CN106554964B - Application of cotton GbABR1 gene in verticillium wilt resistance - Google Patents

Abstract

The application belongs to the technical field of resistance breeding of gene cotton, and particularly relates to cottonGbABR1The application of the gene in the anti-verticillium wilt of cotton. The above-mentionedGbABR1Gene and protein sequence analysis shows that the gene contains a typical highly conserved AP2/EREBP structural domain; the gene belongs to ERF B4 members, and belongs to AP2 family members in sea island cotton; the subcellular localization result shows that the gene is localized in the cell nucleus; when the cotton is infected by verticillium wilt, the expression level of the gene is obviously improved. Application experiments show that after the gene is silenced, cotton plants are sensitive to verticillium wilt, and after the gene is over-expressed, new plant bodies show obvious resistance to verticillium wilt infection. Based on the research result, a certain application foundation can be laid for the research of the molecular mechanism of verticillium wilt resistance and the cultivation of new varieties of new verticillium wilt resistant plants.

Description

Application of cotton GbABR1 gene in verticillium wilt resistance

Technical Field

The application belongs to the technical field of cotton resistance breeding, and particularly relates to cottonGbABR1The application of the gene in the anti-verticillium wilt of cotton.

Background

Cotton is one of the most important commercial crops in the world and China, and verticillium wilt is one of the biotic stresses which have a very serious influence on cotton planting. The verticillium wilt of cotton seriously affects the yield and quality of cotton, the interaction mechanism between the cotton and verticillium wilt is deeply researched, the biological function of the related gene of verticillium wilt resistance of cotton is clarified, and the method is an important subject of the current research on verticillium wilt resistance of cotton.

As the defense mechanisms of plants against biotic stress (such as verticillium wilt diseases and the like) and abiotic stress (such as drought stress, high-salt stress, low-temperature stress and the like) relate to the participation of multiple ways (genes), the mechanism of resisting the adversity stress of crops is analyzed and researched, and then the regulation and control mechanism is applied to increase the yield of the crops, which is one of the common methods in modern breeding. The plant has a large number of transcription factors related to stress resistance, and the transcription factors are mutually combined with action elements on a target gene promoter to play a role, so that a plurality of downstream functional genes related to the stress resistance of the plant are regulated to play a role, and the adaptability of the plant to the stress is regulated; therefore, the regulation and control research of plant transcription factors becomes one of the important research contents of plants.

AP2/EREBP (APETALA 2/Ethylene-responsive element binding protein) is a superfamily of transcription factors, which are transcription factors specific in plants. Transcription factor (Trans-acting factor) refers to a kind of DNA binding protein which is located in eukaryotic cell nucleus and can activate or inhibit the gene transcription process by specific combination and interaction with Cis-element (Cis-acting element) in the 5' end promoter region of the regulated gene. When plants are subjected to abiotic stresses such as external drought and high temperature, the plants can excite transcription factors through a series of signal transmission. The activated transcription factor will, in combination with the corresponding cis-acting element, initiate the expression of a specific gene and thus respond to abiotic stress. Transcription factors play an important role in the growth and development of higher plants and in the response to biotic and abiotic stresses.

Common features of members of the gene family AP2 are: all have a DNA binding domain of AP 2. In plant arabidopsis thaliana, only one AP2 domain is the DREB subgroup and the ERF subgroup, because the structural domains can be differentiated into AP2, RAV, ERF, DREB and an orphan gene; the subgroup AP2 has two AP2 domains. The RAV subgroup contains two domains, AP2 and B3.

In 1995, 4 ERF1/2/3/4 transcription factors capable of binding to GCC-box in tobacco were found as proteins by using ethylene response element as probe, named Ethylene Response Factor (ERF). the ERF subgroup has a AP2 domain, each AP2 binding domain contains 2 conserved elements YRG, RADY element, YRG element with a basic hydrophilic region consisting of 19 to 22 amino acid residues as its conserved amino acid motif, RAYD conserved sequence is composed of 42 to 43 amino acids, analysis of three-dimensional structure of ERF protein found that alanine at position 14 and aspartic acid at position 19 in β -fold can specifically bind to GCC-box cis-acting element.

Goldack et al indicate that the flexibility of the regulation process and the post-transcriptional modification determine the functional specificity of transcription factors in environmental adaptation, ERF transcription factors directly regulate the expression of disease-process related genes such as prb-1b (PR1), β -1, 3-glucanase (PR2), chitinase (PR3) and osmotin (PR5) through the combination with GCC-box (GCCGCC) in disease-process related gene promoters, and the characteristics of ERF family transcription factors, so that the ERF gene can regulate the disease resistance and the stress resistance of cotton in a certain aspect, and the gene has a DNA binding domain, so that certain PR genes can be expressed, and the defense capability of the cotton is improved.

The promoter of PR gene has GCC-box action element, which has important action in resisting stress and disease, because the gene can be induced by ethylene treatment, also called ethylene response element.

The research finds that the essential condition for binding the domain of ERF and GCC-box element is conserved amino acid at the N terminal of the domain, and the research researches the ERF domain of a plurality of proteins, and experiments show that 11 key bases in GCC-box can stably interact with the ERF domain, and the conserved N terminal in the ERF domain plays a role in stably binding with GCC-box, and the different C terminals can distinguish different specific binding.

In conclusion, the ERF protein can be combined with different cis-elements, thereby regulating the response of some environmental stresses. In addition, the ERF structure domain can play a role in the ERF stress resistance if some phosphorylation sites can be found in the ERF structure domain.

Based on the functional diversity of ERF transcription factors, thereforeGbABR1The action mechanism of the verticillium wilt disease also needs to be researched, so that a certain reference and reference are provided for the prevention and control of the verticillium wilt disease.

Disclosure of Invention

Based on screening of the anti-verticillium wilt genes of cotton in the prior stage, the invention mainly aims to provide the anti-verticillium wilt gene with certain application prospectGbABR1The gene lays a certain application foundation for the cultivation of new verticillium wilt resistant cotton varieties.

The detailed technical scheme adopted by the application is as follows.

CottonGbABR1Use of a gene for combating verticillium wilt, said methodGbABR1The gene, GenBank numbering is KP259809, ORF thereof has 1113bp, and protein sequence analysis shows that the gene contains a typical highly conserved AP2/EREBP structural domain; the gene belongs to ERF B4 members, and belongs to AP2 family members in sea island cotton;

the subcellular localization result shows that the gene is localized in the cell nucleus;

the gene expression detection result shows that the gene has expression in root, stem and leaf of cotton, and the expression amount of the gene is obviously increased after the cotton is infected by verticillium wilt.

Further application results of the VIGS technology show that the method is toGbABR1After the gene is silenced, the incidence rate and disease index of the verticillium wilt of the plant are improved, and after the gene is over-expressed, the incidence rate and disease index of the verticillium wilt of the plant are reduced, therefore,GbABR1the gene is a positive regulatory factor for cotton verticillium wilt resistance.

Based on the previous research on the gene related to the induction of cotton verticillium wilt, the inventor sequences EST sequences with obvious difference of expression amount, namely sequences in the applicationGbABR1A gene. Furthermore, the gene is successfully cloned in the sea island cotton by an electronic cloning technology, and the biological information of the gene is analyzed in detail. Further application experiments show that after the gene is silenced, the disease resistance of the plant is reduced, and overexpression is carried out, the plant shows the characteristic of disease resistance to verticillium wilt infection, so that the gene is highly related to the resistance of verticillium wilt. Based on the research result, a certain application foundation can be laid for the research of the molecular mechanism of verticillium wilt resistance and the cultivation of new varieties of new verticillium wilt resistant plants, and meanwhile, a new reference is provided for the screening of new disease-resistant genes and the cultivation of resistant plants.

Drawings

FIG. 1 shows the detection of PCR products by electrophoresis, wherein M is 4500bp ladder Marker; 1-2: PCR products;

FIG. 2 isGbABR1Predicting the three-dimensional structure of the protein;

FIG. 3 is an alignment analysis of the amino acid sequence of the AP2/EREBP domain of the ABR1 protein;

FIG. 4 is a phylogenetic tree analysis of the cotton AP2 transcription factor;

FIG. 5 shows cottonGbABR1Analyzing a subfamily evolutionary tree of the transcription factors;

FIG. 6 shows the PCR detection of the bacterial liquid after the fusion expression vector p35S-GFP is transferred into Agrobacterium GV3101 by GbABR 1; wherein 2K: 2000bp ladder Marker; 1-13: PCR detection of the bacteria liquid is carried out, wherein N is negative control P and positive control;

FIG. 7 shows the expression of the fusion protein GbABR1 after Agrobacterium-mediated tobacco leaf transient transformation for 48 h;

FIG. 8 is a drawing showingGbABR1qRT-PCR results of expression levels in different cotton tissues (roots, stems, leaves);

FIG. 9 shows that after the root system of sea 15 of island cotton is infected by verticillium dahliae V991,GbABR1RT-PCR analysis results of gene expression patterns; wherein GbUB7 is used as an internal reference gene;

FIG. 10 is a control (TRV:00)AndGbABR1(pTRV: GbABR1) analysis of cotton material for verticillium wilt resistance, (A) TRV:00 and pTRV: GbABR1 of Ji cotton 11 are inoculated with verticillium wilt V991 spore liquid (10)⁷conidia/mL)16 days, and counting disease fingers 16 days after verticillium wilt bacteria are inoculated to Ji cotton 11; (B) TRV:00 and pTRV: GbABR1 of New sea 15 are inoculated with verticillium dahliae V991 spore liquid (10) by root dipping method⁷conidia/mL)16 days, and counting disease fingers 16 days after the new sea 15 is inoculated with verticillium wilt; GbCLA is plant phenotype of cotton CLA gene (Cloroplastos alternados) after transgene silencing, so that related phenotype change conditions can be visually compared;

FIG. 11 is a schematic view ofGbABR1Transgenic Arabidopsis vector construction, homozygote assay, Arabidopsis phenotype (A)GbABR1PCR detection of the LR reacted bacterial liquid; 2K is 2000bp ladder Marker; 13-17: PCR detection of bacterial liquid; n is negative control, P is positive control; (B) in transgenic Arabidopsis thalianaGbABR1Detecting the RNA transcription level; 11, 3, 6, 4, 8 and 9 in the drawings represent the differences, respectivelyGbABR1Overexpression of transgenic lines and wild type Arabidopsis; (C) growth status of transgenic arabidopsis;

FIG. 12 is a disease resistance identification of transgenic Arabidopsis inoculated with Verticillium dahliae V991, wherein (A) the phenotype of transgenic Arabidopsis inoculated with Verticillium dahliae V991; (B) the disease after the transgenic arabidopsis is inoculated with the couple bacteria V991 refers to statistical conditions;

FIG. 13 shows germination stage and late germination stageGbABR1The transgenic arabidopsis thaliana is sensitive to salt stress, mannitol and ABA, wherein a graph (A) isGbABR1The germination rates of the transgenic arabidopsis seeds are compared with those of WT wild type seeds under the conditions of salt stress, mannitol, ABA and 1% MS treatment respectively; FIG. B isGbABR1And (3) comparing the growth phenotypes of the transgenic arabidopsis seeds and WT wild types under the conditions of MS, ABA, mannitol and salt stress treatment.

Detailed Description

The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some of the materials in the following embodiments.

The cotton material comprises: the upland cotton-induced verticillium wilt variety Ji Cotton 11 is provided by the researchers of Zhu Hol musical instrument, Cotton research institute of Chinese academy of agricultural sciences;

new sea of anti-verticillium wilt gossypium barbadense 15, offered by Duxiongming researchers of Cotton institute of Chinese academy of agricultural sciences;

verticillium wilt strain: the cotton verticillium wilt strong pathogenic bacterium line V991 is provided by a Jianguiliang researcher of plant protection research institute of Chinese academy of agricultural sciences;

arabidopsis thaliana material: the wild type Arabidopsis seed (Columbia-0) is provided by the An national courage subject group of the Henan university plant adversity laboratory, and the culture conditions are as follows: at 21 ℃, 16 h of light and 8 h of dark;

the primer sequence is as follows: all provided by Hua Dai technology synthesis;

other biological materials include:

escherichia coli strain DH5 α, agrobacterium strain GV3101, TA cloning Vector pMD18-T Vector, BP reaction entry Vector pDONOOR 221, gene overexpression Vector PK7WG2.0, gene subcellular localization Vector PK7FWG2.0, VIGS interference technology Vector TRV1 and TRV2, which are all commercial strains or plasmids and are not described one by one;

experimental reagent:

plasmid extraction kit, oligo (dT)18, RNAase inhibitor, dNTP, pMD18-T Vector, T4-DNA ligase and endonucleaseBamH I andKpnI、ExTaqthe enzyme, the PCR product recovery kit, the fluorescent quantitative PCR kit and the like are all products of TaKaRa company;

the special fluorescent quantitative PCR plate is a product of Labwards company;

RNA extraction kits were purchased from Aidlab Biotech (Beijing, China);

the reverse transcriptase M-MLV is a product of Promega company;

the culture media and solutions used were: LB liquid (solid) medium, YEP liquid (solid) medium, 0.6 MS medium (MS 0.443 g, sucrose 3 g, water 100mL, agar powder 0.6 g, pH 5.8-6.0), Czapek's (Czapek) medium, PDA medium, 50 XTAE Buffer (Na)₂EDTA∙2H₂O37.2 g, glacial acetic acid 57.1 mL, pH =8.3 adjusted with NaOH, water added to a constant volume of 1L), etc.,all prepared according to the conventional preparation method in the field;

other undescribed antibiotics, hormones and other reagents are common in the field and are not described in detail.

Example 1

Based on earlier studies, the inventors found that cotton infected with verticillium wiltGbABR1The expression level of the gene is significantly changed, and therefore, the present inventors have considered that the expression level of the gene is significantly changed based on Gossypium barbadense (New sea 15)GbABR1The genes were analyzed in detail, and the specific procedures are briefly described below.

Sea island cottonGbABR1Obtaining of genes

Firstly, planting cotton, extracting RNA of cotton seedlings, and carrying out reverse transcription to obtain cDNA;

in the RNA extraction step, an EASYspin Plus plant RNA rapid extraction kit is referred, and the reverse transcription step is as follows:

total RNA, 1000/RNA concentration;

Oligo dT18 (10 μM)，1μL；

random primer, 1 μ L;

ddH₂supplementing O (RNase-free) to 13 μ L;

running the PCR instrument at 65 ℃ for 5min, and rapidly placing the PCR instrument on ice for 2 min;

adding the following reagents into the reaction system:

5×RT Buffer，4μL；

50U/μL RNasin，1μL；

10 mM dNTPs，1μL；

200U/L M-MLV，1μL；

the program was run in a PCR instrument: 60 min at 42 ℃; 85 ℃ for 5 min.

Using cDNA as a template, designing primers and amplifying genes, wherein the primers are designed as follows:

GbABR1F: 5’-CGCCAAGGATCATTGATGTATC-3’，

GbABR1R: 5’-CGGTGACTTTATTTAGTGGTTGG-3’；

PCR amplification System:

taq enzyme (2.5U/. mu.L), 10. mu.L;

Primer F（GbABR1F），0.2μL；

Primer R（GbABR1R），0.2μL；

cDNA，1μL；

ddH₂O，8.6μL；

PCR amplification program, 95 ℃, 5 min; 30 cycles of 95 deg.C, 30s, 54 deg.C, 30s, 72 deg.C, 1min20 s; 72 ℃ for 10 min.

The PCR products were electrophoretically detected, and the results are shown in FIG. 1. As can be seen from the figure, the geneGbABR1The size was 1113bp, consistent with the expected results.

II,GbABR1Basic bioinformatics information analysis of genes

And (3) recovering the PCR amplification product in the step one, purifying by using an agarose gel kit (Tiangen purification kit), connecting with a pMD18-T vector, carrying out PCR detection on the bacterial liquid, and further sequencing the positive plasmid.

（1）GbABR1Prediction and analysis of tertiary structure of protein

Preliminary analysis considered that in the present applicationGbABR1Gene and Arabidopsis thalianaAtERF1The DNA binding domains of the genes are relatively similar, thereforeAtERF1(PDB ID: 1gcCA) as a control model by http:// www.sbg.bio.ic.ac.uk// phyre2/html/page.cgiid = index pairGbABR1The spatial structure of the protein of (2) was analyzed, and the results are shown in FIG. 2.

As can be seen from an analysis of figure 2,AtERF1、GbABR1are essentially identical and consist of 1 α helices and 3 β folds.

(2) For conjectureGbABR1Sequence analysis of encoded amino acids

To pairGbABR1The deduced amino acid sequence of the gene was subjected to Blastp homology search and aligned using DNAMAN software, and the results are shown in fig. 3.

Analysis shows that:GbABR1the amino acid sequences of some homologous proteins of Arabidopsis are very similar to the sequences of some segments, and all contain an AP2 structural domain. Contains AP2/EREBP domain containing two conserved sequence elements YRG and RAYD, and WLG conserved sequence element, and has amino acid sequenceThe columns conform to the characteristics of a typical AP2/ERF transcription factor.

(3) GeneGbABR1Sequence alignment and evolutionary tree analysis of

Because the AP2/ERF family research in arabidopsis and rice is complete, the typical AP2/ERF transcription factors in arabidopsis and rice are selected for carrying out homologous evolution comparison to genesGbABR1A homoevolutionary tree is constructed. The results are shown in FIG. 5.

The protein of the gene and AP2 protein of other species were subjected to evolutionary tree analysis using MEGA5.0 software, and the analysis showed that:

as can be seen from the evolutionary tree, the genesGbABR1Evolutionarily belonged to the ERF class in the AP2/ERF transcription factor (FIG. 4);

will be provided withGbABR1The results of the evolutionary tree analysis of ERF B1-B6 families in the AP2 transcription factor in arabidopsis thaliana show that the genesGbABR1Belonging to ERF class B4 subfamily (FIG. 5);GbABR1the amino acid similarity to Arabidopsis AT5G64750 with the closest homology rate was 31.62%.

GbABR1Gene promoter sequence analysis

The cis-acting elements were predicted using the plantarcase online site, and the results are shown in the table below.

TABLE 1 predictedGbABR1Promoter cis-element:

。

analysis of the cis-element of the promoter is shown inGbABR1The upstream of the gene contains TC-richrepeats which play an important role in responding to stress resistance, disease resistance and the like. At the same time, the user can select the desired position,GbABR1the cis-elements of the gene promoter both contain ARE motif which responds to and regulates hypoxic or anoxic conditions. In addition, the genes all contain elements relevant to light and phytohormone response. These results show that it is possible to determine,GbABR1the expression pattern of (A) can be regulated by various factors, and is involved in the regulation of cotton response to biotic stress and abiotic stress and the regulation of hormone signal paths.

III,GbABR1Subcellular localization of proteins in cells

Firstly, the Gateway technology by homologous recombination refers to the existing method for constructing and identifying the vectorGbABR1The PCR product of the ORF segment (without stop codon) of (2) was ligated into the target vector pK7WGF2.0 to obtain the fusion expression vector p35S-GFP:: GbABR 1.

Next, Agrobacterium was transformed with the vector identified as correct, and positive clones were verified by PCR using the bacterial suspension (the results are shown in FIG. 6).

Finally, referring to the existing agrobacterium transfection method, the correctly identified agrobacterium is osmotically injected into the tobacco leaves; after 2-3 days, a part of the leaf was cut, and the GFP green fluorescence was detected by confocal laser microscopy after tabletting. The results are shown in FIG. 7.

The detection result shows that the GFP protein converted by the p35S-GFP vector has stronger expression at each part of the tobacco cell; preliminary explanation of the results that GbABR1 is expressed in the nucleusGbABR1Possibly localized to the nucleus.

Example 2

For the genes in the foregoing example 1GbABR1Based on the basic characteristic understanding, the inventor further specifically analyzes the tissue expression pattern of the gene under the natural condition in the cotton tissue and the tissue expression condition after being stressed, and related experiments are briefly described as follows.

One, natural (wild type) geneGbABR1Tissue expression pattern of

Respectively taking tissues of roots, stems and leaves of the Xinhai 15, extracting RNA, obtaining cDNA by using a reverse transcription kit, and performing qPCR amplification by adopting the following quantitative PCR primers:

QRT-PCR-GbABR1F: 5’- TCCTTATCGTCGTCATCGCCTG-3’，

QRT-PCR-GbABR1R: 5’- CTCCAATACCGCCACCTTGACTAC-3’；

the reference gene is Ubiquitin protein 7 (Ubiquitin7, UB7), and the primer sequence is as follows:

UBQ7F: 5’-GAAGGCATTCCACCTGACCAAC-3’，

UBQ7R: 5’-CTTGACCTTCTTCTTCTTGTGCTTG-3’。

qRT-PCR was performed on ABI 7500 Real-time PCR sequence detection system and software (Applied Biosystems, USA). The 10 mu L reaction system is designed as follows:

cDNA (which may be diluted appropriately), 4.4 μ L;

10×SYBR Green PCR Master Mix，5 µL；

0.2 muL of primer (0.1 muL of forward primer and reverse primer respectively);

ROXⅡ，0.2 µL；

the PCR procedure was: denaturation at 95 ℃ for 1min, followed by 40 cycles (denaturation at 95 ℃ for 5s, renaturation extension at 60 ℃ for 40 s).

The specificity of the amplified products after 40 cycles was detected by a dissolution curve analysis. Each reaction included at least three replicates. The single template is diluted to different concentrations, and the amplification efficiency of the primers is detected by plotting the log value of the dilution multiple of the template to the Ct value of each diluted sample.

The detection results are shown in fig. 8. Analysis shows that:GbABR1the expression level was high in roots and low in leaves. This result indicates thatGbABR1Has certain tissue variability.

Secondly, under the condition of biotic stress (verticillium wilt infection)GbABR1Expression pattern of

The inventors found out in the preliminary basic research that,GbABR1the expression quantity is obviously changed before and after cotton is inoculated with verticillium wilt bacteria, in order to further analyze the expression mode of the gene after the cotton is infected by the verticillium wilt bacteria, the inventor further utilizes RT-PCR and qRT-PCR technology to analyze the gene, and the specific process is as follows.

Selecting a disease-resistant variety of sea island cotton Xinhai 15 with two leaves and one heart for root-damaging inoculation, wherein the concentration of conidium solution of verticillium dahliae V991 used for inoculation is 1 multiplied by 10⁷spores/mL, water treatment as control. Selecting cotton roots at the time points of inoculation of 1 hour, 12 hours, 24 hours, 48 hours, 120 hours and the like, respectively, sampling leaves at the time points of 0 hour, 1 hour, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours and the like, extracting RNA, and performing reverse transcription to obtain cDNA for expression pattern analysis.

In the case of RT-PCR analysis, analysisGbABR1The primers for gene design are as follows:

F: 5’-TCCTTATCGTCGTCATCGCCTG-3’，

R: 5’-CTCCAATACCGCCACCTTGACTAC-3’；

the reaction system is designed as follows:

taq enzyme (2.5U/. mu.L), 10. mu.L;

Primer F，0.2μL；

Primer R，0.2μL；

cDNA，5.0μL；

ddH₂O，4.6μL；

PCR amplification procedure: 95 ℃ for 5 min; 94 ℃, 30s, 58 ℃, 30s, 72 ℃, 30s, 30 cycles; 72 ℃ for 10 min.

Ubiquitin protein 7: (Ubiquitin7，UB7) As an internal reference gene, primers were designed to perform parallel PCR reactions (see the above-mentioned "natural (wild type) gene for the relevant experimental procedures)GbABR1The tissue expression pattern of (1) 'partial content').

After the sample is analyzed for homogeneity, the PCR amplification product is subjected to electrophoresis separation detection on 1% agarose gel.

The experimental results are shown in fig. 9, and it can be seen from the analysis that: the RT-PCR result shows that after V991 conidium liquid and water are treated,GbABR1the expression level of the gene is up-regulated at 1 hour, 12 hours, 24 hours, 48 hours and 120 hours after the cotton root system is inoculated with pathogenic bacteria.

The above results indicate that the gene is infected by verticillium dahliae in the new sea 15GbABR1The expression level of (B) is changed, which indicatesGbABR1Participating in the biotic stress process.

Example 3

Further, the inventors constructed a VIGS interference vector using the VIGS technology to target genesGbABR1Silencing is performed. The results further prove that the gene is infected by verticillium wilt bacteria after gene silencing through observing the phenotype change condition of the cottonGbABR1Highly correlated with verticillium wilt resistance, the relevant experimental conditions are briefly described below.

(1) Construction of VIGS interference vector pTRV GbABR1

In the geneGbABR1Designing a primer according to the non-conservative region:

F: 5’- CGCGGATCCCCCAATCATTCACC-3’，

R: 5’- CGGGGTACCGGCGATGACGAC-3’；

the design principle is as follows: upstream primer plus enzymeKpnI restriction site and protective base, downstream primer plus enzymeBamH I and a protecting base;

PCR amplifying a target sequence (about 200 bp);

the PCR product was recovered and the empty vector pTRV2 was enzymatically isolatedBamH I and enzymesKpnI, carrying out double enzyme digestion under the condition of 37 ℃ water bath;

the enzyme cutting system is as follows:

10×K Buffer，5 μL；

Bamh I andKpni, 2.5 μ L each;

30. mu.L of DNA fragment (20. mu.L of DNA fragment when the pTRV2 plasmid is digested simultaneously);

ddH2O, 10. mu.L (when pTRV2 plasmid was digested simultaneously, ddH₂Adding 20 mu L of O),

carrying out 1% agarose gel electrophoresis on the enzyme digestion product; the target sequence is recovered and is connected by T4 DNA ligase to construct a VIGS interference vector pTRV:: GbABR 1.

(2) Ji cotton 11 and Xinhai 15 geneGbABR1After silencing, cotton responds to verticillium wilt

Separately transforming pTRV1, pTRV2 and pTRV2 GbABR1 into Agrobacterium, and separately shake-culturing to OD₆₀₀About 0.6-0.8;

the infection liquid of pTRV1 is respectively mixed with infection liquid of pTRV2 and pTRV2 and GbABR1 according to the volume ratio of 1: 1 (when preparing the infection liquid, firstly, the thalli is obtained by centrifugation, then the thalli is resuspended, when the thalli is resuspended, the formula of the suspension is 100mmol/L and 200 muL, MES 1 mol/L and 1000 muL, MgCl₂，1 mol/L 、1000 μL；ddH₂O added to 100 mL).

When injecting the bacteria liquid (the infection liquid), a2 mL injector is adopted for injection, and the specific process is as follows:

when the cotyledon is flat and no true leaf is grown, lightly scratching an inclined plane on the back of the cotyledon of the cotton seedling, slowly pumping the bacterial liquid into the cotyledon, and filling the cotyledon as full as possible;

and then, keeping the cotton in the dark for 24 hours, taking the leaves to extract RNA (preferably a new long true leaf) when the cotton seedlings grow to have two leaves and one heart, and then carrying out RT-PCR (reverse transcription-polymerase chain reaction) to detect whether the target gene is reduced and expressed.

The method for infecting cotton adopts root injury inoculation method, and adopts Verticillium dahliae V991 spore liquid (1 × 10)⁷conidia/mL) infested cotton. Counting the disease incidence condition of the cotton verticillium wilt by the time when the real leaves begin to turn yellow and wilting.

According to a Chinese method of quarantine inspection and identification of verticillium dahliae of cotton, the disease index of cotton is counted (standard number: GB/T28084-2011):

disease index = (number of diseased plants × representative value) × 100/number of total plants investigated × representative value of the most serious grade of disease onset.

The results indicated that the disease index of the gene-silenced cotton plants was about 20% higher than that of the control cotton plants (FIG. 10A, B).

Morphological observation showed that the yellowing of leaves and the falling of leaves of the cotton plants in the gene silencing group were all more severe than those in the cotton in the control group (FIG. 10A, B).

The above results show that the disease-resistant variety Ji cotton 11 or the disease-resistant variety Xinhai 15 is silentGbABR1After the gene is expressed, the incidence rate and disease indication of the cotton are obviously improved when the cotton is infected by verticillium wilt, which indicates thatGbABR1Silencing of the gene reduces the resistance of cotton to verticillium wilt, i.e., cotton is more susceptible to disease.

Example 4

Further, in the above pairsGbABR1On the basis of preliminary understanding of gene functions, the inventor constructs an over-expression plant by transforming arabidopsis thaliana, and preliminarily evaluates the verticillium wilt resistance and other resistance of the over-expression plant, and a specific experimental process is briefly introduced as follows.

(A) construction ofGbABR1Overexpression vectors

In the geneGbABR1The two ends of the ORF of (1) are designed and added with joint primers attBl and attB2, and the primer sequences are specifically designed as follows:

F: 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTCATGTATCAGTTGATGATG-3’，

R: 5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTTAGTGGTTGGAAT-3’；

performing PCR amplification, namely performing BP reaction on a PCR product to recombine the PCR product to a vector pDONR221, and then performing LR reaction to recombine the PCR product to a vector pK2GW7.0 in a homologous manner;

in the BP reaction process, a 2.5 mu L reaction system is designed as follows:

TE8.0，1.0μL；

BP enzyme, 0.5. mu.L;

pDONR221，0.5μL；

PCR product, 0.5. mu.L;

reacting for 2-3 hours in water bath at 25 ℃;

in the LR reaction process, a 2.5 muL reaction system is designed as follows:

TE8.0，1.0μL；

LR enzyme, 0.5. mu.L;

pK2GW7.0，0.5μL；

intermediate plasmid (i.e., the aforementioned BP reaction product), 0.5. mu.L;

reacting for 2-3 hours in water bath at 25 ℃.

The PCR product of the ORF segment of gene GbABR1 was ligated to the target vector pk7wg2.0 by the homologous recombination Gateway technique described above, and finally the overexpression vector pk7wg2.0-GbABR1 was constructed (the identification results are shown in fig. 11A).

(II) screening and obtaining of transgenic Arabidopsis homozygous

Taking 200 mu L of agrobacterium tumefaciens competent cells, placing the competent cells on ice for natural dissolution, adding 4-5 mu L of the overexpression vector pKK7WG2.0-GbABR 1 constructed in the step (I), gently mixing uniformly, and standing on ice for 30 minutes;

quickly freezing the mixture in liquid nitrogen for 70 seconds, quickly placing the mixture in a water bath kettle at 37 ℃ for 5-6 minutes, and then placing the mixture on ice for 2 minutes;

adding 1 mL of YEP liquid culture solution, and carrying out shaking culture at 28 ℃ and 180 rpm for 4-5 hours;

centrifuging at 4000 rpm for 4-5 minutes at room temperature, discarding part of YEP liquid culture solution, transferring the bacterial solution to a YEP resistant plate, uniformly coating, air-drying, and performing inverted culture in an incubator at 28 ℃ for 2-3 days.

Selecting positive bacterial plaque, adding the positive bacterial plaque into 5 mL YEP liquid culture solution containing rifampicin and Spe (spectinomycin), carrying out shaking culture at 28 ℃ and 220 rpm, adding 1-2 mL bacterial liquid into 200 mL YEP liquid culture solution containing rifampicin and Spe, and carrying out overnight culture until OD is achieved₆₀₀1.2 to 1.6;

transferring the bacterial liquid into a 50 mL bacterial collecting tube, centrifuging at 4000 rpm for 20 min, collecting thallus, adding infection liquid (5% sucrose, 0.5% MS), blowing, mixing, and adjusting OD₆₀₀The value is set to be between 0.8 and 1.0, and the volume of the added staining solution is calculated. Finally, arabidopsis transformation auxiliary reagent L-77, 300. mu.L/L, is added.

Pouring the prepared infection liquid containing the over-expression vector pKK7WG2.0-GbABR 1 into a container with a wide caliber, then immersing the arabidopsis thaliana ears which bloom and cut off other immature fruit pods into the infection liquid for 30 seconds, and taking out;

after all the seedlings are infected, the seedlings of the arabidopsis are flatly placed, covered by a preservative film and placed in a dark place, and normal culture is recovered after 24 hours.

In order to improve infection conversion efficiency, secondary conversion can be carried out after seven days.

After the plants are mature, arabidopsis seeds of T1 generation are collected.

The dried T1 seed was soaked with 0.1% mercuric chloride for 4 to 5 minutes and then rinsed 4 to 5 times with sterile water, the first two times requiring rapid rinsing. And (3) dibbling the disinfected and cleaned seeds on a 0.6% MS solid culture medium containing kanamycin and cefamycin by using a sterilized large gun head, and sealing the plate after the moisture on the surfaces of the seeds is dried. The petri dish was placed in a refrigerator and vernalized at 4 ℃ for 2 to 3 days. Then the culture dish was placed in a culture room (illumination intensity 150 mmol. m)^-2s^-1Light/dark is 16 h/8 h, temperature is 18 to-22 ℃, relative humidity is about 80 percent). After further culturing for 1-2 weeks, selecting the yang which can grow normallyPerforming single-plant culture and seed harvest on the sexual seedlings; culturing, maturing and harvesting seeds. Until screening to T3 generation to obtain homozygous plant.

(III) Arabidopsis thaliana transgenic plantsGbABR1The condition of gene expression

In thatGbABR1Before over-expressed Arabidopsis T1 generation plants do not bolting, 3-4 pieces of Arabidopsis leaf tissue RNA are extracted.

The results of qRT-PCR showed that: the expression levels of L3, L9 and L11 (L3, L9 and L11 are strain numbers and have no special meaning) in the overexpression strain are high (FIG. 11B). Subsequent experiments therefore carried out some related studies with third generation seeds of two lines L9, L11.

The two pure-line arabidopsis thaliana and the wild type arabidopsis thaliana are transplanted into a small pot, plant phenotypes are observed, and the over-expression arabidopsis thaliana has some differences in various aspects such as plant height, early and late bolting, flowering and fructification and the like compared with the wild type arabidopsis thaliana, and the transgenic arabidopsis thaliana has the characteristics of early bolting, early flowering, short and small plant, early maturity and the like compared with the wild type arabidopsis thaliana (fig. 11C). Description of overexpression of genes in ArabidopsisGbABR1Under the condition of normal growth, the growth of arabidopsis thaliana is influenced to a certain extent.

(IV) identification of verticillium wilt resistance in transgenic Arabidopsis plants

Seedlings grown for 8-12 days were removed from the petri dish, and the root tips were removed by about 1mm to try to damage the roots equally. Soaking the root of the seedling to 1 × 10⁶Treating conidia/mL V991 bacterial liquid for 5-6 min, and transplanting the seedling back to the pot. The onset of Arabidopsis thaliana was examined two weeks or so after inoculation.

Compared with the wild type Arabidopsis thaliana,GbABR1the over-expression arabidopsis thaliana is more resistant to verticillium wilt bacteria V991, and the leaf of the over-expression arabidopsis thaliana strain is less yellow and has good growth vigor. Wild type arabidopsis plants die more. The specific results are shown in FIG. 12.

As can be seen from a combination of the above results,GbABR1after overexpression of the gene, the disease indication of the transgenic plant is significantly lower than that of the wild plant, namely,GbABR1after the gene is over-expressed, the resistance of arabidopsis thaliana to verticillium wilt bacteria can be obviously improved.

(V) resistance of transgenic Arabidopsis plants to abiotic factor salts

Seeds of wild type arabidopsis thaliana and seeds of L11 and L9 strains are respectively dibbled on a solid culture medium which is not added with NaCl and is added with 1% MS of 120 mMNaCl, are vernalized at 4 ℃ for 2-3 days, are placed in a culture room for growth, and the germination rate is counted.

The results are shown in fig. 13, and analysis shows that:

on a solid culture medium with 1% MS, compared with wild Arabidopsis thaliana, L11 and L9 strains have no great difference in germination rate, and all germination only needs 1-2 days;

on a solid culture medium with NaCl and 1% MS, the germination rates of over-expression strains L11 and L9 in seeds are obviously faster than those of wild type strains; differences in germination rates were observed between germination growth up to days 2, 3, and 4, with differences in germination rates of the various materials being most pronounced up to day 3. The germination rate of the over-expression strain L11 is about 30-50%, and the germination rate of the wild type is 10%;

on a solid culture medium with NaCl and 1% MS, the germination grows by about 10, and the over-expression strains L11 and L9 have larger crowns, longer roots, more lateral roots and more green cotyledons, and the overall growth vigor is better than that of wild type.

By combining the phenotype results and the germination rate statistical results, the over-expression material is considered to be insensitive to high salt stress; that is to say that the first and second electrodes,GbABR1after the gene is over-expressed, the salt tolerance of the plant can be improved.

(VI) resistance of transgenic Arabidopsis plants to abiotic factor drought (osmotic stress)

Seeds of wild type arabidopsis thaliana and seeds of L11 and L9 are respectively dibbled in a solid culture medium of 1% MS without adding mannitol and with adding 240mM mannitol, and are placed in a culture room for growth after vernalization treatment at 4 ℃ for 2-3 days, and the germination rate is counted.

The results are shown in fig. 13, and analysis shows that:

on a solid medium of 1% MS, the L11, L9 strains did not differ much in germination rate compared to wild type Arabidopsis thaliana, and all germination required only 1-2 days. However, on a solid medium containing 1% MS of mannitol, the germination rate of the over-expression lines L11, L9 in seeds is significantly faster than that of the wild type; differences in germination rates were observed between germination growth to days 3, 4, and 6, with differences in germination rates of the various materials being most pronounced by growth to day 4. The germination rate of the over-expression strain L11 is about 55%, and the germination rate of the wild type is 20%.

On a solid culture medium containing 1% MS of mannitol, the germination grows by about 10, and overexpression strains L11 and L9 have larger crowns and longer roots, grow better than wild type strains, and can better tolerate hypertonic stress than the wild type strains.

(VII) dependence of transgenic Arabidopsis plants on ABA Signal pathway

Seeds of wild arabidopsis thaliana and seeds of over-expression strains L11 and L9 are respectively dibbled in solid culture media of 1% MS and 1% MS added with 1 mu MABA, and are placed in a culture room for growth after vernalization treatment at 4 ℃ for 2-3 days, and the germination rate is counted. The results are shown in fig. 13, and analysis shows that:

on normal 1% MS solid medium, the L11, L9 strains did not differ much in germination rate compared to wild type Arabidopsis, and all germination required only 1-2 days. However, on a solid medium containing 1% MS of 1uM ABA, the germination rates of the over-expression lines L11 and 1L 9 in seeds are obviously faster than those of the wild type; differences between germination rates were observed between germination growth up to days 2 and 3, with differences in germination rates of the various materials being most pronounced by growth up to day 3. The germination rate of the over-expression strain L11 is about 40 percent, and the germination rate of the wild type is 20 percent;

10 days on normal MS, there was no difference in phenotype of the different materials; however, on a solid culture medium containing 1uM ABA and 1% MS, the growth vigor of over-expression strains L11 and L9 is obviously superior to that of a wild type, and the over-expression strains are represented by longer main roots, larger crowns and more green cotyledons; this result indicates that the over-expressed material is insensitive to ABA during germination of the seeds and early seedling growth.

It can therefore be judged that:GbABR1the method is involved in the response of plants to exogenous ABA in the processes of seed germination and early seedling growth, and has a negative regulation effect in the physiological process.

Claims (3)

1. CottonGbABR1Use of a gene for combating verticillium wilt, characterized in thatGbABR1The gene, GenBank numbering is KP259809, ORF thereof has 1113bp, and protein sequence analysis shows that the gene contains a typical highly conserved AP2/EREBP structural domain; the gene belongs to ERF B4 members, and belongs to AP2 family members in sea island cotton;

the subcellular localization result shows that the gene is localized in the cell nucleus;

the gene is expressed in root, stem and leaf of cotton, and when cotton is infected by verticillium wilt, the expression level of the gene is obviously increased.

2. Cotton as claimed in claim 1GbABR1The application of the gene in the verticillium wilt resistance is characterized in that the gene is expressed by the VISG technologyGbABR1After gene silencing, cotton is susceptible to verticillium wilt.

3. By using cottonGbABR1A method for genetically constructing a new plant species having high verticillium wilt resistance, which comprises the steps ofGbABR1The gene is over-expressed, and the verticillium wilt disease resistance of the transgenic plant after over-expression is enhanced.

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