CN117447572A

CN117447572A - Sea island cotton fusarium wilt resistance protein GbOFP7 and encoding gene and application thereof

Info

Publication number: CN117447572A
Application number: CN202311536930.4A
Authority: CN
Inventors: 华金平; 赵楠; 王为然; 朱家辉; 阿里甫·艾尔西; 孔杰; 王保亮; 王萌
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: 2024-01-26

Abstract

The invention discloses a sea island cotton fusarium wilt resistance protein GbOFP7, and a coding gene and application thereof. The invention relates to the field of botanic, in particular to a cotton fusarium wilt resistance protein GbOFP7 in islands as well as 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 plant with GbOFP7 expression is silenced by the VIGS technology, the disease incidence of the wilt is obviously reduced, and the GbOFP7 protein can negatively regulate and control the wilt resistance of island cotton, so that the method has wide application prospect.

Description

Sea island cotton fusarium wilt resistance protein GbOFP7 and encoding gene and application thereof

Technical Field

The invention relates to the field of botanic, in particular to a cotton fusarium wilt resistance protein GbOFP7 in islands as well as a coding gene and application thereof.

Background

Wilt is one of the main diseases threatening global cotton production, and can occur throughout the growth period of island cotton. The sea island cotton fiber has excellent quality, long, thin and strong, and is an indispensable raw material for high-count combed yarn and special fabrics. The occurrence of fusarium wilt seriously affects the yield and quality of island cotton, but the research on the fusarium wilt resistance of island cotton is very few, and the molecular mechanism of the island cotton for resisting fusarium wilt is not clear. Identification and cloning of the island cotton fusarium wilt resistance gene helps us to understand the molecular mechanism of island cotton fusarium wilt resistance.

Oval Family Proteins (OFPs) are plant-specific growth regulators that regulate cell elongation and secondary cell wall formation by inhibiting the expression of downstream target genes. In addition, the OFP gene is also related to drought resistance, cold resistance and salt resistance. However, there is no report on OFP protein to regulate and control the resistance of island cotton to fusarium wilt.

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 GbOFP7.

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 nucleotide sequence encoding the protein GbOFP7 of the present invention can be easily mutated by a person skilled in the art using a known method, for example, directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the protein GbOFP7 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 GbOFP7 and have the function of the protein GbOFP7.

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 (GbOFP 7 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 GbOFP7 (CDS).

The nucleotide sequence shown in SEQ ID No.2 is the nucleotide sequence of the genome of the protein GbOFP7.

The GbOFP7 gene of the present invention may be any nucleotide sequence capable of encoding the protein GbOFP7. 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 GbOFP7 gene can be constructed 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 GbOFP7 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 GbOFP7 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 changed resistance to plant blight can be obtained. Expression vectors carrying the GbOFP7 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 recombinant vector pCLCrVA-GbOFP7. The recombinant vector pCLCrVA-GbOFP7 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, gbOFP7-GFP.

The recombinant vector p35S is GbOFP7-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.

Recombinant plasmid p35S is GbOFP7 protein shown in sequence 3 in the GbOFP7-GFP expression sequence table. The recombinant plasmid p35S is an expression cassette containing GbOFP7-GFP fusion protein, and the promoter for promoting the transcription of GbOFP7 gene in the expression cassette is a 35S promoter.

The recombinant microorganism can be specifically recombinant Agrobacterium GV3101/GbOFP7-GFP.

The recombinant agrobacterium GV3101/GbOFP7-GFP is obtained by introducing the recombinant vector p35S, gbOFP7-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 GbOFP7 or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein as described above 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 that regulates the activity and/or content of the protein may be a substance that regulates the expression of a gene encoding the protein GbOFP7.

In the above application, the substance for regulating the expression of the gene or the substance for regulating the activity or content of the protein is a biological material related to the protein, and the biological material may be any 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);

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 quantity 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 GbOFP7 in the target plant, or/and the expression level of the encoding gene of the protein comprises introducing the encoding gene GbOFP7 of the protein into a receptor plant to obtain the target plant with altered resistance to plant blight; the GbOFP7 encoding gene encodes the protein GbOFP7.

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 a GbOFP7 protein in a 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 GbOFP7 protein in the plant of interest can be achieved in particular by introducing into the plant of interest an interfering vector targeting the nucleic acid molecule encoding the GbOFP7 protein. The inhibition of the expression of the nucleic acid molecule encoding the GbOFP7 protein in the target plant can be achieved specifically by introducing a gene silencing vector targeting the nucleic acid molecule encoding the GbOFP7 protein into the target plant.

The gene silencing vector may be recombinant vector pCLCrVA-GbOFP7. The recombinant vector pCLCrVA-GbOFP7 is obtained by replacing a fragment between the recognition sites of the sequence SpeI and Pac I of a pCLCrVA vector (starting vector) with a DNA molecule shown as SEQ ID No.4 and keeping other nucleotides of the pCLCrVA vector (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 utilizes natural populations of island cotton to identify the incidence rate of fusarium wilt, locates the genetic locus of island cotton for resisting fusarium wilt at 1110567 th position of D03 chromosome, identifies a gene GbOFP7 at 22.6kb at the downstream of the genetic locus, and has a G-to-C nonsensical mutation (Gbar_D03_ 1134219) on a unique exon. Wherein, the incidence rate of the wilt of the sea island cotton variety carrying the C mutation type is obviously lower than that of the sea island cotton variety carrying the G mutation type. The expression of GbOFP7 in the infected island cotton variety is higher than that in the disease-resistant island cotton variety, and after the expression of GbOFP7 in the infected island cotton is reduced by utilizing virus-induced gene silencing (virus-induced gene silencing, VIGS) technology, the resistance to wilt is obviously enhanced and the plant height is increased. The gene codes a transcription inhibitor of oval family proteins, is mainly positioned on membranes and cores, is a novel type of island cotton anti-wilt gene, and has certain theoretical and application values.

Drawings

Fig. 1 is a distribution diagram of average wilt disease incidence of island cotton groups investigated in the green district nursery of singugar in china in 2015, 2016, 2018 and 2019.

FIG. 2 is a graph of global (a) and local Manhattan for the incidence of wilt disease in island cotton population materials. a is an overall Manhattan diagram of the incidence rate of the fusarium wilt of island cotton group materials, and the arrow marks the localized extremely significant associated SNP (Gbar_D03_ 1110567); b is a local Manhattan plot of the incidence of wilt in the island cotton population material, the arrow marks a non-synonymous SNP (Gbar_D03_ 1134219) within a very significantly associated gene.

FIG. 3 is a diagram showing the location of a nonsensical mutation in the GbOFP7 gene. Blue and yellow rectangles represent untranslated region (UTR) and coding region (CDS), respectively, and black lines represent introns (Intron). Ref GGT (G) represents a reference codon GGT which codes glycine (abbreviated as G), and a key mutation site is G at a second position in the middle; alt GCT (A) means that the variant codon GCT codes for alanine (abbreviated as A), the key mutation site is the second middle position C, i.e. mutation from guanine (G) to cytosine (C), resulting in the amino acid being changed from glycine to alanine.

FIG. 4 shows the incidence of wilt in island cotton varieties carrying two types of GbOFP7 mutations.

FIG. 5 is a bar graph showing the detection of the expression difference of GbOFP7 in the blast resistant cultivar T10-280 and the blast susceptible cultivar II15-3464 by qRT-PCR, and the expression level of GbOFP7 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 No. 7) after silencing GbOFP7 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 shows strain height phenotype values 25 days after inoculation with cotton fusarium wilt (physiological race No. 7) after silencing GbOFP7 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. 8 is a schematic representation of the onset of disease 25 days after inoculation with Botrytis cinerea (physiological race 7) after silencing GbOFP7 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. 9 is subcellular localization of GbOFP7. Panel a shows the cellular localization of recombinant plants p35S:: gbPP2C80-GFP carrying the GbOFP7-GFP fusion vector. Panel b shows the p35S of the recombinant plant carrying the GFP empty vector, the cellular localization of GFP, as a control.

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 (8):1075-1089. 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 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.

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 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 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 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 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

Uses 336 parts of island cotton which is combined with the national institute of field agriculture and the national institutes of Uygur autonomous region agricultural science and is used for finishing genome sequencing by combining the national institute of field crop of China university crop molecular breeding International education department as a positioning group, and usesSNP variation with minimum allele frequency less than 0.05 and deletion rate less than 20% is subjected to genome-wide association analysis. 2015. The wilt disease incidence data of 336 parts of island cotton in the kurla disease nursery of the Xinjiang Uygur autonomous region in 2016, 2018 and 2019 exhibited continuous quantitative genetic characteristics (fig. 1). The trait association analysis was performed using a mixed linear model using the genome-wide efficient mixed model association software GEMMA 0.94.1 (http:// www.xzlab.org/software.html). The GEMMA software parameter is set to "get-bfile file-k kineship-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 markers was detected by F-test and multiple assays were corrected using Bonferroni correction, and SNPs significantly associated with resistance to wilt were screened by the associated significance (P-value). BLUP values based on the four year wilt morbidity phenotype are mapped to a threshold (-log) on the D03 chromosome ₁₀ P-value) is 11.57, a significant cognate site Gbar_D03_1110567 (a in FIG. 2), at 22.6kb downstream of which is the gene Gbar_D03G001640 (designated GbOFP 7), within the unique exon of the gene GbOFP7 there is a critical nonsensical mutation (Gbar_D03_ 1134219, b in FIG. 2), i.e., a guanine G mutation to cytosine C, resulting in a glycine (G) mutation to alanine (A) (FIG. 3). The GbOFP7 gene is derived from a sea island cotton anti-fusarium wilt variety T10-280, the coding sequence (CDS) of the GbOFP7 gene is SEQ ID No.1, the genomic sequence is SEQ ID No.2, the coded protein is named GbOFP7 protein or protein GbOFP7, and the amino acid sequence of the coded protein is shown as SEQ ID No. 3. Wherein, the incidence of the blight of the sea island cotton variety with the genotype C at the 1007 th position of the coding sequence (sequence 1) of the GbOFP7 gene is extremely lower than that of the sea island cotton variety with the genotype G at the 1007 th position (figure 4), namely the incidence of the blight of the sea island cotton variety with the alanine at the 336 th position of the amino acid sequence (sequence 3) of the GbOFP7 protein is extremely lower than that of the sea island cotton variety with the glycine at the 336 th position, so that the GbOFP7 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 GbOFP7 on island cotton fusarium wilt resistance

1. Differential expression analysis of GbOFP7 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, inoculating fusarium wilt bacteria by a root injury method after cotyledon flattening for about two weeks, wherein the concentration of bacterial liquid 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 CGACGTTGGAGTTTCACTGGGA; reverse primer TGGTGGTGGGAATGAATCGTCG), and detecting the expression difference of GbOFP7 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 GbOFP7 in the disease-resistant island cotton material II15-3464 was extremely higher than that in the disease-resistant island cotton material T10-280 (FIG. 5), and therefore, the VIGS conversion was performed in the disease-resistant material II 15-3464.

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

According to the coding sequence of the gene GbOFP7, a primer is designed for constructing a VIGS vector, a SpeI enzyme cutting site (ACTAGT) and a protecting base GG are added at the 5' end of a forward primer, and the nucleotide sequence of the forward primer is as follows: 5'-GGACTAGTTAATCTCCCATCAAATCCTGTC-3'; the nucleotide sequence of the reverse primer added with Pac I restriction enzyme site (TTAATTAA) and protective base CC is as follows: 5'-CCTTAATTAAGCTTTCGCTGCTGAACAA-3'.

The structure of the recombinant plasmid pCLCrVA-GbOFP7 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, a recombinant vector was obtained which kept the other nucleotides of the pCLCrVA vector (starting vector) unchanged.

The recombinant vector pCLCrVA-GbOFP7 is constructed and then is transferred into an agrobacterium GV3101 strain (manufacturing bioengineering Co., ltd., B528430) through heat shock to obtain agrobacterium tumefaciens bacterial liquid containing pCLCrVA-GbOFP7 plasmid and agrobacterium GV3101 containing pCLCrVA-GbOFP7 plasmid, which is named as agrobacterium tumefaciens GV3101/pCLCrVA-GbOFP7. 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.

Acquisition of GbOFP7 Gene-silenced transgenic island Cotton plants S_pCLCrVA-GbOFP7

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-GbOFP7, 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 ) Resuspension of the bacterial cells and adjusting the bacterial liquid concentration to OD ₆₀₀ =about 1.0; GV3101/pCLCrVA-GbOFP7 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 at room temperature for 3 hours and then used for transforming cotton leaves. As an empty vector control, a mixed bacterial solution of GV3101/pCLCrVA and GV3101/pCLCrVB 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, a fusarium wilt No. 7 physiological race (Fusarium oxysporum f.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'-CGACGTTGGAGTTTCACTGGGA-3'; reverse primer 5'-TGGTGGTGGGAATGAATCGTCG-3').

The results show that: after VIGS treatment, transcription of GbOFP7 was significantly inhibited (fig. 5), and the incidence of wilt of the GbOFP 7-silenced island cotton plant s_pclcrva-GbOFP7 was significantly reduced (fig. 6 and fig. 8 b), thus it was seen that GbOFP7 could negatively regulate wilt resistance of island cotton. In addition, the GbOFP 7-silenced, diseased island cotton plants S_pCLCrVA-GbOFP7 were significantly increased in plant height, close to that of the disease-resistant material R_WT (T10-280) (a in FIGS. 7 and 8).

Example 3 subcellular localization of GbOFP7

According to the gene ID of GbOFP7, a CDS sequence is obtained, a primer is designed, a Kpn I enzyme cutting site (GGTACC) is added at the 5' end of a forward primer, and the nucleotide sequence of the forward primer is (5 ' -3 '): 5'-GGTACCATGGCGAAACGATTCAAGTTCAC-3'; the Xba I cleavage site (TCTAGA) is added to the 5' -end of the reverse primer, and the nucleotide sequence of the reverse primer is (5 ' -3 '): 5'-TCTAGAATTTAACCCACAAGAAACTCGAAA-3'. The coding sequence of the gene GbOFP7 is obtained by amplifying a target sequence by taking the leaf cDNA of the sea island cotton as a template (the nucleotide sequence is 1 st to 1056 th positions of the sequence 1 in the sequence table (namely, the final stop codon TGA is not included).

And (3) carrying out double enzyme digestion on the coding sequence of the purified GbOFP7 gene and p35S:: GFP plasmid DNA, and then connecting enzyme digestion products to construct p35S:: gbOFP7-GFP recombinant plasmid, and transforming agrobacterium GV3101 to obtain recombinant agrobacterium GV3101/GbOFP7-GFP.

The structure of the GbOFP7-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 plasmid p35S:: GFP was replaced by the DNA molecule shown in sequence 1 of the sequence listing (excluding the final stop codon TGA). Recombinant plasmid p35S is GbOFP7 protein shown in sequence 3 in the GbOFP7-GFP expression sequence table. The recombinant plasmid p35S is an expression cassette containing GbOFP7-GFP fusion protein, and the promoter for promoting the transcription of GbOFP7 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.

Recombinant Agrobacterium GV3101/GbOFP7-GFP positive bacteria liquid is added into YEP liquid culture medium containing 50 mug/mL Kan and 50 mug/mL Rif, the strain is activated in a shaking table (28 ℃ C., 160 rpm), 1mL activated bacteria liquid is added into 20mL YEP liquid culture medium containing 50 mug/mL Kan and 50 mug/mL Rif, 28 ℃ C., 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.

Selecting good-growth Nicotiana benthamiana (Nicotiana benthamiana), lightly injecting recombinant Agrobacterium GV3101/GbOFP7-GFP suspension containing GbOFP7-GFP into the back of tobacco leaves by using a syringe, simultaneously injecting the Agrobacterium GV3101/GFP suspension containing GFP empty vector 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 GbOFP7 protein by using a Zeiss LSM 880 inverted confocal fluorescence microscope, and exciting light 488nm and emitted light 510nm.

The results are shown in fig. 9 a and b, where the GFP signal of the GbOFP7-GFP fusion protein is localized to the cell membrane and nucleus, indicating that GbOFP7 may play a role on the cell membrane and in the nucleus.

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 or 2 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 or 2, to obtain a plant with increased resistance to wilt;

2) Increasing, enhancing and/or upregulating the expression level of a gene encoding a protein according to claim 1 or 2 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 or 2, 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.