt of plants

Technical Field

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

Background

The Verticillium wilt of cotton is mainly caused by Verticillium dahliae (Verticillium dahliae), and the pathogenic bacteria can be used as soil-borne fungi to contaminate the cotton in the whole growth period of the cotton, so that the cotton leaves are green and yellow, wilted, withered and shed, plants are short, cotton bolls are small and the like, even the cotton is dead, and the yield, the quality and the economic benefit of cotton planting are seriously affected. Due to the irreversibility of verticillium wilt, it is called "cancer" of cotton. Therefore, the method is particularly urgent for breeding new varieties of cotton upland cotton with verticillium wilt resistance. Due to the complexity of the pathogenic mechanism of cotton verticillium wilt and the difficulty and long period of screening disease-resistant varieties by the traditional hybridization method, upland cotton cultivars with excellent resistance to cotton verticillium wilt are not cultivated at present. The molecular improvement of cotton for resisting the verticillium wilt has become an urgent need for the continuous development of high-quality cotton bases. The development of the disease-resistant related genes of cotton to analyze the disease-resistant mechanism and the creation of new resistant germplasm by using transgenosis has become an important means for cotton verticillium wilt resistance breeding.

During the evolution process, plants form a series of complex signal transmission mechanisms to respond to stresses such as diseases, low temperature, drought, salt damage and the like, so that the environmental adaptability of the plants is improved, and damages caused by adversity are reduced to the greatest extent. Ca ²⁺ Is a second messenger commonly existing in plant cells and is widely involved in the growth and development, environmental response, stress response and the like of plantsAnd (6) processing. The calcium-dependent protein kinase CPKs (CPKs) are one of the first calcium-dependent protein kinases found in plants, belong to serine/threonine kinases, and can sense Ca in plant cells ²⁺ The concentration change is specifically combined with specific target cells, can be directly activated by calcium signals without the action of calmodulin, and plays an important role in regulating the growth and development of plants and adapting to the stress.

CPKs are encoded by multiple genes in plants and are involved in regulating plant responses to various environmental stresses (Chen et al, 2021). High temperature, drought, salt stress, exogenous hormone treatment and pathogenic microorganism infection can all cause the expression of CPKs gene by Ca ²⁺ Further regulating the response of the plant to various abiotic stress signals so as to resist the stress and enhance the stress resistance of the plant (Atif et al, 2019; Zhang et al, 2018). With the completion of cotton sequencing work, 41 CPKs genes have been identified from the diploid cotton Gossypium raimonidii genome, and 98 predicted CPKs among them were identified from upland cotton TM-1, with widely distributed CPKs expression in different organs, 19 of which respond rapidly to salt stress at the transcriptional level, and silencing of GhCPK8, GhCPK38, GhCPK54 and GhCPK55 severely affected cotton salt tolerance (Gao et al, 2018). The GhCPK 1gene is involved in regulating cotton fiber development (Huang et al, 2008; Wang et al, 2011). The GhCPK4 gene is involved positively in responding to drought and salt stress (Hu et al, 2019; margosa et al, 2022). The GhCPK5 gene is possibly located in a cell nucleus, can respond to the induction of salt stress and plays a role in a salt stress signal transduction pathway (screening military waves and the like, 2011). Since the calcium-dependent protein kinase is Ca ²⁺ An important component in the signal transmission pathway, and therefore, the identification of the calcium-dependent protein gene and the functional research thereof have important significance on the role of the calcium-dependent protein gene in the growth and development of plants and the response of the plants to environmental stimuli.

Disclosure of Invention

In view of the above, the invention provides the application of the calcium-dependent protein kinase gene GhCPK4 in the plant verticillium wilt resistance, and lays an application foundation for the cultivation of a new verticillium wilt-resistant cotton variety.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of protein or substance for regulating and controlling the expression of the protein coding gene in plant verticillium wilt resistance, wherein the amino acid sequence of the protein is shown as SEQ ID NO: 2 is shown in the specification;

or in the SEQ ID NO: 2 linked with protein tags at the N terminal and/or the C terminal of the amino acid sequence shown in the sequence table;

or the sequence of SEQ ID NO: 2 by substitution and/or deletion and/or addition of more than one amino acid residue, and obtaining the protein with the same function.

Preferably, the protein is derived from cotton.

Another object of the present invention is to provide the use of the protein-related biomaterial as described above for plant verticillium wilt resistance, wherein the protein-related biomaterial is any one of the following materials:

a: a nucleic acid molecule encoding the protein of claim 1;

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

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

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

The term "expression cassette" refers to a DNA capable of expressing a protein as described in the above application 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.

Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 1 is shown in the specification;

or with said SEQ ID NO: 1 nucleotide sequence having a homology of 90% or more and encoding the protein of claim 1.

Preferably, the plant is any one of:

a: a plant of the genus gossypium; b: cotton; c: and (3) upland cotton TM-1.

It is still another object of the present invention to provide a method for improving verticillium wilt resistance in plants by silencing or inhibiting the expression level of a gene encoding the protein or the activity of the protein in plants.

The silencing or suppressing of the expression level of the protein-encoding gene in the plant can be achieved by any means known in the art, such as deletion mutation, insertion mutation or base change mutation of the gene, thereby achieving the reduction or loss of the function of the gene.

Preferably, the amino acid sequence of the protein is as shown in SEQ ID NO: 2, respectively.

Preferably, the nucleotide sequence of the coding gene of the protein is shown in SEQ ID NO: 1 is shown.

According to the technical scheme, compared with the prior art, the gene is amplified by utilizing the sequence information of the GhCPK4 gene, the VIGS plant expression vector is constructed to transform upland cotton TM-1, and the obtained transgenic cotton shows the resistance to verticillium wilt after being inoculated with cotton verticillium wilt bacteria V991, so that the GhCPK4 gene is highly related to the resistance to cotton verticillium wilt. The invention lays an application foundation for the research of molecular mechanism of verticillium wilt resistance and the cultivation of new varieties of new verticillium wilt resistant plants, and simultaneously provides new reference for the screening of new disease-resistant genes and the cultivation of resistant plants.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing an electrophoresis diagram of a PCR product of the GhCPK4 gene in example 1 of the present invention, wherein the Marker is a 2000bp Ladder Marker;

FIG. 2 is a diagram showing an evolutionary tree of CPK related gene families;

FIG. 3 shows the qRT-PCR results of GhCPK4 gene expression level in different cotton tissues (leaf, root, stem, petal, sepal, and developing 5d fiber);

FIG. 4 shows signal molecules (jasmonic acid JA, salicylic acid SA, H) ₂ O ₂ Dip dyeing) induction condition of qRT-PCR result of the expression amount of the GhCPK4 gene;

FIG. 5 shows the qRT-PCR analysis result of expression pattern of GhCPK4 gene induced by verticillium dahliae V991;

FIG. 6 is a table of the plant phenotype of Gossypium hirsutum TM-1 after injection of VIGS vector, wherein: a is the phenotype of the plant after 2 weeks of injecting the VIGS vector of the GhCLA1 gene; b is TRV:00 (control) of upland cotton TM-1 and TRV: GhCPK4 inoculated with verticillium dahliae V991 spore liquid (10) by root dipping method ⁷ conidia/mL) disease incidence for 21 days;

FIG. 7 shows the qRT-PCR results of the expression level of the GhCPK4 gene in the leaf blade of upland cotton TM-1 at the three-leaf stage of the plant TRV:00 and TRV: GhCPK 4;

FIG. 8 is the result of statistics of disease index after transgenic upland cotton TM-1 is inoculated with Verticillium dahliae;

FIG. 9 is a longitudinal cut of cotton stalks of the default and control plants of VIGS-interfered upland cotton TM-1 plants for resistance analysis of verticillium wilt;

FIG. 10 is a graph showing the results of a fungus recovery experiment conducted on silenced plants of VIGS interference upland cotton TM-1 plants with stem segments sterilized on the surface of control plants;

FIG. 11 is a graph showing the statistical results of the cotton verticillium wilt recovery rate of silenced plants and control plants of VIGS interference upland cotton TM-1 plants for resistance analysis of verticillium wilt;

FIG. 12 is the relative abundance of Verticillium oxysporum DNA of the silenced plant and a control plant of VIGS interference upland cotton TM-1 plant for resistance analysis of Verticillium oxysporum.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The cotton material comprises:

upland cotton TM-1, provided by Cotton research institute of Chinese academy of agricultural sciences;

plant VIGS silencing expression vector:

verticillium wilt V991: the laboratory is reserved, and the public can obtain the laboratory from the Nuclear biotechnological research institute of agricultural academy of sciences (the biotechnological research center of Uygur autonomous region of Xinjiang);

the primer sequence is as follows: are all provided by Shanghai biological synthesis;

other biological materials include: escherichia coli strain DH5 alpha, agrobacterium strain GV3101, BP reaction entry vector, VIGS interference technology vector pTRV1 and pTRV2 and the like belong to commercial strains or vectors and are not described again;

experimental reagent: the plasmid extraction kit, oligo (dT)18, an RNAase inhibitor, dNTP, pMD18-T Vector, T4-DNA ligase, endonucleases EcoRI and KpnI, ExTaq enzyme, a PCR product recovery kit and the like are all products of TaKaRa company, and the fluorescent quantitative PCR kit is a product of TOYOBO company;

the special fluorescent quantitative PCR plate is a product of Labwards company;

RNA extraction kits were purchased from TIANGEN (Beijing, China);

the culture media and solutions used were: LB liquid (solid) medium, YEP liquid (solid) medium, Czapek's (Czapek) medium, PDA medium, 50 XTAE Buffer (Na) ₂ EDTA·2H ₂ 37.2g of O, 57.1mL of glacial acetic acid, pH value adjusted by NaOH to be 8.3, and water is added to reach a constant volume of 1L), and the like, and the preparation method is conventional in the field;

other undescribed antibiotics, hormones and other reagents are common in the field and are not described in detail.

Example 1

1. Acquisition of Gossypium hirsutum GhCPK4 gene

Planting upland cotton TM-1, extracting RNA of cotton seedling leaves, and performing reverse transcription to obtain cDNA;

in the RNA extraction step, a TIANGEN plant RNA rapid extraction kit is referred, and a reverse transcription system is as follows:

executing the program: 30min at 42 ℃ and 5s at 85 ℃.

A cDNA (complementary deoxyribonucleic acid) with an ID number of a gene Gh _ A03G1505.1 (a nucleotide sequence is shown as SEQ ID NO: 1, and an amino acid sequence is shown as SEQ ID NO: 2) in a cottonFGD website (https:// CottonFGD. org) is used as a template, a primer is designed, and the gene is amplified, wherein the primer sequence is as follows:

GhCPK4-F:5’-GGGGTACCATGGGCAATACATGCCGTG-3’，SEQ ID NO：3；

GhCPK4-R:5’-GCTCTAGATTACATAGCACCTGGGGCA-3’，SEQ ID NO：4。

the PCR amplification system is as follows:

PCR amplification procedure: 2min at 95 ℃; 35 cycles of 95 ℃ for 20s, 52 ℃ for 30s, 72 ℃ for 30 s; 5min at 72 ℃.

The PCR products were detected by electrophoresis on 1% agarose gel, and the results are shown in FIG. 1. As can be seen from the figure, the size of the gene GhCPK4 is 1707 bp.

2. Biological information analysis of GhCPK4 gene

The physicochemical properties of the GhCPK4 protein were analyzed by an online website (http:// web. expasy. org/protparam /), and the protein contained 568 amino acids, had a molecular weight of 63.5kD, and had an isoelectric point pI of 5.81.

The obtained sequence is used for carrying out online sequence alignment at NCBI website, and the GhCPK4 protein belongs to the STKc _ CAMK protein family. The secondary structure of the GhCPK4 protein was predicted using an online site (http:// ffas. burn. org/Xtal Pred-cgi), and it was found that about 42% of the amino acids formed α -helices and 10% of the amino acids formed β -pleated sheets.

The result of the evolutionary tree analysis of GhCPK4 and the CPK family in Arabidopsis thaliana, maize, rice, tobacco and wheat is shown in figure 2, and the GhCPK4 has the closest genetic distance with the CPK4 and the CPK11 of Arabidopsis thaliana in the evolutionary relationship.

Example 2

1. Tissue expression pattern of GhCPK4 gene under natural condition

Respectively taking leaves, roots, stems, flowers, sepals and fibers developing for 5d from cotton in the full-bloom stage of the cotton, extracting RNA of the fibers, obtaining cDNA by using a reverse transcription kit, and carrying out qPCR amplification by adopting the following quantitative PCR primers:

QRT-PCR-GhCPK4F:5’-GTTAACAAGGATGATGATTTC-3’，SEQ ID NO：5；

QRT-PCR-GhCPK4R:5’-GCTATTACCCGTAAAGCCAT-3’，SEQ ID NO：6；

the reference gene selects Ubiquitin protein 7(Ubiquitin7, UB7), and the primer sequence is designed as follows:

UBQ7F:5’-GAAGGCATTCCACCTGACCAAC-3’，SEQ ID NO：7；

UBQ7R:5’-CTTGACCTTCTTCTTCTTGTGCTTG-3’，SEQ ID NO：8。

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

the 20 μ L reaction system was designed as follows:

the reaction system was 10. mu.L LSYBR Green Realtime PCR Master Mix, 1. mu.L cDNA product, 0.4. mu.L (10. mu.M) of each of the upstream and downstream primers, and nucleic-free Water was supplemented to a total reaction volume of 20. mu.L. There were 4 technical replicates per gene.

The corresponding procedure was a quantification procedure at 94 ℃ for 30 s; 95 ℃ for 5s, 57 ℃ for 15s, 72 ℃ for 31s, 40 cycles.

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

The detection result is shown in figure 3, and the analysis shows that the GhCPK4 gene is expressed in all tissues of cotton, but the transcription level is significantly different, wherein the expression of the gene in the flower is higher than that of other tissues, and sepals are arranged in the second; expression levels in leaves, roots, stems and developing 5d fibers were significantly lower than other tissues, with transcript levels in roots slightly higher than those in leaves, stems and developing 5d fibers.

2. Stressed by signal molecules (jasmonic acid JA, salicylic acid SA, H) ₂ O ₂ Dip-dyeing) of the gene GhCPK4

Soaking TM-1 cotton seeds in water for 24h, sowing in nutrient soil, and culturing under illumination at 26-28 deg.C for 12h in light/12 h in dark. Maintaining humidity at 60% or above, watering once for 4-5d, selecting cotton seedling with uniform growth and size when two leaves and one heart of seedling are planted, and respectively adding 200 μ M jasmonic acid, 2mM salicylic acid, and 1mM H ₂ O ₂ Soaking cotton seedling root system for 10min, and then continuously planting the cotton seedling in the nutrition pot. And respectively taking cotton roots at 0.5h, 1h, 3h and other time points after treatment, extracting RNA, and performing reverse transcription to obtain cDNA for expression pattern analysis.

The primer and PCR reaction procedure for qRT-PCR analysis were the same as in section 1 of this example, "tissue expression pattern of the natural GhCPK 4".

The detection result of qRT-PCR is shown in figure 4, the expression level of GhCPK4 is highest when the JA is treated for 1h and then is reduced; expression level of GhCPK4 in SA and H ₂ O ₂ The treatment trend is downward. The expression level of GhCPK4 is shown to be influenced by signal molecule stress.

3. Expression pattern of GhCPK4 under verticillium wilt pathogen infection condition

Selecting upland cotton TM-1 with two leaves and one heart, and using verticillium dahliae V991 conidium solution with concentration of 1 × 10 ⁷ spores/mL, water treatment as a negative control. Soaking cotton seedling root in the bacterial liquid for 10min, planting in nutrition bowl, and taking cotton at time points of 0h, 0.5h, 2h, 6h, 12h, 24h, 48h, etc. after inoculationRoot system, extracting RNA and reverse transcribing cDNA for expression pattern analysis.

The primer and PCR reaction procedure for qRT-PCR analysis were the same as in section 1 of this example, "tissue expression pattern of the natural GhCPK 4".

The detection result of qRT-PCR is shown in figure 5, after V991 conidium liquid and water are treated, the expression level of GhCPK4 is significantly reduced after 12h of inoculation of verticillium wilt pathogenic bacteria, and the expression level tends to be lowest after 48 h. The result shows that the expression quantity of the gene GhCPK4 is obviously changed after the root system of the TM-1 is infected by verticillium dahliae.

Example 3

VIGS interference vector is constructed by using VIGS technology, so that the gene GhCPK4 is silenced. The phenotypic change of cotton infected with verticillium dahliae after gene precipitation is observed, and the gene GhCPK4 is further proved to be highly related to verticillium wilt resistance.

1. Construction of recombinant expression vectors

(1) Primer design and PCR amplification

Designing a primer in a non-conservative section of the gene GhCPK4, wherein the design principle of the primer is as follows: adding a restriction site and a protection base of a restriction enzyme EcoRI to an upstream primer, and adding a restriction site and a protection base of a restriction enzyme KpnI to a downstream primer;

GhCPK4-1F:5’-GGAATTCGTTAACAAGGATGATGATTTC-3’，SEQ ID NO：9，

GhCPK4-1R:5’-GGGGTACCGCTATTACCCGTAAAGCCAT-3’，SEQ ID NO：10。

extracting total RNA of upland cotton TM-1, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification by using a primer pair consisting of a primer GhCPK4-1F and a primer GhCPK4-1R and taking the cDNA as a template to obtain a PCR amplification product.

The PCR amplification system is as follows:

PCR amplification procedure: 2min at 95 ℃; 35 cycles of 95 ℃ for 20s, 52 ℃ for 30s, 72 ℃ for 30 s; 5min at 72 ℃.

PCR amplifies the target sequence (about 488 bp); the amplification product was subjected to 1% agarose gel electrophoresis.

(2) Obtaining recombinant expression vector after sequencing

PCR amplification products were recovered from the gel, ligated with pEASY-Blunt Zero Cloning Kit (Cloning vector), and positive transformants identified by PCR using GhCPK4-1F/R primers were sequenced.

Selecting a positive transformant with successful sequencing to extract a plasmid, and carrying out double enzyme digestion on the plasmid and an empty vector pTRV2 by using enzymes EcoRI and KpnI respectively under the condition of water bath at 37 ℃, wherein the enzyme digestion system is as follows:

enzyme digestion program: 1h at 37 ℃; 10min at 80 ℃.

Carrying out 1% agarose gel electrophoresis on the enzyme digestion product, utilizing T4 DNA ligase to carry out connection, transforming agrobacterium GV3101 by the plant expression vector recombinant plasmid TRV2-GhCPK4, and obtaining the VIGS interference vector TRV2: GhCPK4 after PCR screening and identification by using GhCPK4-1F/R primer.

2. Obtaining transgenic cotton

(1) Culture of bacterial liquid

Agrobacterium was transformed with pTRV1 and TRV2-GhCPK4, respectively, to kanamycin (50. mu.g. mL) ^-1 ) Gentamicin (50. mu.g. mL) ^-1 ) And rifampicin (25. mu.g.mL) ^-1 ) Resistant LB flasks were grown to OD at 28 ℃ ₆₀₀ Reaching 0.6-0.8.

(2) Obtaining a resuspension

Centrifuging at 4000rpm for 5min to collect thallus cells, and adding resuspension (formula: 10 mmol. L) ^-1 MgCl ₂ ，10mmol·L ^-1 MES and 200. mu. mol. L ^-1 Acetosyringone) was resuspended to a final concentration of OD ₆₀₀ Is 1.5. Standing the re-suspension at room temperature in dark place for more than 3 h. Obtaining recombinant plasmid TRV2-GhCPK 4. Resuspension containing pTRV1 vector, resuspension of TRV2-GhCLA1 vector containing GhCLA 1gene fragment, and resuspension containing TRV2 empty vector were prepared in a similar manner.

Uniformly mixing a heavy suspension containing pTRV1 vector and a heavy suspension containing a recombinant plasmid TRV2-GhCPK4 containing a target gene fragment according to the volume ratio of 1:1 to obtain a TRV/GhCPK 4 solution, and injecting the TRV/GhCPK 4 solution into cotton cotyledons to obtain a gene silencing transformant; uniformly mixing the resuspension containing the pTRV1 vector and the resuspension containing the TRV2-GhCLA1 vector containing the GhCLA 1gene fragment according to the volume ratio of 1:1 to obtain a TRV: GhCLA1 solution, and injecting the TRV: GhCLA1 solution into cotton cotyledons for detecting whether a gene silencing system is correct or not; and mixing the heavy suspension containing the pTRV1 vector and the heavy suspension containing the pTRV2 empty vector according to the volume ratio of 1:1 to obtain a TRV:00 solution, and injecting the TRV:00 solution into cotton cotyledons to serve as a genetic transformation control strain.

(3) Receptor culture

Soaking TM-1 cotton seeds in water for 24h, sowing in nutrient soil, and culturing under illumination at 26-28 deg.C for 12h in light/12 h in dark. Keeping humidity at 60% or above, watering once for 4-5 days, and performing VIGS operation when two leaves are spread and true leaves are not developed.

(4) Acquisition of GhCPK4 Gene-silenced transformant

Slightly puncturing the back of the cotyledon by using a syringe needle to cause micro-wound, and injecting the prepared re-suspension prepared in the step (2) and uniformly mixed according to the volume ratio of 1:1 from the wound by using the syringe needle to obtain the cotton GhCPK4 gene silencing transformant. And (3) keeping out of the light for 24h, and culturing under the light at the temperature of 26-28 ℃ in 12h light/12 h dark.

The ghcell 1gene silencing test strain and pTRV2 empty vector genetic transformation control strain were obtained in a similar manner.

Agrobacteria VIGS specific method reference: gao, X.Shan, L.functional genetic analysis of cotton genes with an Agrobacterium-mediated gene cloning Methods Mol Biol 2013,975: 157-.

3. Detection of VIGS genetic transformation System

(1) The phenotype of the different treated cotton was observed after 2 weeks and the VIGS system assay was performed using the upland cotton ghcia 1gene (cloroplasts alterados 1gene) as marker gene. The gene participates in the development process of the chlorophyll body, encodes 1-deoxyxylulose 5-phosphate synthsase protein, is highly conserved in evolution, and cotton plants have obvious albino phenotype after the GhCLA 1gene is silenced, so that the gene is easy to identify marker characters. As shown in FIG. 6, after 2 weeks of VIGS infection, the true leaves of the TRV1 and TRV2-GhCLA1 injected plants almost completely whitened (FIG. 6A), while the leaves injected with the empty vectors pTRV1 and pTRV2 as controls (TRV:00) did not change (FIG. 6B TRV: 00). The successful establishment of the TRV-mediated VIGS system in Gossypium hirsutum TM-1 is demonstrated.

(2) Fluorescent quantitative Real time-PCR detection

Extracting RNA (preferably a new long true leaf) from the treated cotton leaf, then carrying out qRT-PCR, detecting whether the target gene is reduced in expression or not, and detecting the expression condition of the target gene. 30 individuals were treated for each material.

And extracting total RNA of the VIGS-impregnated cotton plant leaves by using an RNA extraction kit. And detecting the interfered and silenced expression condition of the silenced GhCPK4 gene by using cotton UBQ7 as an internal reference gene and through fluorescent quantitative Real time-PCR.

Primers and PCR reaction procedure for qRT-PCR analysis in reference to section 1 of example 2, "tissue expression Pattern of Gene GhCPPK 4 in nature".

The qRT-PCR result is shown in figure 7, compared with the empty vector (TRV:00) control, the expression level of the GhCPK4 gene is obviously reduced and the silencing effect is obvious in randomly selected GhCPK4 gene VIGS-impregnated plants.

4. Cotton verticillium wilt resistance inoculation and resistance identification

(1) Inoculation of verticillium wilt of cotton

Activating the stored verticillium dahliae strain V991 which is a pathogen of verticillium wilt on a PDA culture medium. The selected thalli are cultured in Czapek, s culture solution for 3-5 d at 25 ℃ and 200 rpm. Filtering the culture solution of pathogenic bacteria with 4 layers of gauze, counting the concentration of pathogenic bacteria with a blood count plate, and adjusting the final concentration to 1.0 × 10 with sterilized double distilled water ⁷ spores/mL, and Tween-20 was added to a final concentration of 0.001% (volume percent). The resulting transformant in which the VIGS gene was silenced for 2 weeks was inoculated by the root injury methodVerticillium dahliae V991 spore liquid.

(2) Statistics of verticillium wilt disease onset

And (5) investigating and counting the disease incidence condition of the verticillium wilt when the real leaves begin to turn yellow and wither, and counting the disease level by adopting a 0-4 level method. 30 individuals were treated for each material. Set 3 biological replicates.

Grade index statistical reference: xue Li, Zhu Long Pai, Zhang Dong Long, research progress of anti-verticillium wilt mechanism of cotton, journal of crops, 2012,38: 1553-; xu L, Zhu L F, Zhang X L.research on resistance mechanism of cotton to Verticillium wilt.acta Agron Sin 2012,38: 1553-.

Disease index ═ number of diseased plants at each stage x corresponding disease stage)/number of total investigated plants x highest disease stage of disease (4) ] x 100

And (4) statistics of test results: compared with a control (TRV:00) plant with an empty carrier, a silencing plant of TRV: GhCPK4 infected with VIGS for 2 weeks is inoculated with verticillium dahliae strain V991, and the verticillium wilt is caused after 21 d. As can be seen in FIG. 6B, the control (TRV:00) plants showed more yellowish plaques and larger areas on the leaves and more pronounced curling down the leaf margins than the GhCPK4 gene-silenced plants. Through statistical analysis of 3 biological repeated observations, the disease index results are shown in figure 8, the average disease index of the control plants injected with the empty vectors reaches 52%, while the disease resistance of the plants subjected to GhCPK4 gene silencing is remarkably enhanced, and the average disease index is 27%. The GhCPK4 gene is shown to participate in the allergic reaction induced by verticillium wilt. After the GhCPK4 gene is silenced in upland cotton TM-1, the morbidity and the disease indication of cotton are obviously reduced when the cotton is infected by verticillium wilt bacteria, which shows that the resistance of the cotton to the verticillium wilt is improved by the silencing of the GhCPK4 gene.

(3) Verification that GhCPK4 gene participates in verticillium wilt induced anaphylactic reaction

In order to verify the accuracy of the phenotype results, the research continues to perform a verticillium wilt recovery culture experiment, diseased plant stem cutting treatment and plant verticillium wilt DNA relative abundance detection.

The experimental method for recovering and culturing verticillium dahliae comprises the following steps: cutting stems of cotton seedlings 21d after verticillium wilt treatment and 6cm away from cotyledons into 1cm long segments by using scissors, soaking the segments in 70% alcohol for 1min, soaking the segments in 30% hydrogen peroxide for 30min, washing the segments with sterile water for 4-5 times, and finally placing the treated cotton stem segments on a PDA culture medium for culture at 25 ℃ for 2d to observe the growth condition of verticillium wilt recovery culture.

The method for treating the diseased plant dissected stems comprises the following steps: taking a plurality of cotton stalks which are inoculated with verticillium wilt bacteria for 21d, longitudinally cutting the cotton stalks by using a sharp blade to expose longitudinal sections of the cotton stalks, observing the phenotype of vascular bundle tissues of the stalks, wherein if the vascular bundle tissues of the stalks are brown, the cotton stalks are infected with the verticillium wilt bacteria and are attacked, and if the vascular bundle tissues of the stalks are not brown and the phenotype is normal, the cotton stalks are not attacked.

The relative abundance method of the verticillium wilt bacteria DNA of the plant comprises the following steps: and extracting RNA of the stem of the cotton seedling after the verticillium wilt treatment, reversing the RNA into cDNA, and performing qPCR amplification.

The primers used were:

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

ST-VE1-R:5’-CTTGGTCATTTAGAGGAAGTAA-3’，SEQ ID NO：12。

reverse transcription and qPCR reaction procedure conditions and procedures reference example 2 section 1 "tissue expression pattern of the gene GhCPK4 in nature".

The experimental results are shown in the attached figures 9-12, and after the stems are longitudinally cut, the stem vascular bundles of the control plant injected with the empty carrier are changed into light brown, while the stem vascular bundles of the plant subjected to GhCPK4 gene silencing are normal in color, and the browning degree of the stem cutting material of the plant material with the TRV: GhCPK4 is lower than that of the control plant material with the TRV:00 injected with the empty carrier (shown in the attached figure 9); the verticillium wilt germ colony of TRV: GhCPK4 plant material cultured in PDA culture medium is less than that of TRV:00 plant material (figure 10); the verticillium wilt recovery rate of TRV:00 control plants reaches 90%, and the verticillium wilt recovery rate of plants after TRV: GhCPK4 gene silencing reaches 30% (shown in figure 11). The relative abundance of verticillium wilt bacteria DNA of TRV:00 plants is 76 times that of TRV: GhCPK4 plants after gene silencing (figure 12).

These results also indicate that cotton resistance to verticillium wilt bacteria is improved after silencing the GhCPK4 gene, indicating that the GhCPK4 gene is involved in regulating verticillium wilt resistance.

In the present specification, the embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

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

Sequence listing

<110> institute of Nuclear technology and Biotechnology of academy of agricultural sciences in Xinjiang (center for Biotechnology research in autonomous region of Uygur autonomous region in Xinjiang)

<120> application of calcium-dependent protein kinase gene GhCPK4 in plant verticillium wilt resistance

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1707

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgggcaata catgccgtgg atctttaaag gggaaacttc ataagggcga caatcagccc 60

aaagaccatt gttctagccg caacaacact tcttccggcc gttcaaccac caccaccgat 120

tattcccctt ctactttaaa ctctcaacaa ctgattgctc aagaattctc caaagaaacc 180

aaccaaaaag aaacccattt tcctgtcatt aaccctacca agaaagacaa caacaataac 240

accatgagac gtggtattga tcaccaggct tactatgttt tgggtcataa aacacccaac 300

atccgtgacc tttacacttt aggtcgtaag ttaggacaag gacagtttgg gactacttac 360

ttgtgtactg agatctctac aggcactgag tatgcctgta agtctatatc caaaaggaag 420

ttgatctcca atgaggatgt ggaggatgtt cggagggaga ttcagataat gcaccatttg 480

gctggtcata agaatattgt gaccattaaa ggtgcatacg aggatacttt gtatgtgcat 540

attgtgatgg agctttgctc tggaggtgaa ttgtttgatc ggattatcca gagggggcat 600

tactccgaga ggaaagcagc tgaattgact aagatcattg ttggggttgt cgaggcttgt 660

cattcgctcg gggttatgca tagagattta aagcctgaga atttcttgtt agttaacaag 720

gatgatgatt tctctctgaa ggccattgat tttggactct cagtcttctt taaacccggc 780

caagttttta ccgacgtggt tggcagtcca tattacgttg ctccggaagt acttctgaaa 840

cattatggac cagaagcaga tgtatggact gcaggagtta tactctatat attgctaagt 900

ggtgtgccac cattttgggc agaaacacag caaggaatat ttgatgcagt gttaaaagga 960

catattgact ttgattcaga cccttggccc ctaatatctg atagtgcaaa agacctaatt 1020

cgaaagatgt tatgctctcg accttcagag cgactgactg ctcatgaagt attatgtcat 1080

ccttggattt gtgaaaatgg ggttgctcct gatagggccc tggatccagc tgtactttct 1140

cgcctcaaac aattctctgc aatgaataaa ctaaagaaga tggctttacg ggtaatagct 1200

gaaagtcttt ctgaggagga gattgctggt ttaagagaaa tgtttacgtc tatggatact 1260

gataacagtg gtgcaatcac atttgatgaa ctcaaagctg gtttgcgaag atacggctct 1320

accttgaaag atacagagat aagggacctt atggatgcag ctgatgtcga taatagtggg 1380

accattgatt atggagaatt catagctgca acagttcacc ttaataaact agagcgtgag 1440

gagcatctcg ttgctgcatt ccgatacttt gataaggatg gaagtgggta tattacagtt 1500

gatgagcttc aacaagcttg tgctgagcat aacatgacag atgttttgct cgaggatata 1560

atcagagaag ttgatcaaga taatgatgga agaatcgatt atggtgaatt tgttgccatg 1620

atgcaaaaag gcaatgcagg aatcggtaga cgaaccatgc gaaacagtgt gaatataagc 1680

atgagagatg ccccaggtgc tatgtaa 1707

<210> 2

<211> 568

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Gly Asn Thr Cys Arg Gly Ser Leu Lys Gly Lys Leu His Lys Gly

1 5 10 15

Asp Asn Gln Pro Lys Asp His Cys Ser Ser Arg Asn Asn Thr Ser Ser

20 25 30

Gly Arg Ser Thr Thr Thr Thr Asp Tyr Ser Pro Ser Thr Leu Asn Ser

35 40 45

Gln Gln Leu Ile Ala Gln Glu Phe Ser Lys Glu Thr Asn Gln Lys Glu

50 55 60

Thr His Phe Pro Val Ile Asn Pro Thr Lys Lys Asp Asn Asn Asn Asn

65 70 75 80

Thr Met Arg Arg Gly Ile Asp His Gln Ala Tyr Tyr Val Leu Gly His

85 90 95

Lys Thr Pro Asn Ile Arg Asp Leu Tyr Thr Leu Gly Arg Lys Leu Gly

100 105 110

Gln Gly Gln Phe Gly Thr Thr Tyr Leu Cys Thr Glu Ile Ser Thr Gly

115 120 125

Thr Glu Tyr Ala Cys Lys Ser Ile Ser Lys Arg Lys Leu Ile Ser Asn

130 135 140

Glu Asp Val Glu Asp Val Arg Arg Glu Ile Gln Ile Met His His Leu

145 150 155 160

Ala Gly His Lys Asn Ile Val Thr Ile Lys Gly Ala Tyr Glu Asp Thr

165 170 175

Leu Tyr Val His Ile Val Met Glu Leu Cys Ser Gly Gly Glu Leu Phe

180 185 190

Asp Arg Ile Ile Gln Arg Gly His Tyr Ser Glu Arg Lys Ala Ala Glu

195 200 205

Leu Thr Lys Ile Ile Val Gly Val Val Glu Ala Cys His Ser Leu Gly

210 215 220

Val Met His Arg Asp Leu Lys Pro Glu Asn Phe Leu Leu Val Asn Lys

225 230 235 240

Asp Asp Asp Phe Ser Leu Lys Ala Ile Asp Phe Gly Leu Ser Val Phe

245 250 255

Phe Lys Pro Gly Gln Val Phe Thr Asp Val Val Gly Ser Pro Tyr Tyr

260 265 270

Val Ala Pro Glu Val Leu Leu Lys His Tyr Gly Pro Glu Ala Asp Val

275 280 285

Trp Thr Ala Gly Val Ile Leu Tyr Ile Leu Leu Ser Gly Val Pro Pro

290 295 300

Phe Trp Ala Glu Thr Gln Gln Gly Ile Phe Asp Ala Val Leu Lys Gly

305 310 315 320

His Ile Asp Phe Asp Ser Asp Pro Trp Pro Leu Ile Ser Asp Ser Ala

325 330 335

Lys Asp Leu Ile Arg Lys Met Leu Cys Ser Arg Pro Ser Glu Arg Leu

340 345 350

Thr Ala His Glu Val Leu Cys His Pro Trp Ile Cys Glu Asn Gly Val

355 360 365

Ala Pro Asp Arg Ala Leu Asp Pro Ala Val Leu Ser Arg Leu Lys Gln

370 375 380

Phe Ser Ala Met Asn Lys Leu Lys Lys Met Ala Leu Arg Val Ile Ala

385 390 395 400

Glu Ser Leu Ser Glu Glu Glu Ile Ala Gly Leu Arg Glu Met Phe Thr

405 410 415

Ser Met Asp Thr Asp Asn Ser Gly Ala Ile Thr Phe Asp Glu Leu Lys

420 425 430

Ala Gly Leu Arg Arg Tyr Gly Ser Thr Leu Lys Asp Thr Glu Ile Arg

435 440 445

Asp Leu Met Asp Ala Ala Asp Val Asp Asn Ser Gly Thr Ile Asp Tyr

450 455 460

Gly Glu Phe Ile Ala Ala Thr Val His Leu Asn Lys Leu Glu Arg Glu

465 470 475 480

Glu His Leu Val Ala Ala Phe Arg Tyr Phe Asp Lys Asp Gly Ser Gly

485 490 495

Tyr Ile Thr Val Asp Glu Leu Gln Gln Ala Cys Ala Glu His Asn Met

500 505 510

Thr Asp Val Leu Leu Glu Asp Ile Ile Arg Glu Val Asp Gln Asp Asn

515 520 525

Asp Gly Arg Ile Asp Tyr Gly Glu Phe Val Ala Met Met Gln Lys Gly

530 535 540

Asn Ala Gly Ile Gly Arg Arg Thr Met Arg Asn Ser Val Asn Ile Ser

545 550 555 560

Met Arg Asp Ala Pro Gly Ala Met

565

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggggtaccat gggcaataca tgccgtg 27

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gctctagatt acatagcacc tggggca 27

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gttaacaagg atgatgattt c 21

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gctattaccc gtaaagccat 20

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gaaggcattc cacctgacca ac 22

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cttgaccttc ttcttcttgt gcttg 25

<210> 9

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggaattcgtt aacaaggatg atgatttc 28

<210> 10

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggggtaccgc tattacccgt aaagccat 28

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aaagttttaa tggttcgcta aga 23

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cttggtcatt tagaggaagt aa 22

Claims (8)

1. The application of protein or substance for regulating and controlling the expression of 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 is shown in the specification;

or in the SEQ ID NO: 2, the N terminal and/or the C terminal of the amino acid sequence is linked with a protein tag to obtain a fusion protein;

or converting said SEQ ID NO: 2 by substitution and/or deletion and/or addition of more than one amino acid residue, and obtaining the protein with the same function.

2. Use according to claim 1, wherein the protein is derived from cotton.

3. Use of a protein-related biological material as claimed in claim 1 or 2 for combating verticillium wilt in plants, wherein the material is any one of the following:

a: a nucleic acid molecule encoding the protein of claim 1;

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

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

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

4. The use according to claim 3, wherein the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO: 1 is shown in the specification;

or with said SEQ ID NO: 1 nucleotide sequence having more than 90% homology and encoding the protein of claim 1.

5. The use according to any one of claims 1 to 4, wherein the plant is any one of the following:

a: a plant of the genus gossypium; b: cotton; c: 1, upland cotton TM-1.

6. A method for improving verticillium wilt resistance in plants, which comprises silencing or inhibiting the expression level of a gene encoding the protein of claim 1 or the activity of the protein of claim 1 in plants to thereby improve verticillium wilt resistance.

7. The method of claim 6, wherein the amino acid sequence of the protein is as set forth in SEQ ID NO: 2, respectively.

8. The method of claim 6, wherein the nucleotide sequence of the gene encoding the protein is as set forth in SEQ ID NO: 1 is shown.

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