CN117586985A

CN117586985A - Sea island cotton fusarium wilt resistance protein GbPP2C80, and coding gene and application thereof

Info

Publication number: CN117586985A
Application number: CN202311536933.8A
Authority: CN
Inventors: 华金平; 赵楠; 王为然; 王萌; 阿里甫·艾尔西; 孔杰; 刘志清
Original assignee: Cash Crop Research Institute Of Xinjiang Academy Of Agricultural Sciences Xinjiang Uygur Autonomous Region Sugar Beet Improvement Center Of Xinjiang Uygur Autonomous Region Cotton Research Institute; China Agricultural University
Current assignee: Cash Crop Research Institute Of Xinjiang Academy Of Agricultural Sciences Xinjiang Uygur Autonomous Region Sugar Beet Improvement Center Of Xinjiang Uygur Autonomous Region Cotton Research Institute; China Agricultural University
Priority date: 2023-11-17
Filing date: 2023-11-17
Publication date: 2024-02-23

Abstract

The invention discloses a sea island cotton fusarium wilt resistance protein GbPP2C80, and a coding gene and application thereof. The invention relates to the field of botanic, in particular to island cotton fusarium wilt resistance protein GbPP2C80 and a coding gene and application thereof. The protein of the present invention is any one of the following: a1 The amino acid sequence is shown as SEQ ID No. 3; a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the protein of A1), has more than 80 percent of identity with the protein shown in A1) and has the function of regulating and controlling plant fusarium wilt resistance; a3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2). The disease-sensitive island cotton plants with GbPP2C80 silencing by the VIGS technology has obviously reduced disease incidence rate, and the GbPP2C80 protein can negatively regulate and control the disease resistance of island cotton, thereby having wide application prospect.

Description

Sea island cotton fusarium wilt resistance protein GbPP2C80, and coding gene and application thereof

Technical Field

The invention relates to the field of botanic, in particular to island cotton fusarium wilt resistance protein GbPP2C80 and a coding gene and application thereof.

Background

Fusarium wilt is a vascular bundle disease caused by fusarium oxysporum, and seriously affects cotton production. In China, the physiological race No. 7 of fusarium wilt is most widely distributed and has the highest virulence. In the cotton area with wilt, the leaves and bolls drop out in large quantity, the yield is reduced by 10% when the cotton is light, the yield is reduced by 30% -50% when the cotton is heavy, and the quality of the cotton is obviously reduced. Island cotton is susceptible to fusarium wilt, but the island cotton fusarium wilt resistance genes identified and cloned at present are few. The important island cotton fusarium wilt resistance genes are mined and molecular mechanisms of the island cotton fusarium wilt resistance are analyzed, so that the method has important theoretical guiding significance and practical application value.

Protein phosphatase 2C (PP 2C) is a monomeric serine/threonine residue protein phosphatase capable of dephosphorylating proteins. Arabidopsis thaliana protein phosphatases PLL4 and PLL5 and tobacco protein phosphatase Pic1 are negative regulators of pattern-triggered immunity. However, there is no report on the regulation of the cotton fusarium wilt resistance of islands in the sea by the PP2C protein.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate and control the wilt resistance of plants.

In order to solve the problems existing in the prior art, the invention provides a protein.

The protein provided by the invention can be any one of the following proteins:

a1 Protein with the amino acid sequence shown as SEQ ID No. 3;

a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the protein of A1), has more than 80 percent of identity with the protein shown in A1) and has the function of regulating and controlling plant fusarium wilt resistance; for example, according to the amino acid sequence shown as SEQ ID No.3 and the conventional technical means in the art such as conservative substitution of amino acid, one or more amino acids can be substituted, deleted and/or added by a person skilled in the art on the premise of not affecting the activity of the protein mutant, so that the protein mutant with the same function as the amino acid sequence shown as SEQ ID No.3 is obtained;

A3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2).

The protein described in A1) above is named GbPP2C80.

In order to facilitate purification or detection of the protein of A1), 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.3 of the sequence Listing.

The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.

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), eFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

The mutation of the nucleotide sequence encoding the protein GbPP2C80 according to the invention can be easily carried out by a person skilled 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 GbPP2C80 isolated according to the invention are all nucleotide sequences derived from the invention and are equivalent to the sequences of the invention, as long as they encode the protein GbPP2C80 and have the function of the protein GbPP2C80.

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, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

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 90% identity or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In the above, the protein is derived from gossypium barbadense (gossypium barbadense l.).

The present invention also provides a biological material related to the above protein, which may be any one of the following:

b1 A nucleic acid molecule encoding a protein as described above;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2);

c1 A nucleic acid molecule that inhibits or reduces or silences the expression of a gene encoding a protein as described above;

c2 Expression of the gene encoding the nucleic acid molecule of C1);

c3 An expression cassette containing the coding gene of C2);

c4 A recombinant vector comprising the coding gene of C2) or a recombinant vector comprising the expression cassette of C3);

C5 A recombinant microorganism containing the gene encoding C2), or a recombinant microorganism containing the expression cassette of C3), or a recombinant microorganism containing the recombinant vector of C4);

c6 A transgenic plant cell line containing the coding gene of C2), or a transgenic plant cell line containing the expression cassette of C3), or a transgenic plant cell line containing the recombinant vector of C4);

c7 A transgenic plant tissue containing C2) said coding gene, or a transgenic plant tissue containing C3) said expression cassette, or a transgenic plant tissue containing C4) said recombinant vector;

c8 A transgenic plant organ containing the coding gene of C2), or a transgenic plant organ containing the expression cassette of C3), or a transgenic plant organ containing the recombinant vector of C4).

In the above biological material, the nucleic acid molecule of B1) is a gene represented by E1) or E2) as follows:

e1 A cDNA molecule or a DNA molecule having a coding sequence of SEQ ID No. 1;

e2 Nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No. 2.

The DNA molecule shown in SEQ ID No.1 (GbPP 2C80 gene for regulating plant disease resistance) encodes a protein whose amino acid sequence is SEQ ID No. 3.

The nucleotide sequence shown in SEQ ID No.1 is the nucleotide sequence of the gene encoding the protein GbPP2C80 (CDS).

The nucleotide sequence shown in SEQ ID No.2 is the nucleotide sequence of the genome of the protein GbPP2C 80.

The GbPP2C80 gene of the invention can be any nucleotide sequence capable of encoding protein GbPP2C 80. 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.

B1 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. 1.

B1 The nucleic acid molecule may also include a nucleic acid molecule having a nucleotide sequence identity of 95% or more with the nucleotide sequence shown in SEQ ID No.1 and being of the same species.

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 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, the vectors pCLCrVA and p35S are GFP vectors.

Recombinant expression vectors containing the GbPP2C80 gene can be constructed by using existing plant expression vectors. Such plant expression vectors include, but are not limited to, vectors such as binary Agrobacterium vectors and vectors useful for microprojectile bombardment of plants, and the like. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to untranslated regions transcribed from the 3' end of plant genes including, but not limited to, agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase Nos genes), plant genes (e.g., soybean storage protein genes).

When the GbPP2C80 gene is used for constructing a recombinant plant expression vector, any one of an enhanced promoter or a constitutive promoter can be added before the transcription initiation nucleotide, including, but not limited to, a cauliflower mosaic virus (CAMV) 35S promoter, a ubiquitin promoter (ubiquitin) of corn, which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, such as by adding genes encoding enzymes or luminescent compounds that produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

The GbPP2C80 gene or the fragment of the gene provided by the invention is introduced into plant cells or receptor plants by using any vector capable of guiding the expression of exogenous genes in plants, so that transgenic cell lines and transgenic plants with altered resistance to plant blight can be obtained. Expression vectors carrying the GbPP2C80 gene can be used to transform plant cells or tissues by conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, and the like, and the transformed plant tissues are cultivated into plants.

As a specific example, the recombinant vector is the recombinant vector pCLCrVA-GbPP2C80. The recombinant vector pCLCrVA-GbPP2C80 is obtained by replacing a fragment between the recognition sites of the SpeI and Pac I sequences of a pCLCrVA vector (a starting vector) with a DNA molecule shown as SEQ ID No.4 and keeping other nucleotides of the pCLCrVA vector (the starting vector) unchanged.

As a specific example, the recombinant vector is recombinant vector p35S, gbPP2C80-GFP.

The recombinant vector p35S is GbPP2C80-GFP is a recombinant expression vector obtained by replacing a small fragment between recognition sequences of restriction enzymes Kpn I and Xba I of plasmid p35S and GFP with a DNA molecule shown in a sequence 1 in a sequence table (removing a stop codon TGA) and keeping other sequences of the p35S and GFP vector unchanged.

The recombinant plasmid p35S is GbPP2C80 protein shown in a sequence 3 in a GbPP2C80-GFP expression sequence table. The recombinant plasmid p35S is an expression cassette containing GbPP2C80-GFP fusion protein, wherein the promoter for promoting the transcription of GbPP2C80 gene in the expression cassette is a 35S promoter.

The recombinant microorganism can be specifically recombinant agrobacterium GV3101/GbPP2C80-GFP.

The recombinant agrobacterium GV3101/GbPP2C80-GFP is obtained by introducing the recombinant vector p35S, gbPP2C80-GFP into the agrobacterium tumefaciens GV3101.

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, agrobacterium tumefaciens GV3101 can be used.

The invention also provides the use of the protein GbPP2C80 or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in any of the following:

u1) the use of the protein or the substance regulating the expression of a gene or the substance regulating the activity or the content of the protein in regulating the resistance of plants to blight;

U2) the use of the protein or the substance regulating the expression of a gene or the substance regulating the activity or the content of the protein in the preparation of a product regulating the resistance to plant blight;

u3) the use of the protein or of the substance regulating the expression of a gene or of the substance regulating the activity or the content of the protein in the cultivation of plants resistant to plant blight;

u4) the use of the protein or of a substance regulating the expression of a gene or of a substance regulating the activity or the content of said protein for the preparation of a product for the cultivation of plants resistant to blight;

u5) the use of the protein or the substance regulating the expression of a gene or the substance regulating the activity or the content of the protein in plant breeding.

Herein, the substance regulating the activity and/or content of the protein may be a substance regulating the expression of a gene encoding the protein GbPP2C80.

In the above application, the substance regulating the expression of the gene or the substance regulating the activity or content of the protein is a biological material related to the protein, and the biological material may be any one of the following B1) to B7):

b1 A nucleic acid molecule encoding a protein as described above;

b2 An expression cassette comprising the nucleic acid molecule of B1);

c2 Expression of the gene encoding the nucleic acid molecule of C1);

c3 An expression cassette containing the coding gene of C2);

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 invention also provides a method for regulating and controlling plant fusarium wilt resistance, which comprises regulating and controlling the activity and/or content of the protein in target plants or/and the expression level of the encoding gene of the protein to regulate and control the plant fusarium wilt resistance.

In the method, the regulation of the activity and/or content of the protein GbPP2C80 in the target plant, or/and the expression level of the encoding gene of the protein comprises introducing the encoding gene GbPP2C80 of the protein into a receptor plant to obtain the target plant with altered plant blight resistance; the GbPP2C80 encoding gene encodes the protein GbPP2C80.

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

In the above applications and methods, the modulation may be enhancement, enhancement or upregulation.

In the above applications and methods, the modulation may be inhibition, reduction or silencing.

To facilitate identification and selection of transgenic cells or plants, the recombinant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color response, antibiotic markers or chemical resistance markers which are expressed in plants, etc. The transformed plants can also be screened directly in adversity without adding any selectable marker gene. The plants obtained by the above method may be transgenic plants, or plants obtained by conventional breeding techniques such as crossing. In the above methods, the transgenic plants are understood to include not only first to second generation transgenic plants but also their progeny. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.

The present invention also provides a method of growing plants with altered resistance to plant blight, comprising: 1) Inhibiting or reducing or silencing the expression level of the gene encoding the protein described above in the plant of interest, or/and inhibiting or reducing or silencing the activity and/or content of the gene encoding the protein described above, to obtain a plant with increased wilt; 2) Increasing, enhancing and/or upregulating the expression of a gene encoding a protein as defined above in a plant of interest, or/and increasing, enhancing and/or upregulating the activity and/or content of a gene encoding a protein as defined above, to obtain a plant with reduced resistance to wilt.

As an embodiment of the present invention, the method for growing a plant resistant to blight comprises the steps of:

(1) Constructing a recombinant expression vector for inhibiting or reducing or silencing the coding gene of the protein (the amino acid sequence of the protein is the sequence 3);

(2) Introducing the expression vector constructed in the step (1) into a plant;

(3) And screening and identifying to obtain the wilt-resistant plant.

In a specific embodiment, a method of growing plants with increased resistance to wilt comprises the steps of: suppression of expression of a nucleic acid molecule encoding the GbPP2C80 protein in the plant of interest results in a transgenic plant with increased resistance to wilt. The inhibition of the expression of the nucleic acid molecule encoding the GbPP2C80 protein in the target plant can be achieved specifically by introducing an interference vector targeting the nucleic acid molecule encoding the GbPP2C80 protein into the target plant. The inhibition of the expression of the nucleic acid molecule encoding the GbPP2C80 protein in the target plant can be specifically realized by introducing a gene silencing vector targeting the nucleic acid molecule encoding the GbPP2C80 protein into the target plant.

The gene silencing vector may be recombinant vector pCLCrVA-GbPP2C80. The recombinant vector pCLCrVA-GbPP2C80 is obtained by replacing a fragment between the recognition sites of the SpeI and Pac I sequences of a pCLCrVA vector (a starting vector) with a DNA molecule shown as SEQ ID No.4 and keeping other nucleotides of the pCLCrVA vector (the starting vector) unchanged.

The invention also provides a method for cultivating plants with improved resistance to wilt, comprising the following steps: inhibiting or reducing or silencing the expression level of a gene encoding a protein as described above in a plant of interest, or/and inhibiting or reducing or silencing the activity and/or content of a gene encoding a protein as described above, to obtain a plant with increased resistance to wilt.

In the present invention, the object of plant breeding includes growing plants that are resistant to wilt.

In the above application or method, the plant is any one of the following:

n1) dicotyledonous plants;

n2) plants of order malvaceae;

n3) malvaceae plants;

n4) cotton plants;

n5) cotton.

In the above, the cotton may be gossypium barbadense L.A. of the susceptible variety II15-3465.

The invention locates the genetic locus of sea island cotton for resisting fusarium wilt on the 1159232 th position of D03 chromosome based on the identification of the incidence rate of fusarium wilt of natural population of sea island cotton and the whole genome association analysis, the mutation is located 1.9kb upstream of gene GbPP2C80, and GbPP2C80 contains a nonsensical mutation T/C (Gbar_D03_ 1162571) which is obviously related to the incidence rate of sea island cotton. The gene GbPP2C80 codes an IIC type protein phosphatase, the expression quantity in island cotton which is easy to be infected by the fusarium wilt is obviously higher than that in sea island cotton which is resistant to the fusarium wilt, and the resistance to the fusarium wilt is obviously enhanced after the GbPP2C80 is silenced in the island cotton which is infected by the virus-induced gene silencing and VIGS technology. GbPP2C80 is mainly located on the cell membrane and interacts with a sea island cotton anti-wilt protein GbWAKL14 on the cell membrane. GbPP2C80 is used as a novel island cotton fusarium wilt resistance gene and has certain theoretical and application values.

Drawings

Fig. 1 is a correlation heat map of the incidence of wilt of island cotton groups investigated in the Xinjiang nursery of China in 2015, 2016, 2018 and 2019.

Fig. 2 is a partial Manhattan plot and LD heatmap of the incidence of wilt disease in a sea island cotton population material. a is a local Manhattan diagram of the incidence rate of the fusarium wilt of the island cotton group material, and the arrows respectively mark the localized extremely significant associated SNP (Gbar_D03_ 1159232) and the non-synonymous SNP (Gbar_D03_ 1162571) inside the associated gene; b is a local LD heatmap around a significantly non-synonymous SNP within a candidate gene.

FIG. 3 shows the location of non-synonymous mutations in the GbPP2C80 gene. Blue and yellow rectangles represent untranslated region (UTR) and coding region (CDS), respectively, and black lines represent introns (Intron). Ref TTG (L) represents a reference codon TTG which codes leucine (abbreviated as L), and a key mutation site is T at a second position in the middle; alt TCG (S) means that the variant codon TCG encodes serine (abbreviated S), the key mutation site is the C in the second position in the middle, i.e.mutation from thymine (T) to cytosine (C), resulting in the amino acid being changed from leucine to serine.

FIG. 4 shows the incidence of wilt in island cotton varieties carrying both GbPP2C80 mutations.

FIG. 5 is a bar graph showing the detection of the expression difference of GbPP2C80 in the blast resistant cultivar T10-280 and the blast susceptible cultivar II15-3464 by qRT-PCR, and the expression level of GbPP2C80 after VIGS in the blast susceptible cultivar. R_WT represents a wild-type control of the blast resistant variety T10-280, and S_WT represents a wild-type control of the blast susceptible variety II 15-3464.

FIG. 6 shows the phenotype values of the disease after 25 days of inoculation with Botrytis cinerea (physiological race 7) after silencing GbPP2C80 using VIGS. R_WT represents a wild-type control of the blast resistant variety T10-280, and S_WT represents a wild-type control of the blast susceptible variety II 15-3464.

FIG. 7 is a schematic representation of the onset of disease 25 days after inoculation with Botrytis cinerea (physiological race 7) after silencing GbPP2C80 using VIGS. Wherein a is the plant disease phenotype; b is the leaf disease phenotype. R_WT represents a wild-type control of the blast resistant variety T10-280, and S_WT represents a wild-type control of the blast susceptible variety II 15-3464.

FIG. 8 shows subcellular localization of GbPP2C 80. a is p35S in a recombinant plant carrying a GbPP2C80-GFP fusion vector, wherein the cell localization of the GbPP2C 80-GFP; b is the p35S of the recombinant plant carrying GFP empty vector, which is the cell location in GFP, as a control.

FIG. 9 is a two-molecule fluorescence complementation (BiFC) demonstrating the protein interactions of GbWAKL14 and GbPP2C 80. a is the interaction of GbWAKL14-YFPN and GbPP2C 80-YFPC; b is a control of YFPN and YFPC empty vector.

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 quantitative experiments in the following examples were performed in triplicate unless otherwise indicated.

The GFP plasmid has been described in the following examples as p35S: zhang W, et al natural variations at TIG1 encoding a TCP transcription factor contribute to plant architecture domestication in rice. Mol plant.2019;12 1075-1089 the public may obtain the biomaterial from the applicant, which is used only for repeated experiments of the invention and not as other uses.

The pCLCrVA plasmids in the examples below have been described: zhao N, et al genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypiumbarbadensein China and selection for fibre length, lint percentage and Fusariumwilt resistance. 20 (4):691-710. The biological material is available to the public from the applicant and is only used for repeated experiments of the invention and is not available for other uses.

The pCambia 1300-YFPC plasmid in the examples below has been described: gu Z, et al, chalcone synthase is ubiquitinated and degraded via interactions with a RING-H2 protein in petals of Paeonia 'HeXie'. J Exp Bot.2019;70 (18):4749-4762. The biological material is available to the public from the applicant and is only used for repeated experiments of the invention and is not available for other uses.

The pCambia 1300-YFPN plasmid in the examples below has been described: gu Z, et al, chalcone synthase is ubiquitinated and degraded via interactions with a RING-H2 protein in petals of Paeonia 'HeXie'. J Exp Bot.2019;70 (18):4749-4762. The biological material is available to the public from the applicant and is only used for repeated experiments of the invention and is not available for other uses.

336 parts of the island cotton cultivar of the following examples have been described: zhao N, et al genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypiumbarbadense in China and selection for fibre length, lint percentage and Fusariumwilt resistance. 20 (4):691-710. The biological material is available to the public from the applicant and is only used for repeated experiments of the invention and is not available for other uses.

The island cotton susceptible variety (S) II15-3464 and the disease resistant variety (R) T10-280 in the following examples have been described in: zhao N, et al genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypiumbarbadensein China and selection for fibre length, lint percentage and Fusarium wilt resistance. 20 (4):691-710. The biological material is available to the public from the applicant and is only used for repeated experiments of the invention and is not available for other uses.

The physiological race 7 fusarium wilt bacteria in the following examples have been described in: zhao N, et al genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypium barbadense in China and selection for fibre length, lint percentage and Fusariumwilt resistance. 20 (4):691-710. The biological material is available to the public from the applicant and is only used for repeated experiments of the invention and is not available for other uses.

The tobacco of Benshi (Nicotiana benthamiana) of the following examples was given away from the China agricultural university Sun Chuanqing laboratory and has been described in: zhang W, et al natural variations at TIG1 encoding a TCP transcription factor contribute to plant architecture domestication in rice. Mol plant.2019;12 1075-1089 the public may obtain the biomaterial from the applicant, which is used only for repeated experiments of the invention and not as other uses.

The following examples were run on IBM spss_statistics_25 statistical software and the experimental results were expressed as averages using the T-Test, with P < 0.05 (x) for significant differences, P < 0.01 (x) for very significant differences, and P < 0.001 (x) for very significant differences.

EXAMPLE 1 Whole genome correlation analysis excavation of island cotton anti-wilt Gene

The method is characterized in that 336 parts of island cotton which is subjected to genome sequencing by combining a national institute of agricultural university crop molecular breeding international cooperation education department with a national institute of industrial crops of Xinjiang agricultural sciences are used as a positioning group, SNP variation is filtered by using a standard that the minimum allele frequency is less than 0.05 and the deletion ratio is less than 20%, and the filtered SNP is used for whole genome association analysis. The correlation analysis is carried out by using the genome-wide efficient hybrid model correlation software GEMMA 0.94.1 (http:// www.xzlab.org/software.html) and taking the morbidity of each material wilt disease in the Xinjiang Korla disease nursery in 2015, 2016, 2018 and 2019 as a phenotype, and the correlation of data in four years is extremely high and the phenotype is reliable (figure 1). Carrying out character association analysis by adopting a mixed linear model, wherein the population genetic structure is used as a fixed effect, and the individual relationship is used as a random effect so as to correct the influence of the population structure and the individual relationship:

y＝Xα+Sβ+Kμ+e

y is a phenotypic character, X is an indication matrix of a fixed effect, and alpha is an estimated parameter of the fixed effect; s is an indication matrix of SNP, and beta is the effect of SNP; k is the only matrix of random effects, μ is the predicted random individuals, e is the random residual, subject to e- (0, δe2). In addition, S matrixes are established by using the first 3 PCs to carry out population structure correction, and K matrixes are established by using the simple matching coefficient matrixes. The GEMMA software parameter is set to "get-bfile file-kkoship-lmm 1-o outfile-MISS 0.2-maf 0.05-c covariates (GCTA: PCA)". Manhattan plots were made using R-packet qqman. The effect value of the gene marker was detected by F-test and Bonferroni was usedCorrection multiple assays were corrected and SNPs significantly associated with resistance to wilt were screened by the associated significance (P-value). A threshold (-log) was detected on the D03 chromosome in all circumstances ₁₀ P-value) is greater than 6, especially the threshold reaches 8.52 in the 2019 kohler environment; the SNP site is located 1.9kb upstream of the gene Gbar_D03G001670 (designated GbPP2C 80), and the GbPP2C80 contains a T-to-C nonsensical mutation site Gbar_D03_1162571, which changes the amino acid from leucine to serine (FIGS. 2 a and b and 3). The GbPP2C80 gene is derived from a sea island cotton fusarium wilt-resistant variety T10-280, the coding sequence (CDS) of the GbPP2C80 gene is SEQ ID No.1, the genomic sequence of the GbPP2C80 gene is SEQ ID No.2, the coded protein is named GbPP2C80 protein or protein GbPP2C80, and the amino acid sequence of the coded protein is shown as SEQ ID No. 3. Wherein, the incidence rate of the fusarium wilt of the sea island cotton variety with the genotype C at 209 th position of the coding sequence of the GbPP2C80 gene is extremely lower than that of the sea island cotton variety with the genotype T at 209 th position (figure 4), namely, the incidence rate of the fusarium wilt of the sea island cotton variety with serine at 70 th position of the amino acid sequence of the GbPP2C80 protein is extremely lower than that of the sea island cotton variety with leucine at 70 th position, so that the GbPP2C80 is presumed to be a main effective gene for regulating and controlling the fusarium wilt resistance of the sea island cotton.

Example 2, VIGS validation of the effect of GbPP2C80 on island cotton fusarium wilt resistance

1. Differential expression analysis of GbPP2C80 and determination of VIGS conversion receptor

Respectively in nutrient soil and vermiculite 1:1, planting the susceptible fusarium wilt island cotton variety (S) II15-3464 and the high-resistance fusarium wilt island cotton variety (R) T10-280 in a uniformly mixed basin, and inoculating fusarium wilt pathogen No. 7 physiological seed by root injury method after cotyledon flattening for about two weeks, wherein the bacterial liquid concentration reaches 1 multiplied by 10 ⁷ Individual/cm ² After 25 days of inoculation, taking sea island cotton leaves, extracting RNA, reversely transcribing the RNA into cDNA by reverse transcriptase, taking the cDNA as a template, designing a specific primer (forward primer 5'-TCAGTTGACCCGGCCAGAGTAT-3'; reverse primer 5'-TGTTGCTGCACTGGGGAATGAA-3'), and detecting the expression difference of GbPP2C80 in the disease-resistant and disease-sensitive sea island cotton materials by Real-time PCR.

As a result, it was found that the expression level of GbPP2C80 in the disease-resistant sea island cotton material was extremely higher than that in the disease-resistant sea island cotton material (FIG. 5), and therefore, the VIGS conversion was performed in the disease-resistant material.

2. Construction of VIGS vector and identification of resistance to wilt

According to the coding sequence of the candidate gene GbPP2C80, designing a primer for constructing a VIGS vector, adding a Spe I restriction enzyme cutting site (ACTAGT) and a protecting base GG at the 5' end of a forward primer, wherein the nucleotide sequence of the forward primer is as follows: 5'-GGACTAGTTGTCATTTGATGGCAGTTCT-3'; the nucleotide sequence of the reverse primer added with Pac I restriction enzyme site (TTAATTAA) and protective base CC is as follows: 5'-CCTTAATTAATCACCATTGTTACCGCTCT-3'.

The structure of the recombinant plasmid pCLCrVA-GbPP2C80 is described as follows: to replace the fragment between the recognition sites of the pCLCrVA vector sequences SpeI and Pac I with the DNA molecule shown in SEQ ID No.4, the other nucleotides of the pCLCrVA vector were kept unchanged.

The recombinant vector pCLCrVA-GbPP2C80 is constructed and then is transferred into an agrobacterium GV3101 strain (manufactured and bioengineered Co., ltd., B528430) through heat shock to obtain an agrobacterium tumefaciens bacterial solution containing pCLCrVA-GbPP2C80 plasmid and agrobacterium GV3101 containing pCLCrVA-GbPP2C80 plasmid, which is named as agrobacterium tumefaciens GV3101/pCLCrVA-GbPP2C80. The empty vector pCLCrVA was heat-shocked into Agrobacterium GV3101 strain to obtain GV3101/pCLCrVA as empty control. The vector pCLCrVB is thermally shocked and transferred into an agrobacterium GV3101 strain to obtain GV3101/pCLCrVB, and the vector pCLCrVB is mixed with an agrobacterium liquid containing GV3101/pCLCrVA-GbOFP7 and GV3101/pCLCrVA in equal volume before transformation.

3. Acquisition of GbPP2C80 Gene-silenced transgenic island Cotton plants S_pCLCrVA-GbPP2C80

The island cotton disease-sensing material II15-3464 is planted by the soil culture method as a transgenic receptor, and the disease-resistant variety T10-280 is planted as a disease-resistant control. After two weeks, the cotyledons were completely flattened and transformed. Taking strains GV3101/pCLCrVA-GbPP2C80, GV3101/pCLCrVA and GV3101/pCLCrVB, and culturing at 28 ℃ to logarithmic phase; centrifugation at 8000rpm for 5min, collecting the cells, and further using VIGS invader solution (10 mM MES, 200. Mu.M AS,10mM MgCl) ₂ ) ResuspensionThallus and adjust the concentration of the bacterial liquid to OD ₆₀₀ =about 1.0; GV3101/pCLCrVA-GbPP2C80 and GV3101/pCLCrVA are respectively mixed with GV3101/pCLCrVB bacterial liquid according to the volume ratio of 1:1, and the mixture is placed still for 3 hours at room temperature and then used for transforming cotton leaves. As an empty vector control, a mixed bacterial solution of GV3101/pCLCrVA and GV3101/pCLCrVB, respectively, was used. Bacterial liquid was aspirated using a 1mL sterile syringe and inoculated on the back of cotyledons by injection. The cotton plants after injection were placed in a 28℃greenhouse and cultured for 16h/8h of light-dark cycle.

Two weeks later, fusarium oxysporum # 7 physiological race (fusarium oxysporum sp. Vasinefectum race 7) was inoculated by root-wounding, 25 days after inoculation, the onset phenotype was observed, and samples were taken to detect the silencing efficiency of the candidate gene by Real-time PCR (forward primer 5'-TCAGTTGACCCGGCCAGAGTAT-3'; reverse primer 5'-TGTTGCTGCACTGGGGAATGAA-3').

The results show that: after VIGS treatment, transcription of GbPP2C80 was significantly inhibited (fig. 5), the incidence of wilt of the disease-susceptible island cotton plants s_pclcrva-GbPP2C80 silenced by GbPP2C80 was significantly reduced (a and b in fig. 6 and 7), i.e., wilt resistance was significantly enhanced, and thus it was seen that GbPP2C80 was a major gene regulating wilt resistance of island cotton and that GbPP2C80 negatively regulated wilt resistance of island cotton.

Example 3 subcellular localization of GbPP2C80

According to the gene ID of GbPP2C80, a CDS sequence is obtained, a primer is designed, a Kpn I restriction site (GGTACC) is added at the 5' end of a forward primer, and the nucleotide sequence of the forward primer is as follows: 5'-GGTACCATGGCTGTGTCTGGTTCCA-3'; the Xba I restriction enzyme site (TCTAGA) is added to the 5' end of the reverse primer, and the nucleotide sequence of the reverse primer is as follows: 5'-TCTAGACACATCGGCGGAGCTAGT-3'. The target sequence was amplified using leaf cDNA of the sea island cotton variety II15-3464 as a template to obtain the 1 st to 1125 th positions (i.e., not including the final stop codon TGA) of the coding sequence (SEQ ID No. 1) of the gene of GbPP2C 80.

And (3) carrying out double digestion on the purified coding sequence of the GbPP2C80 gene and p35S:: GFP plasmid, and then connecting enzyme digestion products to construct p35S:: gbPP2C80-GFP recombinant plasmid, and transforming agrobacterium GV3101 to obtain recombinant agrobacterium GV3101/GbPP2C80-GFP.

The structure of the GbPP2C80-GFP recombinant plasmid is described as follows: the small fragment between the recognition sequences of the restriction enzymes Kpn I and Xba I of the GFP plasmid p35S was replaced by the DNA molecule shown in SEQ ID No.1 (without the final stop codon TGA). The recombinant plasmid p35S is GbPP2C80-GFP expressing GbPP2C80 protein shown in SEQ ID No. 3. The recombinant plasmid p35S is an expression cassette containing GbPP2C80-GFP fusion protein, wherein the promoter for promoting the transcription of GbPP2C80 gene in the expression cassette is a 35S promoter.

The same transformation method is adopted to directly transform the GFP plasmid into the agrobacterium GV3101 to obtain the recombinant agrobacterium GV3101/GFP. Recombinant Agrobacterium GV3101/GFP served as a control for subsequent experiments.

Adding recombinant Agrobacterium GV3101/GbPP2C80-GFP positive bacteria solution into YEP liquid culture medium containing 50 μg/mL Kan and 50 μg/mL Rif, activating strain in shaking table (28deg.C, 160 rpm), adding 1mL activated bacteria solution into 20mL YEP liquid culture medium containing 50 μg/mL Kan and 50 μg/mL Rif, amplifying culturing at 28deg.C, 160rpm to OD ₆₀₀ =1.0, and the bacterial liquid was collected by centrifugation at 5000rpm for 5min, and the suspension (containing 10mM MES,10mM MgCl) ₂ Ph=5.2, 100 μΜ AS) to OD ₆₀₀ =1.0, standing at room temperature for 2-5h.

Selecting Nicotiana benthamiana (Nicotiana benthamiana) with good growth condition, lightly injecting recombinant agrobacterium GV3101/GbPP2C80-GFP suspension containing GbPP2C80-GFP into the back of tobacco leaves by using a syringe, simultaneously injecting agrobacterium GV3101/GFP bacterial suspension containing GFP empty carrier into the lower epidermis of the leaves of Nicotiana benthamiana as a control, culturing for 24 hours in the dark, culturing for 48 hours in the light, observing subcellular localization of GbPP2C80 protein by using a Zeiss LSM 880 inverted confocal fluorescence microscope, and exciting light at 488nm and emitting light at 510nm.

The results are shown in FIGS. 8 a and b, where the GFP signal of the GbPP2C80-GFP fusion protein is localized to the cell membrane, indicating that GbPP2C80 plays a major role on the cell membrane.

Example 4 interaction protein of GbPP2C80

The national institutes of agricultural university crop molecular breeding International Cooperation education department combined laboratory and Xinjiang national academy of agricultural sciences economic crop institute previously identified a gene GbWAKL14 (Gbar_D03G 001910) that was significantly associated with the resistance to the fusarium wilt of island cotton and verified its resistance to the fusarium wilt of island cotton negatively regulated by VIGS. The coding sequence (CDS) of the GbWAKL14 gene is SEQ ID No.5, the genomic sequence of the GbWAKL14 gene is SEQ ID No.6, and the amino acid sequence of the coded protein is shown as SEQ ID No. 7.

To investigate whether GbPP2C80 interacts with GbWAKL14, the wilt resistance of island cotton was co-regulated, specific primers were designed using island cotton leaf cDNA as a template, and 5' ends of the forward primer and the reverse primer carried cleavage site Kpn I (GGTACC) and cleavage site Spe I (ACTAGT), respectively, and specific primer nucleotide sequences are shown in Table 1.

TABLE 1 primer information for the bimolecular fluorescent complementary vectors of GbPP2C80 and GbWAKL14

Gene name	Forward primer (5 '-3')	Reverse primer (5 '-3')
			GbPP2C80	GGTACCATGGCTGTGTCTGGTTCCA	ACTAGTCACATCGGCGGAGCTAGT
GbWAKL14	GGTACCATGATAAGGATAAAGCTATGCTTC	ACTAGTTTCTCGTTGAGATGCATTATTAC

Cloning to obtain CDS full-length sequences of GbPP2C80 and GbWAKL14 with stop codons removed, and respectively connecting the sequences to pCambia 1300-YFPC and pCambia 1300-YFPN vectors to obtain recombinant plasmids GbPP2C80-YFPC and GbWAKL14-YFPN.

The structure of the recombinant plasmid GbPP2C80-YFPC is described as follows: the fragment between the Kpn I and Spe I recognition sites of the pCambia 1300-YFPC vector was replaced with the DNA molecule shown in SEQ ID No.1 (stop codon TGA was removed) to obtain a recombinant vector keeping the other nucleotides of the pCambia 1300-YFPC vector unchanged.

The structure of the recombinant plasmid GbWAKL14-YFPN is described as follows: to replace the fragment between the Kpn I and Spe I recognition sites of the pCambia 1300-YFPN vector with the DNA molecule shown in SEQ ID No.5 (removal of stop codon TGA), the other nucleotides of the pCambia 1300-YFPN vector were kept unchanged.

The recombinant plasmids GbPP2C80-YFPC and GbWAKL14-YFPN are respectively transformed into agrobacterium GV3101 by a heat shock method to obtain recombinant agrobacterium GV3101/GbPP2C80-YFPC and GV3101/GbWAKL14-YFPN.

The same transformation method is adopted to directly transform the YFPC and YFPN plasmid empty vector into agrobacterium GV3101 to obtain recombinant agrobacterium GV3101/YFPC and recombinant agrobacterium GV3101/YFPN as experimental control.

The positive bacterial liquid of the recombinant Agrobacterium GV3101/GbPP2C80-YFPC, GV3101/GbWAKL14-YFPN is added into a YEP liquid culture medium containing 50 mug/mL Kan and 50 mug/mL Rif, the strain is activated in a shaking table (28 ℃,160 rpm), and 1mL of the activated bacterial liquid is added into 20mL of a YEP liquid culture medium containing 50 mug/mL Kan and 50 mug/mL Rif, 28 ℃,160rpm, and the culture is enlarged to OD ₆₀₀ =1.0, and the bacterial liquid was collected by centrifugation at 5000rpm for 5min, and the suspension (containing 10mM MES,10mM MgCl) ₂ Ph=5.2, 100 μΜ AS) to OD ₆₀₀ =1.0, standing at room temperature for 2-5h.

Mixing the agrobacterium suspension containing GbPP2C80-YFPC and GbWAKL14-YFPN in equal volume, selecting Nicotiana benthamiana (Nicotiana benthamiana) with good growth condition, lightly injecting the suspension to the back of tobacco leaves by a syringe, taking the back of the tobacco leaves subjected to equal volume mixing injection of the bacterial suspension of recombinant Agrobacterium GV3101/YFPC and recombinant Agrobacterium GV3101/YFPN as a control, culturing in the dark for 24h, culturing in the light for 48h by using a Zeiss LSM 880 inverted confocal fluorescence microscope, observing the interaction condition of GbPP2C80 and GbWAK14 proteins, exciting light for 514nm, and emitting light for 527nm.

As a result, as shown in FIGS. 9 a and b, YFP signals of transgenic plants containing GbPP2C80-YFPC and GbWAKL14-YFPN fusion proteins were localized on cell membranes, indicating that GbPP2C80 and GbWAKL14 interacted on cell membranes.

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

Claims

1. A protein which is any one of the following:

a1 Protein with the amino acid sequence shown as SEQ ID No. 3;

a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the protein of A1), has more than 80 percent of identity with the protein shown in A1) and has the function of regulating and controlling plant fusarium wilt resistance;

2. The protein of claim 1, wherein: the protein is derived from cotton.

3. A biomaterial associated with the protein of claim 1 or 2, said biomaterial being any one of the following:

b1 A nucleic acid molecule encoding a protein as claimed in claim 1 or 2;

b2 An expression cassette comprising the nucleic acid molecule of B1);

c1 A nucleic acid molecule which inhibits or reduces or silences the expression of a gene encoding a protein as claimed in claim 1 or 2;

c2 Expression of the gene encoding the nucleic acid molecule of C1);

c3 An expression cassette containing the coding gene of C2);

4. A biomaterial according to claim 3, wherein: b1 The nucleic acid molecule is a gene as shown in E1) or E2) below:

e1 A cDNA molecule or a DNA molecule having a coding sequence of SEQ ID No. 1;

e2 Nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No. 2.

5. The application is characterized in that: the application is any one of the following:

u1) use of the protein or the substance regulating gene expression or the substance regulating the activity or the content of the protein according to claim 1 or 2 for regulating plant blight resistance;

u2) use of the protein or the substance regulating gene expression or the substance regulating the activity or the content of the protein according to claim 1 or 2 for the preparation of a product regulating the resistance to plant blight;

u3) use of the protein or the substance regulating gene expression or the substance regulating the activity or the content of the protein according to claim 1 or 2 for cultivating plants resistant to blight;

u4) use of the protein or the substance regulating the expression of a gene or of the substance regulating the activity or the content of said protein according to claim 1 or 2 for the preparation of a product for growing plants resistant to wilt;

u5) use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of said protein according to claim 1 or 2 in plant breeding.

6. The use according to claim 5, characterized in that: the substance regulating the expression of a gene or the substance regulating the activity or content of the protein is a biological material related to the protein, and the biological material is any one of the following B1) to B7):

b1 A nucleic acid molecule encoding a protein as claimed in claim 1 or 2;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2).

7. A method of modulating resistance to plant blight, comprising: comprising regulating the activity and/or content of the protein of claim 1 or 2 in a target plant, or/and regulating the expression level of the gene encoding the protein of claim 1 or 2, thereby regulating the resistance to plant blight.

8. The method according to claim 7, wherein: the regulation of the activity and/or content of the protein of claim 1 or 2 in a plant of interest, or/and the expression level of the gene encoding the protein of claim 1 or 2, comprises introducing into a recipient plant a nucleic acid molecule that inhibits or reduces or silences the gene encoding the protein, resulting in a plant of interest having a higher resistance to wilt than the recipient plant; the gene encoding the protein encodes the protein of claim 1 or 2.

9. A method of growing plants with altered resistance to plant blight comprising: 1) Inhibiting or reducing or silencing the expression level of a gene encoding the protein according to claim 1 in a plant of interest, or/and inhibiting or reducing or silencing the activity and/or content of a gene encoding the protein according to claim 1, to obtain a plant with increased resistance to wilt;

2) Increasing, enhancing and/or upregulating the expression of a gene encoding a protein according to claim 1 in a plant of interest, or/and increasing, enhancing and/or upregulating the activity and/or content of a gene encoding a protein according to claim 1, to obtain a plant with reduced resistance to wilt.

10. The method according to any one of claims 7-9, characterized in that: the plant is any one of the following: n1) dicotyledonous plants;

n2) plants of order malvaceae;

n3) malvaceae plants;

n4) cotton plants;

n5) cotton.