CN110343157B

CN110343157B - Cotton verticillium wilt related gene GhBONI and encoding protein and application thereof

Info

Publication number: CN110343157B
Application number: CN201910700649.7A
Authority: CN
Inventors: 郝晓燕; 黄全生; 李建平; 高升旗; 足木热木; 常晓春; 胡文冉; 陈果
Original assignee: Xinjiang Academy Of Agricultural Sciences Institute Of Nuclear Technology Biotechnology (xinjiang Uygur Autonomous Region Biotechnology Research Center)
Current assignee: Xinjiang Academy Of Agricultural Sciences Institute Of Nuclear Technology Biotechnology (xinjiang Uygur Autonomous Region Biotechnology Research Center)
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2022-06-28
Anticipated expiration: 2039-07-31
Also published as: CN110343157A

Abstract

The invention discloses a cotton verticillium wilt related gene GhBONI, and a coding protein and application thereof. The invention provides a protein with the amino acid sequence of SEQ ID No.1 or with 99%, 95%, 90%, 85% or more than 80% of identity and the same function through substitution and/or deletion and/or addition of one or more amino acid residues, or a fusion protein formed after connecting a label at the N end and/or the C end of the protein. The GhBONI gene sequence is used for constructing a VIGS plant expression vector to transform upland cotton TM-1, and the obtained transgenic cotton shows the resistance to verticillium wilt after being inoculated with cotton verticillium wilt V991, which indicates that the GhBONI gene and the protein coded by the GhBONI gene can participate in a cotton verticillium wilt resistance mechanism. The invention has important significance for cultivating the verticillium wilt-resistant transgenic cotton.

Description

Cotton verticillium wilt related gene GhBONI and encoding protein and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a cotton verticillium wilt related gene GhBONI, and a coding protein and application thereof.

Background

Cotton is an important commercial crop in the world, being the largest crop of fibre production scale in the world. Cotton (Gossypium hirsutum L) in the genus Gossypium is the most widely cultivated cotton species at present due to its good fiber quality, high yield and short growth cycle, and the yield accounts for about 95% of the total world cotton yield. However, cotton Verticillium wilt (Verticillium wilt), a soil-borne pathogenic fungus, Verticillium dahliae, has a devastating vascular bundle disease in cotton production, severely restricts the growth and development of cotton, and becomes the first disease of cotton worldwide, called "cancer" of cotton. In recent thirty years, the disease occurs in all cotton areas of China, and is outbreak and disasters in partial years and regions. To date, cotton verticillium wilt cannot be effectively controlled in upland cotton, and upland cotton varieties with high verticillium wilt resistance are still unavailable at present. Years of practice prove that the development of disease-resistant genes, the understanding of the reaction mechanism of cotton to verticillium wilt, the acceleration of the breeding process of cotton verticillium wilt resistance and the improvement of the disease resistance of cotton become the main approaches of cotton verticillium wilt resistance breeding work.

With the rapid development of molecular biology, people gradually deepen the understanding of the molecular mechanism of the plant biological stress response, and find that a Disease Resistance gene (R) is a key factor for mediating the differential regulation of defense response among species. The disease-resistant gene R can directly or indirectly identify the nontoxic gene product of pathogenic bacteria, thereby inducing a series of downstream defense reactions. Plants containing the R gene can resist infection of germs carrying the corresponding AVR gene, while plants without the R gene show susceptibility to germs carrying the corresponding AVR gene. Most of NB-LRR proteins encoded by plant R genes are homologous proteins of animal immune receptors (NLRs), and researches show that the R genes have tight regulation and control in plant transcription level, and the regulation and control play an important role in plant defense response and growth balance. Under sterile conditions, the expression level of the R gene is generally lower, the expression of the R gene is activated under the induction of pathogen infection, and constitutive up-regulation expression or over-expression of the R gene can influence the normal growth of plants and even cause the death of the plants. In addition, Reactive Oxygen Species (ROS) serving as signal molecules are found in disease-resistant defense reactions of plants to interact with signal pathways such as calcium ion signals, protein phosphorylation and hormone regulation, so that the disease resistance of the plants is changed. The transgenic research process of cotton is time-consuming and labor-consuming, the Virus-induced gene silencing technology (VIGS) is a ubiquitous genetic immune mechanism in plants, belongs to post-transcriptional gene silencing, plants are subjected to silencing phenotype by silencing target genes, so that the functions of the target genes are judged, and the genetic transformation and gene function redundancy of the plants are avoided. The VIGS technology can invade agrobacterium into plants in a short time, and inoculate a large number of plant seedlings or roots to complete gene transformation to research the functions of target genes.

Disclosure of Invention

The invention aims to provide a cotton verticillium wilt related gene GhBONI, and a coding protein and application thereof.

In a first aspect, the invention claims a protein.

The protein claimed by the invention is derived from cotton (Gossypium hirsutum L.) and can be any one of the following:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In a second aspect, the invention claims nucleic acid molecules encoding said proteins.

The nucleic acid molecules claimed in the present invention may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA, rRNA, or tRNA, among others.

Further, the nucleic acid molecule is a gene encoding the protein, and the gene may be a DNA molecule described in any one of the following:

(B1) DNA molecule shown in SEQ ID No. 2;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes said protein;

(B3) a DNA molecule which has 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity to the DNA sequence defined in (B1) or (B2) and which encodes the protein.

The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In a third aspect, the invention claims recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria comprising the above-described nucleic acid molecules.

The recombinant vector claimed in the present invention may be a recombinant expression vector, and may also be a recombinant cloning vector.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pCAMBIA1300, pCUbi1390, pCHF3, pGreen0029, pCAMBIA3301, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UBIN or other derivative plant expression vectors. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as a cauliflower mosaic virus (CAMV)35S promoter, a Ubiquitin gene Ubiquitin promoter (pUbi), a stress-inducible promoter rd29A and the like, can be added before the transcription initiation nucleotide, and 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 recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant 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 change, antibiotic markers having resistance or chemical resistance marker genes, etc., which are expressed in plants. Or directly screening the transformed plants in a stress environment without adding any selective marker gene.

The expression cassette claimed by the invention consists of a promoter capable of promoting the expression of said gene, and a transcription termination sequence.

In a fourth aspect, the invention claims the use of said protein or said nucleic acid molecule or said recombinant vector, expression cassette, transgenic cell line or recombinant bacterium in any one of:

(a) regulating and controlling plant disease resistance;

(b) breeding plant varieties with enhanced or weakened disease resistance;

(c) and (5) plant breeding.

In (a), the modulating plant disease resistance may be embodied as: in the plant, the expression level of the protein (or the gene) is decreased, and the plant is increased in resistance to diseases.

In the method (b), the method for breeding a plant variety with enhanced disease resistance may specifically comprise the step of crossing a plant with a relatively low expression level of the protein (or the gene) as a parent. The method for breeding a plant variety with reduced disease resistance may specifically include a step of crossing a plant having a relatively high expression level of the protein (or the gene) as a parent.

In (c), the plant breeding may be specifically breeding of a plant variety with enhanced disease resistance.

In a fifth aspect, the invention claims a method of breeding a plant variety.

The method for cultivating plant varieties claimed by the invention can be a method A or a method B:

the method A comprises the following steps: a method for breeding a plant variety having enhanced disease resistance, comprising the step of reducing the expression level and/or activity of the protein in a recipient plant;

the method B comprises the following steps: a method for breeding a plant variety with reduced disease resistance, comprising the step of increasing the expression level and/or activity of the protein in a recipient plant.

In a sixth aspect, the invention claims a method of breeding transgenic plants.

The method for cultivating a transgenic plant claimed in the present invention may be method C or method D:

the method C comprises the following steps: a method of breeding a transgenic plant with enhanced disease resistance comprising the steps of: inhibiting the expression of the coding gene of the protein in the receptor plant to obtain a transgenic plant; the transgenic plant has enhanced disease resistance as compared to the recipient plant;

the method D comprises the following steps: a method of breeding a transgenic plant with reduced disease resistance comprising the steps of: introducing into a recipient plant a nucleic acid molecule capable of expressing said protein, resulting in a transgenic plant; the transgenic plant has reduced disease resistance as compared to the recipient plant.

In method C, inhibiting the expression of the gene encoding the protein in the recipient plant can be specifically achieved by: introducing a pTRV2-GhCLA1 vector and a pTRV1 vector into the recipient plant; the pTRV2-GhCL 1 vector is a recombinant vector obtained by inserting the DNA fragment shown in the 1 st to 417 th sites of SEQ ID No.2 into the restriction enzyme site EcoR I and Kpn I of the pTRV2 vector.

In the method D, the gene can be specifically introduced into the recipient plant through the recombinant expression vector to obtain the transgenic plant. The recombinant expression vector is a recombinant vector which can express the gene and is obtained by inserting the gene into a multiple cloning site of a plant expression vector.

In each of the above aspects, the disease may be verticillium wilt.

In the above aspects, the disease may be a plant disease caused by Verticillium dahliae.

Further, the verticillium wilt disease can be cotton verticillium wilt disease.

Further, the Verticillium dahliae may be Verticillium dahliae strain V991 (i.e., Verticillium dahliae strain V991).

In each of the above aspects, the plant may be a dicot.

Further, the dicot may be a cotton plant.

Further, the cotton plant may be cotton.

The invention provides a cotton calcium-dependent phospholipid-binding protein BONI gene and a coding protein thereof, the gene is amplified by utilizing GhBONI gene sequence information, a VIGS plant expression vector is constructed to transform upland cotton TM-1, and the obtained transgenic cotton shows resistance to verticillium wilt after being inoculated with verticillium wilt cotton V991, which indicates that the GhBONI gene and the coding protein thereof can participate in a verticillium wilt resistance mechanism of cotton. The invention has important significance for culturing transgenic cotton with verticillium wilt resistance.

Drawings

FIG. 1 is a comparison of the successful establishment of TRV-mediated VIGS system in upland cotton TM-1 and the growth of cotton plants inoculated with verticillium dahliae V99112 days after two weeks of VIGS. A is the successful establishment of a TRV mediated VIGS system in upland cotton TM-1. TRV: 00 (control): injection of unloaded pTRV 2; TRV: ghLA 1: albino gene was injected. B is the growth condition comparison of cotton plants inoculated with verticillium dahliae V99112 days after two weeks of VIGS. TRV: 00 (control): injection of unloaded pTRV 2; TRV: BONI: GhBONI gene is injected.

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

FIG. 3 is a statistical analysis of morbidity and disease indices.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Terrestrial cotton TM-1: obtained to the cotton institute of Chinese academy of agricultural sciences. Reference: the fingerprint of the cotton variety is constructed by using a Cotton SNP63K chip, Chinese agricultural science, 2017,50(24), 4692-containing materials 4704, which can be obtained by the public from the applicant, can only be used for repeated invention experiments and cannot be used for other purposes.

Plant VIGS silencing expression vector:

pTRV 1: changsha Yingrun biotechnology, Inc., cat # VRW 0365;

pTRV 2: changsha Yingrun Biotechnology Inc., cat # VRW 0366.

pTRV 2-ghcia 1, reference: liuhui, in Xiu, Huang Xian faithful, establishment of TRV virus-mediated gene silencing system in Xinjiang Gossypium hirsutum and Asian Cotton, Cotton Science 2016,28(5): 485-492. The applicant can obtain the said product, and can only use it for repeating the experiment of the invention, and has no other use.

Agrobacterium GV 3101: the development area of the Wuluqigao new technology Kerui Dai business, AC 1001.

Verticillium wilt bacteria V991: the laboratory is kept and publicly available from the institute of biotechnology, nuclear technology, academy of agricultural sciences (Xinjiang Uygur autonomous area Biotechnology research center). Reference documents: zhanhui, tianxindong, gao wei, cai emergency, longluo, upland Cotton PPO gene genome wide identification and response analysis to verticillium wilt pathogen cottonslaugh Cotton Science 2017, 29 (5): 428-436. The applicant can obtain the said product, and can only use it for repeating the experiment of the invention, and has no other use.

Example 1 obtaining of Cotton verticillium wilt-associated Gene GhBONI and its encoded protein

Extracting the total RNA of the cotton variety Gossypium hirsutum TM-1, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification by using the obtained cDNA as a template and adopting a primer GhBONI ORF-F and a primer GhBONI ORF-R.

Primer GhBONIORFF: 5'-ATGGGAAATTGCTGCTCCGA-3', respectively;

primer GhBONIORFR: 5'-TCAAATCCTGGGTTTAATAT-3' are provided.

Sequencing the amplified product to obtain the cDNA nucleotide sequence of SEQ ID No.2, and naming the gene as GhBONI with the amino acid sequence of the encoded protein of SEQ ID No. 1.

The above cDNA (SEQ ID No.2) can also be obtained by artificial synthesis.

Example 2 application of cotton verticillium wilt related gene GhBONI and encoding protein thereof

Construction of recombinant expression vector

1. Extracting total RNA of cotton (upland cotton standard system TM-1), carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification by using the cDNA as a template and a primer pair consisting of a primer GhBONI-1F and a primer GhBONI-1R to obtain a PCR amplification product.

Primer GhBONI-VIGS-F: 5' -GAATTCATGGGAAATTGCTGCTCCGA-3’；

Primer GhBONI-VIGS-R: 5' -GGGGTACCCCACCAAGAGATTGCTGCTCCT-3’。

In the primer GhBONI, restriction enzyme EcoRI and KpnI sequences are underlined, so that the subsequent connection with a plant silencing expression vector pTRV2 is convenient for cotton genetic transformation.

2. The PCR amplification product obtained in step 1 was ligated with pEASY-Blunt Zero Cloning Kit (Cloning vector) using GhBONI-1F/R primerSequencing the positive transformant after PCR identification. The obtained sequence is' 5GAATTC+ position 1-417 + of SEQ ID No.2GGGGTACCCC-3’”。

3. And (3) carrying out double enzyme digestion on the sequence fragment obtained in the step (2) and a pTRV2 vector to obtain a VIGS plant expression vector recombinant plasmid named pTRV 2-GhBONI. pTRV2-GhBONI structure description: the recombinant plasmid obtained after inserting the DNA fragment shown in the 1 st to 417 th positions of SEQ ID No.2 between the restriction sites EcoRI and KpnI of the pTRV2 vector.

4. And (3) transforming the plant expression vector recombinant plasmid pTRV2-GhBONI obtained in the step (3) into agrobacterium GV3101, and carrying out PCR screening and identification by using a GhBONI-1F/R primer to obtain an agrobacterium positive clone.

Second, obtaining transgenic cotton

1. The obtained positive plant expression vector recombinant plasmid pTRV2-GhBONI Agrobacterium was streaked on LB solid medium containing kanamycin (50. mu.g/ml) and gentamicin (20. mu.g/ml) resistance, and cultured at 28 ℃ for 2 days.

2. The single clone obtained in step 1 was selected and inoculated into 5ml of LB liquid medium containing kanamycin (50. mu.g/ml) and gentamicin (20. mu.g/ml) resistance, and 180 rpm was carried out at 28 ℃ ^-1Culturing for 24 h; inoculating into 50 ml LB liquid culture medium, and rotating for 180 min at 28 deg.C^-1Culturing for 12h at 4000 rpm^-1Centrifuging for 5min to collect thallus cells, and resuspending in a suitable volume of resuspension (formulation: 10 mmol. multidot.L)^–1MgCl₂，10mmol·L^–1MES and 200. mu. mol. L^–1Acetosyringone) was resuspended to a final concentration of 1.5 (OD)₆₀₀). Standing the re-suspension at room temperature for more than 3 h. A resuspension containing the recombinant plasmid pTRV2-GhBONI was obtained. The preparation method is similar to that of the recombinant suspension containing pTRV1 vector, pTRV2-GhCLA1 vector containing CLA1 gene segment and pTRV2 empty vector.

Uniformly mixing a heavy suspension containing a pTRV1 vector and a heavy suspension containing a recombinant plasmid pTRV2-GhBONI containing a target gene fragment according to the volume ratio of 1: 1, and injecting cotton cotyledons for obtaining a gene silencing transformant; uniformly mixing the resuspension containing the pTRV1 vector and the resuspension containing the pTRV2-GhCLA1 vector containing the CLA1 gene fragment according to the volume ratio of 1: 1, and injecting the mixture into cotton cotyledons for detecting whether a gene silencing system is correct or not; the heavy suspension containing pTRV1 vector and the heavy suspension containing pTRV2 empty vector are mixed uniformly according to the volume ratio of 1: 1 and injected into cotton cotyledon to be used as a genetic transformation control strain.

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

4. And (3) slightly puncturing the back of the cotyledon by using a syringe needle to cause a micro wound, and injecting the prepared heavy suspension which is uniformly mixed according to the volume ratio of 1: 1 in the step (2) from the wound by using the syringe with the needle to obtain the cotton GhBONI gene silencing transformant. After 2 weeks, the phenotypes of the cotton treated differently were observed, and the expression of the target gene was examined. 30 individuals were treated for each material.

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

Detection of VIGS genetic transformation system

1. Detection of VIGS system was carried out using the upland cotton CLA 1gene (Cloroplastos alterados 1gene) as marker gene. The gene is involved in the development process of chloroplast, encodes 1-deoxyxylulose 5-phosphate synthsase protein, is highly conserved in evolution, cotton plants have obvious albino phenotype after CLA 1gene silencing, and is a marker character easy to identify. The results are shown in FIG. 1, panel A. After 2 weeks of VIGS infection, the true leaves of the plants injected with pTRV1 and pTRV2-ghcl 1 whitened almost completely, while the leaves injected with empty vectors pTRV1 and pTRV2 as controls (TRV: 00) did not change at all. The successful establishment of the TRV-mediated VIGS system in Gossypium hirsutum TM-1 is demonstrated.

2. Fluorescent quantitative Real time-PCR detection

And extracting the Total RNA of the cotton Plant leaves after 2 weeks infection of VIGS by using a Plant Total RNA Extraction Kit. And (3) detecting the interfered and silenced expression condition of the silenced GhBONI gene by using cotton Histon3 as an internal reference gene through fluorescent quantitative Real time-PCR.

The fluorescence quantitative PCR was performed using an apparatus of Applied Biosystems StepOne (Applied Biosystems, USA) and a reagent of SYBR Premix Ex Taq^TMSYBR Premix Ex Taq from kit (TransGene, Beijing)^TMA kit. The reverse transcription cDNA was used as template, starting at 150ng, and each treatment was repeated 3 times. The reaction procedure is as follows: denaturation (95 ℃, 30 s); (95 ℃, 5 s; 58 ℃, 15 s; 72 ℃, 31s) for 40 cycles of amplification; dissolution (95 ℃, 15 s; 60 ℃, 1 min; 95 ℃, 15 s).

Primer Histon 3-RT-F: 5'-GCCAAGCGTGTCACAATTATGC-3', respectively;

primer Histon 3-RT-R: 5'-ACATCACATTGAACCTACCACTACC-3' are provided.

Primer GhBONI-RT-F: 5'-CATCACTGGCAACCAAAGCG-3', respectively;

primer GhBONI-RT-R: 5'-CCACCAGCATCGGATCACTC-3' are provided.

The results are shown in FIG. 2. Compared with the control of an empty vector (TRV: 00), in two randomly selected GhBONI gene VIGS infected plants (BONI-1 and BONI-2), the expression level of the GhBONI gene is obviously reduced, and the silencing effect is obvious.

Fourth, cotton verticillium wilt resistance inoculation and resistance identification

Stored verticillium dahliae strain V991 (recorded in 'Zhang Hui, Tianxin, Gao Wei, Chua Yixuan, Longluo, upland Cotton PPO gene whole genome identification and response analysis to verticillium dahliae', Cotton Science 2017, 29 (5): 428-436 'Verticillium dahliae V991' in the text) is activated on a PDA culture medium. The selected thalli are cultured in Czapek's culture solution for 3-5 days at 25 ℃ at a speed of 200 r/min. Filtering the pathogenic bacteria culture solution with 4 layers of gauze, counting the concentration of pathogenic bacteria with a blood counting plate, and adjusting the final concentration to 1.0 × 10 with sterilized double distilled water⁷spores/mL, and Tween-20 was added to a final concentration of 0.001% (volume percent). And (3) inoculating verticillium wilt V991 spore liquid to the obtained transformant after 2 weeks of VIGS gene silencing by using a root soaking method, investigating and counting the pathogenesis of verticillium wilt after 12 days of inoculation, and counting the disease grade by adopting a 0-4 grade method. 30 individuals were treated for each material. Set 3 biological replicates.

Grade index statistical reference: Xue-Li, Zhu Long Pai, Zhang Dong Long, research progress of anti-verticillium wilt mechanism of cotton, journal of crops 2012,38:1553 and 1560; xu L, Zhu L F, Zhang X L.research on resistance mechanism of cotton to Verticillium wilt.acta Agron Sin 2012,38: 1553-.

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

The result is shown as B in FIG. 1. The pathogenesis of verticillium wilt after 2 weeks of VIGS infection of pTRV2-GhBONI silent plant and empty vector control (TRV: 00) plant are inoculated with Verticillium dahliae strain V991 and 12 days. As can be seen from B in FIG. 1, the control (TRV: 00) plants showed more yellowish plaques and larger area of leaves and more pronounced curling down the leaf margins than the GhBONI gene-silenced plants. Statistical analysis was performed by 3 biological replicates and the results are shown in figure 3. The average disease index of the control plant injected with the empty vector reaches 41.72, while the disease resistance of the plant after GhBONI gene silencing is obviously enhanced, and the average disease index is 32.18. The GhBONI gene is shown to participate in the allergic reaction induced by verticillium wilt.

The experimental results fully show that the expression level of the GhBONI gene is obviously reduced and the disease resistance is enhanced after VIGS infection.

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

<120> cotton verticillium wilt related gene GhBONI and coding protein and application thereof

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cagtttctca atattgaagt aaagatgctt aagctggagg agcagcaatc tcttggtgag 420

gcaagttgtg cattatcaga gattgtaacc aaaccaaaca ggtctttaac cttggatctt 480

gtacgtagag ttgaatccgt ctcatcaacc cattcccaac accatggaaa acttactgtg 540

catgctgagg aatgctttag ctcaaggact acggcggaga tgatgttaag ttgtttagat 600

ttggaatcta aggatctctt ctcaaaatgc gaccccttct tggtaatatc aaaacttgtg 660

gagagtggga tttcgattcc tgtatgtaaa actgaagtct taaagaatga tcataaccca 720

acatggaagc cagtattttt gaatattcaa caagtaggaa gcaaggatag tccattagtg 780

atagagtgct ttaacttcaa tagcaatggg aagcatgatc tgattggaaa agtccagaag 840

tcactagcag atttggaaaa gattcattct ggaagggaag gagaaaattt atttttgcca 900

actctggttg ggcatgattg cgagaacaag atattaaaaa gcaagctttt cgtggaaaat 960

ttctctgaga ctatccaaca taccttcctg gattacctgg ctgggggagt tgaacttaac 1020

tttatggtgg ctattgattt tacggcttca aatggaaatc cccgtcttcc tgattccttg 1080

cattacattg atccatctgg acggcagaat gcataccaga aagcaatcta tgaggttgga 1140

gaagtattgc agttctatga tacagataag tgttttcctg catggggatt tggagcacgg 1200

cccattgatg gtccagtttc tcattgtttc aacttgaatg gaagcaataa ctactgtaag 1260

gttgaaggaa tccgaggcat tatgatggcc tatacaagcg cgcttttcaa tgtttcgctt 1320

gcaggaccaa cactatttgg gcatgtggtt aacaaagctg cactaattgc cagccaatct 1380

cttgccgatg aagctcaaaa atactttgtt ttgttgatta tcacggatgg agttgtgacc 1440

gatctccaag aaaccaaaga tgcactggta aaagcatcag atctcccact gtcgatcctc 1500

attgttggag ttggaggagc agacttcaaa gaaatggaga ttttagatgc agacaaaggg 1560

gaaagacttg aaagctcgac cggacgtgtt gcttcacgtg atattgttca atttgttcca 1620

tttagggatg tacaaggtgg tgaagtatct attgttcaag cgcttttggc tgaattaccg 1680

acacaatttt taacctatat gcgaagcaga gatattaaac ccaggatttg a 1731

Claims

1. A protein, which is any one of:

(A1) A protein having an amino acid sequence of SEQ ID No. 1;

(A2) and (C) attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in (A1).

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a gene encoding the protein of claim 1, and the gene is a DNA molecule shown in SEQ ID No. 2.

4. A recombinant vector comprising the nucleic acid molecule of claim 2 or 3.

5. An expression cassette comprising the nucleic acid molecule of claim 2 or 3.

6. A recombinant bacterium comprising the nucleic acid molecule of claim 2 or 3.

7. The recombinant vector according to claim 4, wherein: the recombinant vector is a recombinant expression vector or a recombinant cloning vector.

8. Use of the protein of claim 1 or the nucleic acid molecule of claim 2 or 3 or the recombinant vector of claim 4 or 7 or the expression cassette of claim 5 or the recombinant bacterium of claim 6 in any one of:

(a) regulating and controlling the disease resistance of plants;

(b) breeding plant varieties with enhanced or weakened disease resistance;

(c) plant breeding;

the plant is cotton;

The disease is cotton verticillium wilt.

9. A method for breeding a plant variety, method A or method B:

the method A comprises the following steps: a method for breeding a plant variety having enhanced disease resistance, which comprises the step of reducing the expression level and/or activity of the protein of claim 1 in a recipient plant;

the method B comprises the following steps: a method for breeding a plant variety with reduced disease resistance, which comprises the step of increasing the expression level and/or activity of the protein of claim 1 in a recipient plant;

the plant is cotton;

the disease is cotton verticillium wilt.

10. A method of breeding a transgenic plant, method C or method D:

the method C comprises the following steps: a method of breeding a transgenic plant with enhanced disease resistance comprising the steps of: inhibiting the expression of a gene encoding the protein of claim 1 in a recipient plant to produce a transgenic plant; the transgenic plant has enhanced disease resistance as compared to the recipient plant;

the method D comprises the following steps: a method of breeding a transgenic plant with reduced disease resistance comprising the steps of: introducing into a recipient plant a nucleic acid molecule capable of expressing the protein of claim 1 to produce a transgenic plant; the transgenic plant has reduced disease resistance as compared to the recipient plant;

The plant is cotton;

the disease is cotton verticillium wilt.