CN112209997A

CN112209997A - Gossypium hirsutum anti-verticillium wilt-related GhBZR1 protein, and coding gene and application thereof

Info

Publication number: CN112209997A
Application number: CN202011047280.3A
Authority: CN
Inventors: 简桂良; 贺浪; 张华崇
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2021-01-12

Abstract

The invention discloses a greencotton verticillium wilt resistance-related GhBZR1 protein, a coding gene and application thereof, wherein the nucleotide sequence of the protein is shown as SEQ ID No. 1. The invention provides a protein coded by a greencotton Verticillium wilt resistance related gene GhBZR1, which is obtained by cloning from 86-1 leaves of a greencotton variety infected with Verticillium wilt to obtain the Verticillium wilt resistance related gene GhBZR1, and is proved to be synergistically expressed in the leaves and roots under the stress of Verticillium dahliae by RT-PCR. The evaluation of the disease resistance of arabidopsis thaliana which is transformed to over-express the gene proves that the gene can improve the verticillium dahliae resistance of upland cotton varieties which are infected with verticillium wilt, so that the gene can be used for improving the genetic improvement of verticillium wilt resistance of upland cotton or the genetic improvement of verticillium wilt resistance of other verticillium dahliae host crops.

Description

Gossypium hirsutum anti-verticillium wilt-related GhBZR1 protein, and coding gene and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a Gossypium hirsutum anti-greensickness related GhBZR1 protein, and a coding gene and application thereof.

Background

Cotton is an important economic crop and plays an important role in national economy, and the cotton is not only an important raw material for textile, chemical industry, medicine and national defense industry, but also an important export-exchange commodity. Among them, upland cotton (gossypium hirsutum) is the main species of plant, and accounts for over 99% of the cotton area in our country. The verticillium wilt is one of the most important diseases in cotton production, the 1930 s are introduced into China, the 1950 s in the beginning of liberation begin to spread and generate damages in some areas of China's cotton province, the transmission is very rapid along with the regulation and transportation of cotton seeds with bacteria, and in the beginning of 1990, the damages are aggravated year by year in main cotton areas of China and the damages are developed in one year in individual years.

The main pathogen causing Verticillium wilt is Verticillium dahliae (Verticillium dahliae). The verticillium wilt is a main soil-borne disease, the host range of pathogenic bacteria of the verticillium wilt is wide, the verticillium wilt is easy to change, no special parasitic relation exists, and a dormant body, namely microsclerotia and the like which can survive for a long time can be formed under adverse environmental conditions, so that the verticillium wilt is extremely difficult to control and is one of the main obstacles for the sustainable development of cotton in China. Long-term research at home and abroad shows that the comprehensive control is the most economic and effective measure for preventing and treating the verticillium wilt, and the breeding of disease-resistant varieties is the most important, economic and ecological main content of the comprehensive control. However, the disease resistance is controlled by polygene, and the disease resistance is complicated by the epidemic, so that the difficulty in breeding disease-resistant varieties is high. In China, disease-resistant varieties (strains) are lacked all the time, and particularly, the disease-resistant varieties of upland cotton which account for 99.9% of the planting area in China are particularly scare, which is also an important reason for causing the disease to be epidemic and harmful at times.

The immune response (PTI) triggered by pathogen-associated pattern molecules plays an important role in the process of resisting the invasion of pathogens by plants, and the recognition receptors (PRRs) of the plants can recognize the associated pattern molecules (PAMPs) on the surfaces of the pathogens to promote the PTI. PRRs of the leucine-rich repeat receptor kinase (LRR-RKS) class rely on the regulation of LRR-RKBAK1 to transmit signals, BAK1(BRI 1-assisted transcriptional kinase1) also interacts with LRR-RKBRI1, BAK1 and BRI1 (Brassinosteroid-peptide-reactive 1) are the main receptors of Brassinosteroid hormone (BR) and can promote the transmission of BR signals, in the immune mechanism of plants, the signals of exogenous pathogen invasion can be received by BAK1 and transmitted by signal step, BR signals activate phosphatase PP2A, so that BZR1 and BES1 are dephosphorylated and combined with the promoters of a large number of genes, thereby regulating the expression of the genes; BR is also a positive regulator mediating plant growth.

BR is a hormone necessary for wide development and physiological processes in plant life history, and plays an important role in plant growth and development and stress response. In most of the past reports, genes related to biosynthesis or signal transduction of BRs are studied and are related to a series of phenotypes such as plant dwarfing, flowering delay, aging and the like. It has been shown that BES1/BZR1 is the only transcription factor of the BR signal transduction pathway. BR also plays an important role in the process of infecting plants by pathogens, and Albrecht et al find that BR signals play an important role in immune regulation in the process of plant growth by regulating immune signals at the downstream of leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1 and are potential regulation sites in the process of pathogen infection. Physiological studies have also shown that BRs promote cell elongation, enhance tolerance to environmental stress and resistance to pathogen infestation, and thereby increase crop yield. Research on arabidopsis thaliana by Lozano-Duran R and the like discovers that BZR1 can induce the expression of several WRKY transcription factors (WRKY15, WRKY18 and WRKY11) and HBI1, and the transcription factors have negative control effect on early immune response; in addition, BZR1 also binds to WRKY40, mediating antagonism between BR and immune signals, and finally BZR1 mediated transcriptional changes will lead to inhibition of PTI signaling. Therefore, BZR1 is an important regulator of BR signals, can induce the expression of negative regulators of PTI to inhibit the immune defense line of plants, and is favorable for the invasion of pathogens.

The currently published BZR genes are mostly derived from model plants and herbaceous plants, and part of forest trees are also cloned to obtain the BZR genes, such as Chinese red pine, poplar and the like. Upland cotton has important economic value as the main planting species in China, but no research report of BZR homologous genes exists at present.

Disclosure of Invention

In order to solve the technical problems described in the background technology, the invention provides a BZR1 regulatory factor protein of upland cotton, wherein BZR genes related to upland cotton disease resistance are obtained by cloning from upland cotton leaves, the important role of the BZR regulatory factor protein in anti-verticillium wilt is proved through VIGS and over-expression in Arabidopsis, the synergistic expression in leaves and roots of high-susceptibility verticillium wilt varieties is proved according to transgenic Arabidopsis verticillium wilt resistance evaluation, the capability of reducing the verticillium wilt resistance is proved, and the genetic improvement of the verticillium wilt resistance of high-quality high-susceptibility cotton varieties can be improved if the transgenic Arabidopsis thaliana is silenced.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the nucleotide sequence of the invention is shown in SEQ ID NO. 1.

The Gossypium hirsutum anti-greensickness related GhBZR1 protein is coded, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

A primer pair for amplifying the greensickness resistant GhBZR1 protein of the upland cotton is used for cloning a greensickness resistance related gene GhBZR1, and the primer pair comprises:

5’OGhBZR1：5’—GATTACGCCAAGCTTCGGTGGGTAGGACTAGCTGGGGC—3’

3’OGhBZR1：5’—TGCCTCCCCAAAATTTGCAC—3’。

a gene cloning method of GhBZR1 protein related to verticillium wilt of gossypium hirsutum includes

(1) Extracting total RNA of upland cotton leaves;

(2) 3' -RACE experiment of total RNA of upland cotton leaves;

(3) 5' -RACE experiment of total RNA of upland cotton leaves;

(4) and analyzing and splicing the PCR sequencing results of the 3 '-RACE and the 5' -RACE to obtain the GohBZR 1 protein.

The GhBZR1 protein and the coding GhBZR1 protein are applied to the upland cotton in improving the verticillium dahliae resistance of upland cotton varieties infected with verticillium wilt.

The invention has the beneficial effects that:

the invention provides a GhBZR1 protein of a resistance-related gene of upland cotton and verticillium wilt, which is obtained by cloning a GhBZR1 gene related to the verticillium wilt resistance of upland cotton from cotton leaves, and is proved to be synergistically expressed in the leaves and roots of a verticillium wilt-sensitive cotton variety through RT-PCR (reverse transcription-polymerase chain reaction).

Drawings

FIG. 1 shows the secondary structure of the protein of the present invention.

FIG. 2 shows the tertiary structure of the protein of the present invention.

FIG. 3 shows that the disease resistance of susceptible variety is enhanced by silencing GhBZR1 gene of susceptible variety 86-1 by VIGS technology.

FIG. 4 shows that the transgenic Arabidopsis thaliana is more serious under the stress of Verticillium wilt disease compared with the transgenic Arabidopsis thaliana which overexpresses the GhBZR1 gene and is inoculated with a Verticillium wilt disease strong pathogenic bacteria line V991.

Detailed Description

The present invention will now be described in further detail with reference to the accompanying drawings.

A protein structure of a gene GhBZR1 related to resistance of upland cotton related to verticillium wilt;

the nucleotide of the full-length cDNA of the gene GhBZR1 related to the resistance of upland cotton and verticillium wilt is the nucleotide sequence shown in SEQ ID No. 1;

the protein coded by the gene GhBZR1 related to the resistance of upland cotton related to verticillium wilt is an amino acid sequence shown in SEQ ID No. 2.

Specifically, the primer pair for cloning the GhBZR1 gene is as follows:

qGhBZR1-F：5’—GCTTCCCTTTTCATCGTTTTCA—3’

qGhBZR1-R：5’—GCGATTAAGTTTGGATTGGATTG—3’。

the protein of the gene GhBZR1 related to resistance of upland cotton and verticillium wilt is 1) the protein consisting of amino acid shown in SEQ ID No. 2; or 2) the amino acid sequence shown in SEQ ID No. 2; or 3) protein which is derived from the protein of 1) and has equivalent activity by substituting, deleting or adding one or more amino acids.

Depending on the biological properties, one skilled in the art can substitute, delete and/or add one or several amino acids based on the disclosed amino acid sequences without affecting their biological activity to obtain mutated sequences of the proteins.

Therefore, the Gossypium hirsutum and verticillium wilt resistance-related gene GhBZR1 protein also comprises a protein which is obtained by substituting, deleting or adding one or more amino acids in an amino acid sequence shown in SEQ ID No.2, has the same activity as the GohBZR 1 protein related to the GohBZR 1 gene of Gossypium hirsutum and verticillium wilt resistance-related gene GhBZR1 protein, and is derived from the Gossypium hirsutum and verticillium wilt-related gene GhBZR1 protein.

The invention also provides a coding gene sequence of the protein, and the nucleotide sequence is shown as SEQ ID No. 1. Furthermore, it will be appreciated that, given the degeneracy of codons and the preference of codons for different species, one skilled in the art can use codons suitable for expression in a particular species as desired.

The invention separates GhBZR1 gene from upland cotton, and transfers GhBZR1 gene separated from upland cotton into Arabidopsis thaliana by agrobacterium-mediated method, thus obtaining transgenic Arabidopsis thaliana. The transgenic Arabidopsis thaliana differs from the wild type Arabidopsis thaliana in that the transgenic Arabidopsis thaliana has reduced verticillium wilt resistance.

The molecular cloning steps of the upland cotton GhBZR1 gene from a DNA sequence are as follows:

(1) extracting total RNA of upland cotton leaves;

(2) 3' -RACE experiment of total RNA of upland cotton leaves;

(3) 5' -RACE experiment of total RNA of upland cotton leaves;

(4) and analyzing and splicing the PCR sequencing results of the 3 '-RACE and the 5' -RACE to obtain the full-length cDNA sequence of the GohBZR 1 gene of upland cotton, wherein the length is 1515bp, and the sequence is shown as SEQ ID No. 1.

Secondary structure and tertiary structure of GhBZR1 protein

As shown in the attached figure 1, the secondary structure of the GhBZR1 protein mainly consists of an Alpha helix (Alpha helix) and a Random coil (Random coil), wherein the Alpha helix proportion is 19.49%, and the Random coil proportion is 69.33%; the tertiary structure of the protein is shown in figure 2.

Cis-acting element of GhBZR1 gene promoter region

The promoter region (1000bp) of the GhBZR1 gene is a cis-acting element related to pathogenic bacteria resistance. There are three classes of related action elements of GhBZR1 (table 1). At-510, there is a response element in the promoter, TC-rich repeats, that reacts to defense and stress; at +604 and +605, there are two ABRE cis-acting elements that react with abscisic acid; and an MBSI action element of which MYB binding site is involved in flavonoid biosynthesis gene regulation is positioned at-843.

TABLE 1 cis-acting elements of GhBZR1 associated with pathogen resistance

Example 1:

verticillium dahliae stress silences the verticillium wilt resistance state of the verticillium dahliae-infected upland cotton variety 86-1.

(1) Material

The cotton planting 86-1 in wild type greensickness-induced upland cotton varieties and the cotton planting 86-1 in greensickness-induced upland cotton varieties which silence GhBZR1 genes are taken as experimental materials.

(2) Preparation of culture medium

Control group: wild type 86-1;

experimental groups: cotton 86-1 is planted in greencotton variety with greensickness of GhBZR1 gene silencing, and cotton 86-1 is planted in a transformation empty vector (CLCrV-00) to inoculate V991.

(3) Test design and Property measurement

Before sowing, delinting with concentrated sulfuric acid, selecting plump seeds with consistent size for subsequent test, soaking the seeds in 70% ethanol for 5min for surface sterilization, soaking the seeds in 3% hydrogen peroxide for 2h, and finally washing with sterile water. And (3) dibbling the sterilized seeds in a flowerpot (nutrient soil: vermiculite: 2:1), placing the flowerpot in a greenhouse with the temperature of 24 ℃, the light for 16h, the darkness for 8h and the relative humidity of 70 percent for growing, when the cotton plants grow out of true leaves, transferring the cotton plants to a beaker, and culturing the cotton plants by adopting a nutrient culture method.

The bacterial line for measuring the verticillium wilt resistance is deciduous strong pathogenic bacteria V991. Firstly, inoculating the stored V991 into a PDA culture medium, culturing for one week at 26 ℃, and placing the activated V991 into a Chachi culture medium at 26 ℃, 200rpm and 5-6 d; filtering the bacterial solution to the required concentration into a sterilized beaker, measuring the concentration of the spore suspension by using a blood counting plate, and diluting the concentration of the spore suspension to 10⁷one/mL.

(4) Measurement results

The roots of both wild type and transgenic plants growing in the vermiculite culture flowerpot containing nutrient soil can grow well. After cotton seedlings grew for one month, suspension of spores of the strong pathogenic pathogen V991 of the deciduous leaf type (concentration 10) was used⁷seed/mL) and inoculating the germs by a root dipping method, and investigating the pathogenesis of the verticillium wilt after 5, 10 and 15 days of inoculation. The result shows that the disease resistance of the plant can be obviously improved by silencing the GhBZR1 gene, so that the verticillium wilt resistance of the plant can be enhanced. As shown in FIG. 3, after silencing the gene GhBZR1 by VIGS, the disease resistance of the verticillium wilt-susceptible variety is improved under the stress of verticillium wilt. The wild type verticillium wilt-susceptible variety and the empty vector-transferred verticillium wilt-susceptible cotton variety are still highly susceptible to verticillium wilt; their incidence and disease index are shown in table 2. After the gene GhBZR1 is silenced by adopting VIGS, the morbidity and disease index of a verticillium wilt-infected variety are compared under the stress of verticillium wilt.

The GhBZR1 planted with 86-1 in susceptible varieties is silenced, the incidence rate of verticillium wilt is only 26.5 +/-2.1%, the disease index is only 18.1 +/-2.1, and is extremely obviously lower than the incidence rate of 81.3 +/-1.9% and the disease index of 42.57 +/-1.5 of wild type. The capital letters in the table represent significant differences at the 1% level.

Example 2:

and (3) detecting the Verticillium wilt resistance of transgenic over-expression GhBZR1 gene Arabidopsis thaliana under the stress of Verticillium dahliae.

(1) Material

Second generation arabidopsis thaliana and wild type arabidopsis thaliana which are used for trans-overexpression of the GhBZR1 gene are taken as experimental materials.

(2) Culture medium

Culture medium: 1/2MS culture medium; 1/2MS +50mg/L kanamycin medium

A culture medium: vermiculite +1/4MS nutrient solution

(3) Assay design and disease resistance determination

Wild type Arabidopsis thaliana was cultured in 1/2MS medium, and transgenic Arabidopsis thaliana was cultured in 1/2MS +50mg/L kanamycin medium. After the seedlings grow to a certain size, the kanamycin-resistant transgenic arabidopsis thaliana is transplanted to a nutrition pot. Transplanting for 7 days, then carrying out verticillium dahliae stress treatment, and inoculating verticillium dahliae V991 to the over-expression plant by adopting a root dipping method. A total of 3 treatment conditions were set, Control (CK): wild Col-0 type Arabidopsis thaliana and a transformed empty vector Arabidopsis thaliana are inoculated with V991 respectively as negative controls; the Arabidopsis thaliana plants over-expressing GhBZR1 are inoculated with V991 for treatment respectively. Inoculating verticillium dahliae (verticillium dahliae) V991 (the concentration of spore suspension is 10) by adopting a root irrigation method⁵one/mL). Photographing observation and determination of morbidity are carried out 15 days after inoculation.

(4) Measurement results

The difference between the morbidity and the disease index of the wild Col-0 arabidopsis thaliana and the transformation empty vector arabidopsis thaliana inoculated with V991 is not obvious, but the morbidity and the disease index of the verticillium wilt of a plant over-expressing GhBZR1 gene are obviously higher than those of the wild Col-0 arabidopsis thaliana and the transformation empty vector arabidopsis thaliana.

As shown in FIG. 3, the disease resistance of the susceptible variety was enhanced by silencing the GhBZR1 gene of the susceptible variety 86-1 by the VIGS technique. Wherein A is 86-1 of wild type planted cotton of a non-cultured bacterium; b is inoculated wild type planted cotton 86-1; c is cotton 86-1 inoculated strain V991 in a transformation empty vector (CLCrV-00); d is cotton 86-1 inoculated strain V991 planted in GhBZR 1.

TABLE 2 comparison of the disease index of verticillium wilt after silencing the GhBZR1 gene of susceptible variety 86-1 and inoculating the Strong pathogenic strain of verticillium wilt V991

As shown in FIG. 4, the pathogenesis of verticillium wilt disease is compared after arabidopsis thaliana over-expressing GhBZR1 gene is inoculated with verticillium wilt bacterium strong pathogenic bacteria line V991. Wherein, A is V991 inoculated by wild Col-0 type arabidopsis thaliana; b is a transformation empty vector Arabidopsis thaliana inoculation V991; c is inoculated V991 of Arabidopsis thaliana with overexpression GhBZR 1.

TABLE 3 comparison of disease indexes of verticillium wilt after over-expressing GhBZR1 gene Arabidopsis thaliana and inoculating verticillium wilt bacterium strong pathogenic bacterium line V991

Type of silencing	Incidence of disease	Index of disease condition
			Inoculation of wild type Col-0 type Arabidopsis thaliana with V991	32.3±3.9B	26.4±2.9B
Transformation of empty vector Arabidopsis thaliana for V991 inoculation	35.3±2.9B	28.5±1.6B
			V991 inoculated in Arabidopsis thaliana with overexpression of GhBZR1	56.3±1.4A	43.7±0.8A

As shown in figure 4, the incidence of Arabidopsis with overexpression of the GhBZR1 gene is significantly higher than that of the wild type and is higher than that of the wild type by 24.0 percent under the stress of Verticillium dahliae (Table 3). The incidence and disease index of the plants over-expressing GhBZR1 are significantly higher than those of the wild type. This means that GhBZR1 plays an important role in plant response to Verticillium dahliae, and the GhBZR1 gene has the capability of reducing the verticillium wilt resistance of plants. Functionally proves the important function of the GhBZR1 upland cotton in resisting verticillium wilt.

In light of the foregoing description of the preferred embodiment of the present invention, it is to be understood that various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.

Sequence listing

<110> institute of plant protection of Chinese academy of agricultural sciences

<120> upland cotton verticillium wilt resistance-related GhBZR1 protein, and coding gene and application thereof

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cttttaacgt ttttttttgt ttccacccaa gctaaagcgt attcctcgtt actcgtgatc 120

ttctccactt cttccttctt tttctttgtt tggtgttata atcctctaca acgcaccttc 180

acgccggaga gagagagaga gagagagaga gagagagaga gaaatgacat cggatggggc 240

gacgtcgacg ccggctccaa ggaggaaacc gtcgtggagg gagagggaga ataacaggag 300

gagagaaagg aggagaagag ccattgctgc taagatttat actggcctta gagctcaagg 360

gaactacaat ttgcccaagc actgtgataa caatgaggtc ctcaaagctc tttgttcgga 420

ggctggttgg gtcgtagaag atgatggcac cacttatcgc aagggatgta agccacctcc 480

aatagatata ggtggaagtt caagtaagat taccccattt tcatcccaaa atccaagtcc 540

attatcatct gcatttccaa gtccaattcc ttcatgtcaa gtcagccctt cctcctcttc 600

ctaccctagt cccactaggt ttgatgctaa taacccctct actctccttc ccttccttcg 660

aaacgccatt ccttcatctc tgcctccact cagaatctca aacagtgccc ctgttacacc 720

cccactttca tctcctacct caagaaatcc taagcccctt cccaactggg aaaccattgc 780

aaaggaatcc atggcctctt ttaactaccc tttttatgct gtttctgccc cagctagtcc 840

tacccaccgt cattttcatg ccccggctac tataccggaa tgtgatgaat ccgatacatc 900

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tgatttgggc gtaaaagata aaggaagagg tgcagagttt gagtttgaga gtggacatct 1080

gaaaccatgg gaaggagaga ggattcatga cataggaatg gatgatctgg agctcacgct 1140

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Ser Trp Arg Glu Arg Glu Asn Asn Arg Arg Arg Glu Arg Arg Arg Arg

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Ala Ile Ala Ala Lys Ile Tyr Thr Gly Leu Arg Ala Gln Gly Asn Tyr

35 40 45

Asn Leu Pro Lys His Cys Asp Asn Asn Glu Val Leu Lys Ala Leu Cys

50 55 60

Ser Glu Ala Gly Trp Val Val Glu Asp Asp Gly Thr Thr Tyr Arg Lys

65 70 75 80

Gly Cys Lys Pro Pro Pro Ile Asp Ile Gly Gly Ser Ser Ser Lys Ile

85 90 95

Thr Pro Phe Ser Ser Gln Asn Pro Ser Pro Leu Ser Ser Ala Phe Pro

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Ser Pro Ile Pro Ser Cys Gln Val Ser Pro Ser Ser Ser Ser Tyr Pro

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Ser Pro Thr Arg Phe Asp Ala Asn Asn Pro Ser Thr Leu Leu Pro Phe

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Leu Arg Asn Ala Ile Pro Ser Ser Leu Pro Pro Leu Arg Ile Ser Asn

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Ser Ala Pro Val Thr Pro Pro Leu Ser Ser Pro Thr Ser Arg Asn Pro

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Lys Pro Leu Pro Asn Trp Glu Thr Ile Ala Lys Glu Ser Met Ala Ser

180 185 190

Phe Asn Tyr Pro Phe Tyr Ala Val Ser Ala Pro Ala Ser Pro Thr His

195 200 205

Arg His Phe His Ala Pro Ala Thr Ile Pro Glu Cys Asp Glu Ser Asp

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Thr Ser Thr Val Glu Ser Gly Gln Trp Ile Ser Phe Gln Lys Phe Ala

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Pro Ser Thr Ser Gln Val Pro Thr Ser Pro Thr Phe Phe Lys Leu Val

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Lys His Leu Pro Pro Gln Asn Leu His Asn Asp Leu Gly Val Lys Asp

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Lys Gly Arg Gly Ala Glu Phe Glu Phe Glu Ser Gly His Leu Lys Pro

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Trp Glu Gly Glu Arg Ile His Asp Ile Gly Met Asp Asp Leu Glu Leu

290 295 300

Thr Leu Gly Ser Gly Lys Pro Gln Cys

305 310

Claims

1. The upland cotton verticillium wilt resistance-related GhBZR1 protein is characterized in that: the nucleotide sequence is shown in SEQ ID NO. 1.

2. The Gossypium hirsutum anti-greensickness-associated GhBZR1 protein of claim 1, wherein the amino acid sequence of the protein is represented by SEQ ID No. 2.

3. A primer for amplifying the anti-verticillium wilt-associated GhBZR1 protein of upland cotton as shown in claim 1, which is characterized in that: the primer pair for cloning the greensickness-resistant related gene GhBZR1 of upland cotton is as follows:

5’OGhBZR1：5’—GATTACGCCAAGCTTCGGTGGGTAGGACTAGCTGGGGC—3’

3’OGhBZR1：5’—TGCCTCCCCAAAATTTGCAC—3’。

4. a gene cloning method of GhBZR1 protein related to verticillium wilt of gossypium hirsutum, which is characterized by comprising the following steps: comprises that

(1) Extracting total RNA of upland cotton leaves;

(2) 3' -RACE experiment of total RNA of upland cotton leaves;

(3) 5' -RACE experiment of total RNA of upland cotton leaves;

(4) and analyzing and splicing the PCR sequencing results of the 3 '-RACE and the 5' -RACE to obtain the anti-verticillium wilt GhBZR1 protein gene of the upland cotton.

5. The use of the nucleotide sequence of GhBZR1 as claimed in claim 1 and the protein encoding GhBZR1 as claimed in claim 2 in improving the verticillium wilt resistance of upland cotton varieties and other crops.