CN115820722B

CN115820722B - Cotton Verticillium wilt resistance-related gene GhCBL3 and its encoding protein and application

Info

Publication number: CN115820722B
Application number: CN202210897827.1A
Authority: CN
Inventors: 高升旗; 郝晓燕; 胡文冉; 黄全生; 李建平; 常晓春; 陈果; 赵准
Original assignee: Xinjiang Academy Of Agricultural Sciences Institute Of Nuclear Technology Biotechnology (xinjiang Uygur Autonomous Region Biotechnology Research Center)
Current assignee: Xinjiang Academy Of Agricultural Sciences Institute Of Nuclear Technology Biotechnology (xinjiang Uygur Autonomous Region Biotechnology Research Center)
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2024-11-12
Anticipated expiration: 2042-07-28
Also published as: CN115820722A

Abstract

The invention discloses a cotton verticillium wilt resistance related gene GhCBL, and a coding protein and application thereof. The protein is any one of the following: b1 A protein having an amino acid sequence of SEQ ID No. 1; b2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.1, has more than 80% of identity with the protein shown in B1) and has the same function; b3 A fusion protein having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of B1) or B2). According to the invention, disease resistance of the GhCBL gene-silenced transgenic cotton is identified, and the result shows that the morbidity and the disease index of the transgenic cotton are obviously reduced, and the verticillium resistance is obviously enhanced, so that the GhCBL protein and the coding gene thereof can regulate and control the disease resistance of plants, and have important significance in cultivating verticillium resistant transgenic cotton.

Description

Cotton verticillium wilt resistance related gene GhCBL and encoding protein and application thereof

Technical Field

The invention relates to a cotton verticillium wilt resistance related gene GhCBL in the field of plant genetic engineering and a coding protein and application thereof.

Background

Cotton is an important commercial crop in the world, and upland cotton (Gossypium hirsutum l.) in cotton (Gossypium) is the most widely planted main cultivated cotton species at present due to its good fiber quality, high yield and short growth cycle, and the yield is about 95% of the total yield of cotton in the world. Cotton verticillium wilt (Verticillium wilt) is the most important disease in cotton production and is also one of the subjects of national agricultural plant quarantine. Cotton verticillium wilt is a destructive vascular bundle disease caused by the soil-borne pathogenic fungus verticillium dahliae (Verticillium dahliae) in cotton production, severely restricting cotton growth and development, and becoming the first disease of cotton worldwide, called "cancer" of cotton. To date, cotton verticillium wilt cannot be effectively controlled in upland cotton, and upland cotton varieties with high verticillium wilt resistance are still difficult to obtain. Years of practice prove that the development of disease resistance genes, the recognition of the reaction mechanism of cotton to verticillium wilt, the acceleration of the cotton verticillium wilt-resistant breeding process, and further the improvement of the disease resistance of cotton, become the main way of cotton verticillium wilt-resistant breeding work.

With the rapid development of molecular biology, the understanding of plant biotic stress response molecular mechanisms is gradually deepened. As a second messenger ubiquitous in plant bodies, ca ²⁺ is involved in regulating plant growth and development and multiple stress responses. Research shows that adversity stress acts on plant cells to first trigger the change of intracellular calcium ion concentration, and the specific change of Ca ²⁺ concentration appears in time and space, and the change can be sensed by corresponding Ca ²⁺ receptors, and further signals are transmitted to downstream target proteins, so that a series of physiological and biochemical reactions in the cells are triggered to respond to the adversity stress. Currently, ca ²⁺ receptor proteins found in plants are largely divided into three classes: calmodulin (calmodulin, caM), calcineurin-like B subunit proteins (calcineurin B-like proteins, CBL) and caldependent protein kinases (calcium-DEPENDENT PROTEIN KINASE, CDPK). One of the plant-specific Ca ²⁺ receptor proteins, calmodulin B-like protein (CBL), is capable of regulating the expression of the downstream early response gene by signaling with the protein with which it acts. CBL-type calcium receptors are small molecule proteins that are found only in plants that are very similar to the animal calcineurin B subunit (CNB) and central nervous calcium receptors (NCS), i.e., CBL or CBL calcium receptors. The protein kinase CIPK with which CBL receptors interact constitutes a CBL-CIPK signaling system to regulate response to calcium signaling and to regulate expression of downstream genes. In the genomes of dicotyledonous plant Arabidopsis and monocotyledonous plant rice, maize, the CBL gene family contains 10 members, and the 10 CBL genes each contain 6 or 7 introns in the coding region, wherein the positions and arrangement order of 4 introns in the coding region of the 10 genes are highly conserved. at present, researches on CBL genes are focused on plants responding to drought, salt, alkali, low temperature and other adversity stresses, but researches on the resistance function of cotton verticillium have not been reported. CBL proteins consist essentially of two globular domains, the N-and C-terminal, each domain containing a pair of EF-chiral motifs, each consisting of 12 amino acid residues. In comparison to the amino acid sequence of the typical EF hand motif of CaM (DXD (N) XD (N) (S) GXI (V) D (N) (S) XXE), there is a different degree of variation in the EF hand structure of CBL, such as 2 typical EF hand motifs in 10 members of the arabidopsis CBL family, atCBL and AtCBL, atCBL6, atCBL7, atCBL and AtCBL10 contain only one typical EF hand motif, whereas AtCBL2, atCBL3, atCBL and AtCBL5 do not. Cotton CBL3 has 4 EF motifs and can serve as calcium binding sites.

In view of the above, the verticillium wilt-resistant gene is mined and identified, the genetic basis of verticillium wilt resistance is analyzed, and disease-resistant molecular design breeding is carried out by combining biotechnology, so that the damage of verticillium wilt of cotton can be fundamentally prevented and controlled, the breeding of verticillium wilt-resistant cotton varieties is accelerated, the commercial cotton breeding process is promoted, and the problem of lack of disease-resistant varieties in production is effectively solved. The improvement of verticillium wilt resistance of cotton by genetic means is not only the most economical and effective solution, but also has important significance for guaranteeing the quality and high and stable yield of cotton, and has wide application value in the field of cotton molecular breeding.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate and control the disease resistance of plants (such as improving or reducing verticillium wilt resistance of plants). The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

To solve the above technical problems, the present invention provides first an application of a protein or a substance regulating the activity and/or content of the protein, wherein the application may be any of the following:

A1 Use of a protein or a substance regulating the activity and/or content of said protein for regulating plant disease resistance;

a2 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product regulating plant disease resistance;

a3 Use of a protein or a substance regulating the activity and/or content of said protein for growing disease-resistant plants;

A4 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product for growing disease-resistant plants;

A5 Use of a protein or a substance regulating the activity and/or content of said protein in plant breeding or plant germplasm resource improvement;

a6 Protein or the substance for regulating the activity and/or content of the protein is applied to prevention and control of cotton diseases;

the protein is GhCBL, and can be any one of the following:

b1 A protein having an amino acid sequence of SEQ ID No. 1;

B2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.1, has more than 10% of identity with the protein shown in B1) and has the same function;

B3 A fusion protein having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of B1) or B2).

In the above application, the protein GhCBL may be derived from cotton (Gossypium spp).

Further, the protein GhCBL may be derived from upland cotton (Gossypium hirsutum l.).

Further, the protein GhCBL may be cotton calcineurin B subunit protein (calcineurin B-like proteins, CBL) GhCBL3 involved in Ca ²⁺ regulation.

Further, the protein GhCBL may be a cotton disease resistance related protein GhCBL3, and specifically may be a cotton verticillium resistance related protein GhCBL3.

Further, the protein GhCBL may be cotton verticillium wilt resistance (Verticillium wilt) related protein GhCBL3.

In order to facilitate purification or detection of the protein of B1), a tag protein may be attached to the amino-or carboxy-terminus of the protein consisting of the amino acid sequence shown in SEQ ID No.1 of the sequence Listing.

Such tag proteins include, but are not limited to: GST (glutathione-sulfhydryl transferase) tag protein, his6 tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

The nucleotide sequence encoding protein GhCBL of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the protein GhCBL3 isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein GhCBL3 and have the function of the protein GhCBL.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.

Herein, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

Herein, the substance that regulates the activity and/or content of the protein may be a substance that regulates the expression of a gene encoding the protein GhCBL.

In the above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).

The substance regulating gene expression may specifically be a biological material as described in any one of E1) to B4) herein.

Further, the agent that modulates gene expression may be an agent (including a nucleic acid molecule or vector) that inhibits or reduces or down-regulates expression of the gene encoding the protein GhCBL.

Further, the agent that modulates gene expression may also be an agent (including a nucleic acid molecule or vector) that increases or upregulates expression of the gene encoding the protein GhCBL.

The invention also provides the use of a biological material associated with the protein GhCBL, which may be any of the following:

d1 Use of biological material related to said protein GhCBL in modulating plant disease resistance;

D2 Use of a biological material related to said protein GhCBL in the preparation of a product for modulating plant disease resistance;

d3 Use of biological material related to said protein GhCBL in the cultivation of disease-resistant plants;

d4 Use of a biological material related to said protein GhCBL for the preparation of a product for the cultivation of disease resistant plants;

D5 Use of biological material related to said protein GhCBL in plant breeding or plant germplasm resource improvement;

D6 The application of biological materials related to the protein GhCBL in prevention and control of cotton diseases;

The biomaterial may be any one of the following E1) to E8):

e1 A nucleic acid molecule encoding said protein GhCBL;

E2 A nucleic acid molecule that inhibits or reduces expression of a gene encoding the protein GhCBL;

e3 An expression cassette containing the nucleic acid molecule of E1) and/or E2);

E4 A recombinant vector comprising E1) and/or E2) said nucleic acid molecule, or a recombinant vector comprising E3) said expression cassette;

E5 A recombinant microorganism containing the nucleic acid molecule of E1) and/or E2), or a recombinant microorganism containing the expression cassette of E3), or a recombinant microorganism containing the recombinant vector of E4);

E6 A transgenic plant cell line containing E1) and/or E2) said nucleic acid molecule, or a transgenic plant cell line containing E3) said expression cassette, or a transgenic plant cell line containing B4) said recombinant vector;

E7 A) transgenic plant tissue containing E1) and/or E2) said nucleic acid molecule, or a transgenic plant tissue containing E3) said expression cassette;

e8 A transgenic plant organ comprising E1) and/or E2) said nucleic acid molecule, or a transgenic plant organ comprising E3) said expression cassette.

In the above application, the nucleic acid molecule may be any of the following:

F1 A DNA molecule with the coding sequence of SEQ ID No.2 or SEQ ID No. 3;

F2 A DNA molecule with the nucleotide sequence of SEQ ID No.2 or SEQ ID No. 3.

The DNA molecule shown in SEQ ID No.2 (cotton verticillium wilt resistance related gene GhCBL) encodes protein GhCBL whose amino acid sequence is SEQ ID No. 1.

The nucleotide sequence shown in SEQ ID NO.2 is the nucleotide sequence of the gene encoding protein GhCBL (CDS).

E1 The nucleic acid molecules may also comprise nucleic acid molecules which have been modified by codon preference on the basis of the nucleotide sequence indicated in SEQ ID No. 2.

E1 The nucleic acid molecules also include nucleic acid molecules which have more than 95% identity to the nucleotide sequence shown in SEQ ID No.2 and are of the same species.

The gene of protein GhCBL (GhCBL gene) according to the present invention may be any nucleotide sequence capable of encoding protein GhCBL. In view of the degeneracy of codons and the preferences of codons of different species, one skilled in the art can use codons appropriate for expression of a particular species as desired.

The expression cassette includes a promoter, which may be a CaMV35S promoter, a NOS promoter or an OCS promoter, a nucleic acid molecule encoding the protein GhCBL, and a terminator, which may be a NOS terminator or an OCS polyA terminator.

The nucleic acid molecule described herein may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be an RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or an antisense RNA.

Vectors described herein are well known to those of skill in the art and include, but are not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, or viral vectors. Specifically PYL156 vector and/or-Blunt Zero Cloning Vector。

The microorganism described herein may be a yeast, bacterium, algae or fungus. Wherein the bacteria may be derived from Escherichia, erwinia, agrobacterium (Agrobacterium), flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas, bacillus (Bacillus), etc. Specifically, the agrobacterium tumefaciens GV3101 and/or escherichia coli DH5 alpha can be used.

The recombinant vector can be specifically a recombinant vector pEASY-GhCBL3 and/or a recombinant vector PYL156-GhCBL3.

The recombinant vector pEASY-GhCBL (i.e. pEASY-GhCBL plasmid) is prepared by using cohesive end cloning method to connect DNA fragment whose nucleotide sequence is SEQ ID No.3 in sequence table to-Blunt Zero Cloning Vector on a carrier, holdThe other sequences of the Blunt Zero Cloning Vector vector are unchanged, and the recombinant vector is obtained.

The recombinant vector PYL156-GhCBL is a recombinant vector obtained by replacing a fragment (small fragment) between EcoRI and KpnI recognition sites of the PYL156 vector with a DNA fragment shown as SEQ ID No.3 in a sequence table, and keeping other nucleotide sequences of the PYL156 vector unchanged.

The recombinant microorganism can be obtained by introducing the recombinant vector into a starting microorganism.

The recombinant vector carrying GhCBL gene of the present invention may be used to transform plant cells or tissues by using Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium-mediated transformation, etc. conventional biological methods, and the transformed plant tissues are cultivated into plants.

The recombinant microorganism can be specifically recombinant Agrobacterium GV3101/PYL156-GhCBL3.

The recombinant Agrobacterium GV3101/PYL156-GhCBL3 is a recombinant bacterium obtained by introducing the recombinant vector PYL156-GhCBL3 into Agrobacterium tumefaciens GV 3101.

The present invention also provides a method of growing a disease resistant plant, which may include reducing the content and/or activity of the protein GhCBL in a plant of interest to obtain a disease resistant plant having a disease resistance higher than that of the plant of interest.

In the above method, the reduction of the content and/or activity of the protein GhCBL3 in the target plant can be achieved by reducing the expression level of the gene encoding the protein GhCBL3 in the target plant.

In the above method, the reducing the expression level of the gene encoding the protein GhCBL3 in the target plant may be reducing the expression level of the gene encoding the protein GhCBL3 in the target plant genome by using a gene mutation, a gene knockout, a gene editing or a gene knockdown technique.

In the above method, the reduction of the expression level of the gene encoding the protein GhCBL in the genome of the target plant by the gene knockdown technique may be performed using a virus-induced gene silencing vector constructed by forward integrating the nucleic acid molecule shown in SEQ ID No.3 into a tobacco brittle virus (Tobacco rattle virus, TRV) -based plasmid vector.

Further, the tobacco embrittlement virus (Tobacco rattle virus, TRV) -based plasmid vector may be a PYL156 vector.

The method for cultivating disease-resistant plants can comprise the following steps: inhibiting expression of a nucleic acid molecule capable of expressing GhCBL protein in a recipient plant (target plant) to obtain a transgenic plant; the transgenic plant has increased disease resistance as compared to the recipient plant. Wherein, the inhibition of expression of the nucleic acid molecule capable of expressing GhCBL protein in the recipient plant can be achieved by any technical means capable of achieving this objective.

The method for cultivating disease-resistant plants according to the present invention may comprise the steps of:

M1) inserting the nucleic acid molecule shown in SEQ ID No.3 into a plasmid vector containing a tobacco brittle virus sequence, and constructing a recombinant plasmid (namely a gene silencing vector);

m2) introducing the recombinant plasmid into a plant of interest (e.g., crop or cotton);

m3) screening and identifying to obtain the disease-resistant plant.

The introduction refers to transformation by recombinant means including, but not limited to, agrobacterium (Agrobacterium) -mediated transformation, biolistic (biolistic) methods, electroporation, or in planta technology.

Herein, the plant may be a crop (e.g., a crop).

In the above application and/or method, the plant may be any one of the following:

g1 Monocotyledonous or dicotyledonous plants;

g2 Malvaceae plants;

G3 Cotton plant;

G4 Cotton.

Further, the cotton may specifically be upland cotton (Gossypium hirsutum l.).

The protein GhCBL, and/or the biological material are also within the scope of the present invention.

The invention also provides application of the method for cultivating the disease-resistant plant in creating the disease-resistant plant and/or plant breeding and/or plant germplasm resource improvement.

Herein, the disease resistant plant may be a plant with increased disease resistance (upregulation).

In the present invention, the modulation may be up-regulation or down-regulation. The modulation may also be down-regulation or enhancement or elevation.

Modulating plant disease resistance as described herein may be increasing (up-regulating) plant disease resistance or decreasing (down-regulating) plant disease resistance.

Herein, the disease resistance may be verticillium wilt (Verticillium wilt) resistance.

In particular, modulating plant resistance as described herein may be modulating verticillium wilt resistance of a plant, including increasing (up-regulating) verticillium wilt resistance of a plant or decreasing (down-regulating) verticillium wilt resistance of a plant.

Any of the above verticillium wilt can be specifically verticillium wilt caused by verticillium dahliae (Verticillium dahliae).

The plant breeding described herein may be crop disease resistance breeding, and in particular, cotton verticillium wilt resistance breeding, with the goal of breeding cotton with increased verticillium wilt resistance.

The plant breeding described herein may be molecular breeding that utilizes the genes GhCBL and/or proteins GhCBL3 described herein to improve disease resistance in crops.

The cotton disease described herein may be a disease caused by the fungus verticillium dahliae (Verticillium dahliae), and in particular may be a verticillium wilt disease of cotton.

In this context, the term "disease-resistant plant" is understood to include not only the first generation transgenic plant obtained by silencing or knocking out the GhCBL gene, but also its progeny. The transgenic plants include seeds, calli, whole plants and cells.

The invention provides a cotton calcineurin B subunit protein CBL3 gene and its coding protein, which uses GhCBL gene sequence information to amplify the gene and constructs VIGS plant expression vector to transform upland cotton TM-1, the obtained transgenic cotton is further subjected to disease resistance identification, after the cotton verticillium wilt V991 is inoculated, the incidence and disease index of the transgenic cotton are obviously reduced, the resistance to verticillium wilt is shown, the plant disease resistance after GhCBL gene silencing is enhanced, the GhCBL gene participates in verticillium wilt-induced anaphylactic reaction, and GhCBL gene and its coding protein participate in verticillium wilt resistance mechanism of cotton.

In conclusion, inhibiting (down-regulating) GhCBL gene expression in plants (e.g., cotton) significantly increases plant disease resistance (e.g., increases verticillium wilt resistance of cotton). The GhCBL protein and the coding gene GhCBL thereof can regulate and control the disease resistance (such as verticillium wilt resistance) of plants, and can obviously improve the disease resistance of target plants by reducing the content and/or activity (such as inhibiting, silencing or interfering GhCBL gene expression) of GhCBL protein in the target plants. Therefore, the cotton verticillium wilt-resistant related protein GhCBL and the coding gene thereof have important theoretical significance and practical value in regulating and controlling plant disease resistance, and have important significance in cultivating verticillium wilt-resistant transgenic cotton.

Drawings

FIG. 1 shows the detection and disease resistance identification of the VIGS genetic transformation system in example 2. Wherein FIG. 1A and FIG. 1B (control, TRV: 00) are phenotypes of successful establishment of a TRV-mediated VIGS system in upland cotton TM-1; FIG. 1C shows the growth of cotton plants inoculated with verticillium V991 days after two weeks of VIGS infection compared to control (TRV: 00);

FIG. 2 shows the expression level of the gene GhCBL by fluorescent quantitative Real time-PCR.

FIG. 3 is a statistical analysis of the morbidity and disease index of 16d and 21d after inoculating Verticillium dahliae strain V991.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The following examples treat data using GRAPHPAD PRISM statistical software, and experimental results are expressed as mean ± standard deviation, using T-test method, P < 0.05 (x) indicates that there is a statistical difference, P < 0.01 (x) indicates that the statistical difference is significant, and P < 0.001 (x) indicates that the statistical difference is very significant. The quantitative tests in the following examples were carried out in three replicates, and the results were averaged unless otherwise indicated.

The upland cotton genetic Standard line TM-1 (short upland cotton TM-1) in the following examples is given to cotton institute of national academy of agricultural sciences, and is described in the following documents: cultivation of the 18 th chromosome fragment substitution line of sea island cotton in the background of upland cotton and related agronomic trait QTL location [ J ]. Crop theory, 2013,39 (1): 21-28., the public can obtain the biological material from the applicant, and the biological material is only used for repeated experiments of the invention and can not be used for other purposes.

The plant VIGS expression vector PYL156 in the following examples was given to the Baogong teacher at the university of tokyo agriculture as a TRV-mediated gene silencing vector, described in the following literature: yang Xiaomin, wang Junjuan, wang Delong, et al functional verification of cotton GhDMT3 and bioinformatics analysis [ J ]. Biotechnology bulletins 2019,35 (1): 11-16. The biological material is available to the public from applicant and is only used for repeated experiments of the invention and is not used for other purposes.

Agrobacterium tumefaciens GV3101 in the examples described below was purchased from Urufimbria high technology development district Rui Da market.

Verticillium dahliae (Verticillium dahliae) V991 in the examples below is described in the following documents: zhang Xin, jian Guiliang, lin Ling, et al molecular detection methods for verticillium dahliae in soil [ J ]. Jiangsu agricultural journal, 2011,27 (5): 990-995. The biological material is available to the public from applicant and is only used in repeated experiments of the present invention, but not as other uses.

Example 1, acquisition of Cotton disease resistance-associated protein GhCBL3 and Gene encoding the same

Through extensive and intensive studies, the inventor of the application discovers a DNA coding sequence through analyzing a cotton genome sequence, the nucleotide sequence of the DNA coding sequence is shown as SEQ ID No.2 of a sequence table, and the amino acid sequence of the coded protein is shown as SEQ ID No.1 of the sequence table.

The nucleotide sequence shown in SEQ ID No.2 is named GhCBL gene, the encoded protein is named GhCBL protein, and the amino acid sequence is shown in SEQ ID No. 1.

GhCBL3 protein is cotton calmodulin B subunit protein GhCBL.

Example 2, cotton disease resistance-associated protein GhCBL3 and use of coding Gene thereof

1. Acquisition of GhCBL Gene fragment containing the cleavage site

Extracting total RNA of cotton (upland cotton genetic standard line TM-1), reversely transcribing the total RNA into cDNA, and carrying out PCR amplification by using the cDNA as a template and adopting a GhCBL F/R primer, namely a primer pair consisting of a primer GhCBL F and a primer GhCBL R, so as to obtain a PCR amplification product. Wherein the primer sequences are as follows:

Primer GhCBL F:5'-CGGAATTCTAGGTTTCGAAGAGTTTGCTC-3';

primer GhCBL R:5'-GGGGTACCTCAGGTATCATCAACTTGAGAGT-3'.

In primer GhCBL F and primer GhCBL R, restriction enzyme EcoRI and KpnI sequences are respectively underlined, so that subsequent connection of plant expression vector PYL156 for genetic transformation of cotton is facilitated.

The PCR amplified product (393bp,SEQ ID No.3) was a GhCBL gene fragment containing a restriction enzyme site, i.e., a restriction enzyme site in which EcoRI was added at 305bp and KpnI restriction enzyme was added at 681bp of the GhCBL gene shown in SEQ ID No.2 (681 bp). The PCR amplified product (393bp,SEQ ID No.3) was detected by 1% agarose gel electrophoresis and recovered for purification.

2. Construction of virus-induced Gene silencing (VIGS) recombinant expression vector PYL156-GhCBL3

Mixing the PCR amplified product (393bp,SEQ ID No.3) obtained in step 1 with-Blunt Zero Cloning Vector(The composition of the components in-Blunt Zero Cloning Kit,Blunt Zero Cloning Kit is a product of full-scale gold biotechnology Co., ltd., product of product number CB 501-01), the ligation product is transformed into E.coli DH 5. Alpha. And positive transformants identified by PCR using GhCBL F/R primers are sequenced. The colony with correct sequence is preserved, and the plasmid is extracted, so that the plasmid with correct sequence is named pEASY-GhCBL. The pEASY-GhCBL plasmid contains a DNA fragment whose nucleotide sequence is SEQ ID No. 3.

The recombinant vector pEASY-GhCBL (i.e., pEASY-GhCBL plasmid) is a method of using cohesive end cloning to ligate the DNA fragment whose nucleotide sequence is SEQ ID No.3 of the sequence Listing to-Blunt Zero Cloning Vector on a carrier, holdThe other sequences of the Blunt Zero Cloning Vector vector are unchanged, and the recombinant vector is obtained.

The plant VIGS expression vector PYL156 (TRV mediated gene silencing vector) was digested with restriction enzymes EcoRI and KpnI, and the digested backbone vector was recovered.

The plasmid pEASY-GhCBL3 obtained above was digested with restriction enzymes EcoRI and KpnI, and the digested DNA fragment 1 (small fragment) was recovered.

And (3) connecting the DNA fragment 1 with the backbone vector after enzyme digestion to obtain a virus-induced gene silencing (VIGS) recombinant expression vector PYL 156-GhCBL.

The recombinant vector PYL156-GhCBL is obtained by replacing a fragment (small fragment) between EcoRI and KpnI recognition sites of the PYL156 vector with a DNA fragment shown as a forward insertion SEQ ID No.3 in a sequence table, and keeping other nucleotide sequences of the PYL156 vector unchanged.

3. Acquisition of recombinant Agrobacterium

Transforming the plant expression vector recombinant plasmid PYL156-GhCBL3 obtained in the step 2 into agrobacterium tumefaciens GV3101, picking a monoclonal and carrying out colony PCR screening identification on the recombinant plasmid PYL156-GhCBL3 by using GhCBL F/R primers, and confirming that the agrobacterium positive clone (namely the recombinant agrobacterium transformed into PYL 156-GhCBL) is obtained, and is named GV3101/PYL156-GhCBL3.

4. Acquisition of transgenic cotton

4-1, The obtained recombinant Agrobacterium GV3101/PYL156-GhCBL was streaked on LB solid medium containing kanamycin (50 ug/ml) and gentamicin (20 ug/ml) resistance, cultured at 28℃for 2 days.

4-2, Picking the monoclonal obtained in the step 4-1, inoculating the monoclonal into 5ml of LB liquid medium containing kanamycin (50 ug/ml) and gentamicin (20 ug/ml) resistance, and culturing for 24 hours at 28 ℃ for 180 min ^-1; transferring into 50mL LB liquid medium, culturing at 28deg.C for ^-1 min at 180 min, centrifuging at 4000 min ^-1 for 5min to collect bacterial cells, resuspending with appropriate volume of heavy suspension (10 mmol L ^– ¹MgCl₂,10mmol L^–1 - (N-morpholino) ethanesulfonic acid (MES) and 200 μmol L ^–1 acetosyringone), and adjusting OD _600nm value to 1.5; standing the heavy suspension at room temperature for more than 3 hours to obtain recombinant agrobacterium transformation suspension (namely GV3101/PYL156-GhCBL bacterial liquid).

The GhCLA gene (GenBank accession number KJ 123647) was cloned from the upland cotton genetic standard line TM-1. Amplifying a 994-1414 bp gene fragment positioned in the middle of the cotton GhCLA gene ORF, wherein the target amplified fragment length is 421bp. The amplified CLA1 gene fragment is connected with a PYL156 vector to construct a recombinant vector PYL156-CLA1. And transforming the recombinant vector PYL156-CLA1 into agrobacterium tumefaciens GV3101 to obtain GV3101/PYL156-CLA1 bacterial liquid.

PTRV1 bacterial liquid is a bacterial liquid which is compounded with GV3101/PYL156-GhCBL bacterial liquid to complete gene silencing, and is described in the following literature: the establishment and application of TRV virus mediated gene silencing system in cotton, wang Xinyu, lv Kun, cai Caiping, xu Jun, guo Wangzhen, etc., [ J ]. Crop theory, 2014,40 (8): 1356-1363.

GV3101/PYL156-CLA1 bacterial liquid is positive control bacterial liquid.

GV3101/PYL156 bacterial liquid is negative control bacterial liquid, namely PYL156 carrier transformed PYL156 bacterial liquid of the transfer PYL-156 empty carrier obtained by transforming the agrobacterium tumefaciens GV 3101.

The experiment set up was as follows:

1. Uniformly mixing pTRV1 bacterial liquid and GV3101/PYL156-GhCBL bacterial liquid according to a volume ratio of 1:1, injecting cotton cotyledon, and obtaining a gene silencing transformant, which is marked as TRV: ghCBL3.

2. Uniformly mixing pTRV1 bacterial liquid and GV3101/PYL156-CLA1 bacterial liquid according to a volume ratio of 1:1, injecting cotton cotyledon for detecting whether a gene silencing system is correct, and marking as TRV: ghCLA 1A 1.

3. Uniformly mixing pTRV1 bacterial liquid and GV3101/PYL156 bacterial liquid according to a volume ratio of 1:1, injecting cotton cotyledon, and using the mixture as a genetic transformation control strain, and marking the genetic transformation control strain as TRV:00.

4-3, Sowing cotton seeds of upland cotton TM-1 into nutrient soil, and culturing at 26-28 ℃ in 12h of light/12 h of darkness. The humidity is kept at 60% and above, water is poured once for 4-5d, and the two cotyledons can be used for VIGS operation when the two cotyledons are flat and the true cotyledons are not developed yet.

4-4, Infection by VIGS injection: the back of cotyledon is first pricked with the needle to produce micro wound, and the re-suspension in 1 to 1 ratio is injected into the wound with the needle eliminated to obtain cotton GhCBL gene silencing transformant. After 2 weeks the phenotype of the different treated cotton was observed and the expression of the gene of interest was examined. For each material 30 individual plants were treated.

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

5. Detection of VIGS genetic transformation System

5-1, Carrying out VIGS system detection by adopting upland cotton CLA 1gene (cloroplastos alterados 1 gene), namely GhCLA 1gene as a marker gene. The gene participates in chloroplast development process, codes 1-deoxyxylulose 5-phosphate synthase (1-deoxyxylulose 5-phosphate synthase, DXS) protein, is highly conserved in evolution, and GhCLA 1gene silenced cotton plants have obvious albino phenotype and are marked characters easy to identify, and the result is shown in figure 1A. 2 weeks after infection with VIGS injection, TRV: the true leaves of the plants of GhCLA treatment group were almost completely whitened, while the leaves of the control (TRV: 00 treatment group) were unchanged (B in FIG. 1). Illustrating the successful establishment of the TRV-mediated VIGS system in upland cotton TM-1.

5-2, Fluorescent quantitative Real time-PCR detection

Total RNA from leaves of cotton plants after 2 weeks of infection with VIGS was extracted using Plant Total RNA Extraction Kit kit. The expression of the silenced GhCBL gene was detected by fluorescent quantitative Real time-PCR using cotton UBQ7 as a reference gene, and the results are shown in FIG. 2. Compared with empty vector (TRV: 00) control, the expression level of GhCBL3 gene is obviously reduced and the silencing effect is obvious in 1 GhCBL gene VIGS infected plants selected randomly.

The apparatus used for fluorescence quantitative PCR was Applied Biosystems StepOne (Applied Biosystems, U.S.) and the reagents used were SYBR Premix Ex Taq ^TM kit (TRANSGENE, beijing). The reverse transcribed cDNA was used as a template, starting at 150ng, 3 replicates per treatment. The reaction procedure is: denaturation (95 ℃,30 s); (95 ℃,5s;58 ℃,15s;72 ℃,31 s) for 40 cycles; dissolution (95 ℃,15s;60 ℃,1min;95 ℃,15 s). The sequence of the fluorescent quantitative Real time-PCR detection primer is as follows:

primer GhUBQ-RT-F: 5'-GAAGGCATTCCACCTGACCAAC-3';

Primer GhUBQ-RT-R: 5'-CTTGACCTTCTTCTTCTTGTGCTTG-3'.

Primer GhCBL-RT-F: 5'-TACGATCTCAAGCAGCAAGGTT-3';

Primer GhCBL-RT-R: 5'-ATGTCGCAAAACAAGGCTTCTC-3'.

6. Inoculation for resisting verticillium wilt and disease resistance identification of cotton

The identification of resistance by artificial inoculation of verticillium dahliae (Verticillium dahliae) V991 (also referred to herein as verticillium dahliae V991 or verticillium strain V991 or V991) is performed as follows:

The preserved verticillium strain V991 was activated on PDA medium. The thalli are selected in Czapek's culture solution, and are cultured for 3-5 d at 25 ℃ and 200 r/min. Filtering the pathogenic bacteria culture solution with 4 layers of gauze, counting the concentration of pathogenic bacteria by using a blood cell counting plate, regulating the final concentration of pathogenic bacteria to 1.0X10 ⁷ spores/mL by using sterilized double distilled water, and adding Tween-20 to the final concentration of 0.001% (volume percent) to obtain verticillium wilt bacteria V991 spore liquid. And (3) inoculating pathogenic bacteria V991 spore liquid to the obtained transformant after 2 weeks of VIGS gene silencing by a root dipping method, and checking and counting verticillium wilt morbidity after 12d inoculation, and counting disease indexes by a 0-4-level method. For each material 30 individual plants were treated. Let 3 biological replicates.

Statistical index of disease reference: xu Li, zhu Longfu, zhang Xianlong, progress of research on verticillium wilt resistance mechanism of cotton, crop theory report ,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 disease plants at each stage×corresponding disease grade)/survey total number of plants×highest disease grade (4) ]100

TRV after 2 weeks of infection with VIGS: ghCBL3 control of treated silenced plants and empty vector (TRV: 00) plants, verticillium dahliae strain V991, 12d was inoculated and the onset of verticillium was shown in FIG. 1C. The disease resistance identification results of 16d and 21d after inoculating verticillium dahliae V991 by 3 biological repeated observation statistical analysis are shown in FIG. 3. The incidence of empty vector-injected control plants (transgenic plants treated with TRV: 00) was 50.55, the average disease index was 34.55 after 16d inoculation, 72.05 after 21d inoculation, 67.22, and GhCBL3 gene-silenced plants (transgenic plants treated with TRV: ghCBL 3), 36.54 after 16d inoculation, 22.74, 49.05 after 21d inoculation, and 39.97.

The results show that compared with the control, the incidence rate and the disease index of the plant with GhCBL gene silencing are obviously reduced, and the disease resistance of the plant with GhCBL gene silencing is enhanced. The GhCBL gene is shown to be involved in verticillium-induced allergic reactions.

The experimental results fully prove that the expression quantity of GhCBL genes is obviously reduced after VIGS infection, and the disease resistance of plants after GhCBL genes are silenced is enhanced. Experimental results show that inhibiting (down-regulating) GhCBL gene expression in plants (e.g., cotton) can significantly improve plant disease resistance (e.g., improve verticillium wilt resistance of cotton). The GhCBL protein and the coding gene GhCBL thereof can regulate and control the disease resistance (such as verticillium wilt resistance) of plants, and can obviously improve the disease resistance of target plants by reducing the content and/or activity (such as inhibiting, silencing or interfering GhCBL gene expression) of GhCBL protein in the target plants.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. Application for reducing protein content and/or activity, characterized in that the application is any of the following:

A1) Application in improving the resistance of upland cotton to Verticillium wilt;

A2) Application in the preparation of products for improving the resistance of upland cotton to Verticillium wilt;

A3) Application in breeding upland cotton resistant to Verticillium wilt;

A4) Use in the preparation of products for breeding upland cotton resistant to Verticillium wilt;

A5) Application in breeding of upland cotton for resistance to Verticillium wilt or improving upland cotton germplasm resources for resistance to Verticillium wilt;

A6) Application in the prevention and control of Verticillium wilt disease in upland cotton;

The protein is any of the following:

B1) a protein whose amino acid sequence is SEQ ID No. 1;

B2) A fusion protein with the same function as B1) is obtained by connecting a tag to the N-terminus and/or C-terminus.

2. The application of the biomaterial related to the protein according to claim 1, characterized in that the application is any of the following:

D1) Application in improving the resistance of upland cotton to Verticillium wilt;

D2) Application in the preparation of products that improve the resistance of upland cotton to Verticillium wilt;

D3) Application in breeding upland cotton resistant to Verticillium wilt;

D4) Use in the preparation of products for breeding upland cotton resistant to Verticillium wilt;

D5) Application in breeding of upland cotton for resistance to Verticillium wilt or improving upland cotton germplasm resources for resistance to Verticillium wilt;

D6) Application in the prevention and control of Verticillium wilt disease in upland cotton;

The biological material is any one of the following E1) to E7):

E1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein of claim 1;

E2) an expression cassette containing the nucleic acid molecule described in E1);

E3) a recombinant vector containing the nucleic acid molecule described in E1), or a recombinant vector containing the expression cassette described in E2);

E4) a recombinant microorganism containing the nucleic acid molecule described in E1), or a recombinant microorganism containing the expression cassette described in E2), or a recombinant microorganism containing the recombinant vector described in E3);

E5) a transgenic plant cell line containing the nucleic acid molecule described in E1), or a transgenic plant cell line containing the expression cassette described in E2), or a transgenic plant cell line containing the recombinant vector described in E3);

E6) transgenic plant tissue containing the nucleic acid molecule described in E1), or transgenic plant tissue containing the expression cassette described in E2);

E7) A transgenic plant organ containing the nucleic acid molecule described in E1) or a transgenic plant organ containing the expression cassette described in E2).

3. The use according to claim 2, characterized in that the nucleotide sequence encoding the protein is shown as SEQ ID No. 2.

4. A method for cultivating plants resistant to Verticillium wilt, characterized in that the method comprises reducing the content and/or activity of the protein described in claim 1 in a target plant to obtain a disease-resistant plant having higher Verticillium wilt resistance than the target plant, wherein the plant is upland cotton.

5. The method according to claim 4, characterized in that the reduction of the content and/or activity of the protein according to claim 1 in the target plant is achieved by reducing the expression level of the gene encoding the protein in the target plant.

6. The method according to claim 5 is characterized in that reducing the expression level of the gene encoding the protein in the target plant is to reduce the expression level of the gene encoding the protein in claim 1 in the genome of the target plant by using gene mutation, gene knockout, gene editing or gene knockdown technology.

7. The method according to claim 6 is characterized in that the gene knockdown technology is used to reduce the expression level of the gene encoding the protein according to claim 1 in the genome of the target plant by using a virus-induced gene silencing vector, and the virus-induced gene silencing vector is a recombinant vector constructed by forward integration of the nucleic acid molecule shown in SEQ ID No. 3 into a plasmid vector based on tobacco rattle virus.