CN106554964A - Application of the Cotton Gossypii GbABR1 genes in resisting verticillium - Google Patents

Abstract

The application belongs to gene Cotton Resistance breeding technical field, and in particular to Cotton GossypiiGbABR1The patent application of application of the gene in verticillium wilt resistance of cotton by same.It is describedGbABR1Gene, sequential analysis of protein show which contains a typical highly conserved AP2/EREBP domain；The gene belongs to ERF class B4 members, AP2 family members in category sea island cotton；Subcellular Localization result shows that the gene mapping is in nucleus；After Cotton Gossypii is infected by verticillium wilt pathogen, the gene expression amount is significantly improved.Application experiment shows that after gene is carried out silence, cotton plants are more sensitive to verticillium wilt, and after working as the gene overexpression, new plant body infects to verticillium wilt and shows obvious resistance.Based on this result of study, can be that Study on Molecular Mechanism, the new resisting verticillium new variety of plant of resistance to verticillium wilt is cultivated and establish certain application foundation.

Description

Application of the Cotton Gossypii GbABR1 genes in resisting verticillium

Technical field

The application belongs to Cotton Resistance breeding technical field, and in particular to Cotton GossypiiGbABR1Gene is in verticillium wilt resistance of cotton by same Application patent application.

Background technology

Cotton Gossypii is one of the world and Chinese most important industrial crops, affects very serious one kind biological on cotton planting Stress is verticillium wilt.Cotton verticillium wilt has a strong impact on the yield and quality of Cotton Gossypii, between further investigation Cotton Gossypii and verticillium wilt pathogen Coupling effects, understand fully the biological function of verticillium wilt resistance of cotton by same disease related gene, are the important of current verticillium wilt resistance of cotton by same research Problem.

As plant is to biotic（Such as verticillium wilt disease etc.）And abiotic stress（Drought stress, high-salt stress and low Temperature stress etc.）Deng defense mechanism be related to multiple approach（Gene）Participate in, crop of analyzing and researching resists the mechanism of environment stress, enters And the yield for increasing crop with the regulatory mechanism is one of conventional method of modern breeding.There are a large amount of degeneration-resistant phases in plant The transcription factor of pass, many transcription factor by and target gene promoters on functional element be combined with each other and be allowed to play a role, Adjust the degeneration-resistant related functional gene of downstream various plants to play a role, so as to regulate and control the adaptability to adverse circumstance of plant；Thus The study on regulation of plant transcription factor becomes one of primary study content of plant.

AP2/EREBP (APETALA2/ Ethylene-responsive element binding protein) is The superfamily of one transcription factor, it is the distinctive transcription factor in plant.Transcription factor (Transcription Factor), i.e. trans acting factor (Trans-acting factor) refers to and is positioned in eukaryotic cell core, by with quilt There is specific binding and interaction in the cis element (Cis-acting element) of the end of controlling gene 5 ' promoter region, reach A class DBP to the activation or suppression of the gene transcription process.When plant is subject to the non-lifes such as extraneous arid, high temperature When thing is coerced, plant can excite transcription factor by a series of signal transmission.The transcription factor being excited can be suitable with corresponding Formula functional element combines and starts the expression of specific gene, so as to carry out response to abiotic stress.Transcription factor is in high plant The growth promoter of thing and to playing highly important regulating and controlling effect in biological and abiotic stress response.

The common feature of this kind of gene family members of AP2 is：All there is the DNA binding domain of an AP2.Intend south in plant Mustard, because the difference of domain can be divided into AP2, RAV, ERF, DREB and an orphon, only one of which AP2 domain It is DREB subgroups and ERF subgroups；AP2 subgroups have two AP2 domains.RAV subgroups contain two domains of AP2 and B3.

When nineteen ninety-five, in Nicotiana tabacum L., the ERF for having 4 ERF1/2/3/4 transcription factor be combined with GCC-box Class transcription factor, they are the albumen found for probe by element responsive to ethylene, are named as the ethylene responses factor (ERF). ERF class subgroups have the domain of an AP2, include 2 Conserved Elements YRG elements, RADY first in each AP2 binding domain Part, with an alkaline hydrophilic area being made up of 19 ~ 22 amino acid residues as its conserved amino acid motifs in YRG elements； The conserved sequence of RAYD is made up of 42 ~ 43 aminoacid.Three-dimensional structural analysis to ERF protein, find in β-folding The alanine of the 14th in folded and the aspartic acid of the 19th, the knot that they can be special with GCC-box cis acting element Close.Sakuma etc. is divided into 6 subgroups, B1-B6 again ERF subgroups.His division be by it translate albumen in which has Aminoacid.It has carried out functional analyses to the function of ERF transcription gene according to the characteristics of conservative region simultaneously.

Transcriptional control and post-transcriptional control serve important function in terms of plant cell adapts to external environment change. Golldack etc. points out that the motility of regulation process and posttranscriptional modification determine that the function of transcription factor in environment adaptation is special Property.ERF transcription factor by and pathogenesis-related gene promoter in GCC-box (GCCGCC) combination direct regulation and control disease Journey related gene, such as prb-1b (PR1), β -1,3- glucanase (PR2), chitinase (PR3) and osmotin (PR5) expression.The feature of ERF races transcription factor, so gene ERF can adjust cotton disease resistance in terms of certain degeneration-resistant, base Because of the binding domain for having DNA, some PR gene expressions can be made, improve Cotton Gossypii defence capability.

There are GCC-box functional elements in the promoter of PR genes, all play a very important role on degeneration-resistant, disease-resistant etc., because Usually because of ethylene process and then can be induced for this genoid, be also designated as element responsive to ethylene.

Research finds that the domain of ERF and the essential condition combined by GCC-box elements are the conservative ammonia of the N-terminal in domain Base acid, they have studied the ERF domains of many albumen, and experiment has 11 critical bases just stablize in showing GCC-box And in ERF domains interaction and ERF domains guard N-terminal play a part of and GCC-box stable bonds, the C for differing End, and the specific bond for differing can be distinguished.

In sum ERF albumen can with the combination of different cis elements, so as to adjust the response of some environment-stress. In addition if some phosphorylation sites can be had in research discovery ERF domains, then their work(to the anti-environment stresses of ERF Be able to can play a role.

Based on the multiformity of ERF class functional transcription factors, thereforeGbABR1Mechanism of action between verticillium wilt is also necessary Studied, so as to being the prevention of verticillium wilt and controlling to provide certain reference and reference.

The content of the invention

On the basis of early stage verticillium wilt resistance of cotton by same genescreen, present invention is primarily intended to provide that there is necessarily anti-yellowing withering Sick application prospectGbABR1Gene, so as to establish certain application foundation for the cultivation of new verticillium wilt-resistant cotton kind.

The detailed technology scheme taken by the application is as follows.

Cotton GossypiiGbABR1Application of the gene in resisting verticillium, it is describedGbABR1Gene, GenBank are numbered and are KP259809, its ORF have 1113bp, and sequential analysis of protein shows, which contains a typical highly conserved AP2/EREBP knot Structure domain；The gene belongs to ERF class B4 members, AP2 family members in category sea island cotton；

Subcellular Localization result shows that the gene mapping is in nucleus；

Gene expression detection result shows, the gene has expression in the root of Cotton Gossypii, stem, leaf, when Cotton Gossypii is subject to verticillium wilt pathogen Infect after, the gene expression amount is significantly improved, this result show the gene cotton verticillium wilt biotic signal turn There is important function in leading approach.

Further VIGS technology application results show, incite somebody to actionGbABR1After gene silencing, plant verticillium wilt sickness rate and the state of an illness Index is lifted, and by after the gene overexpression, plant verticillium wilt sickness rate and disease index decline, therefore,GbABR1Gene is The positive regulatory factor of verticillium wilt resistance of cotton by same.

In early stage to, on cotton verticillium wilt induction related gene Research foundation, inventor is obvious to wherein expression difference Est sequence is sequenced, i.e., described hereinGbABR1Gene.Further, by electronic cloning technology, on island In cotton, successful clone obtains this gene, and has carried out labor to its biological information.Further application experiment table It is bright, this gene is being carried out into silence, the disease resistance of plant is reduced, and after carrying out overexpression, plant pair verticillium wilt infects performance Go out disease-resistant properties and characteristicses, so as to show the resistance height correlation of this gene and verticillium wilt.Based on this result of study, can be Huang The Study on Molecular Mechanism of disease of withering resistance, new resisting verticillium new variety of plant are cultivated and establish certain application foundation, while being also new Disease-resistant gene screening and resistance plant cultivate there is provided new reference and reference.

Description of the drawings

Fig. 1 be PCR primer electrophoresis detection, wherein M:45000bp ladder Marker；1-2：PCR primer；

Fig. 2 isGbABR1The three dimensional structure prediction of albumen；

Fig. 3 is analyzed for the amino acid alignment of ABR1 protein A P2/EREBP domains；

Phylogenetic analysis of the Fig. 4 for Cotton Gossypii AP2 transcription factor；

Fig. 5 is Cotton GossypiiGbABR1The subtribe phylogenetic analysis of transcription factor；

Fig. 6 is fusion expression vector p35S-GFP::GbABR1 proceeds to bacterium solution PCR detection after Agrobacterium GV3101；Wherein 2K： 2000 bp ladder Marker；1-13：Bacterium solution PCR detects N:Negative control P:Positive control；

Fig. 7 is to observe GFP after agriculture bacillus mediated tobacco leaf instantaneously turns 48 h::GbABR1 expressing fusion protein situations；

Fig. 8 isGbABR1In different cotton tissues（Root, stem, leaf）The qRT-PCR results of middle expression；

Fig. 9 isGbABR1The analysis result of expression pattern, wherein 15 root systems of (A) sea island cotton sea are infected in verticillium wilt pathogen V991 Afterwards,GbABR1The RT-PCR analysis results of gene expression pattern；(B) sea island cotton sea 15 root systems after verticillium wilt pathogen V991 infects,GbABR1The qRT-PCR analysis results of gene expression pattern；

Figure 10 is control (TRV:00) andGbABR1(pTRV:GbABR1) Analysis of Resistance of the cotton material to yellow Pedicellus et Pericarpium Trapae pathogenic bacteria, (A) The TRV of Ji cotton 11:00 and pTRV:GbABR1 root dipping method inoculation verticillium wilt pathogen V991 spore liquid (10⁷Conidia/mL) 16 days Incidence, Ji cotton 11 is inoculated with verticillium wilt pathogen statistics for referring to of disease after 16 days；(B) TRV in new sea 15:00 and pTRV:GbABR1 Verticillium wilt pathogen V991 spore liquid (107conidia/mL) incidence of 16 days is inoculated with root dipping method, new sea 15 is inoculated with verticillium wilt The bacterium statistics that disease refers to after 16 days；

Figure 11 isGbABR1Transgenic arabidopsis vector construction, homozygote inspection, arabidopsiss phenotype (A)GbABR1LR reaction Bacterium solution PCR detection afterwards；2K: 2000bp ladder Marker；AG-6：Bacterium solution PCR detects N:Negative control P:It is positive right According to；(B) in transgenic arabidopsisGbABR1The detection of rna transcription level；In figure 11,3,6,4,8 and 9 represent respectively it is differentGbABR1Overexpression transgenic line and wildtype Arabidopsis thaliana；(C) upgrowth situation of transgenic arabidopsis；

Figure 12 is that transgenic arabidopsis are inoculated with yellow Pedicellus et Pericarpium Trapae pathogenic bacteria V991 anti-disease enzymes, wherein (A) transgenic arabidopsis inoculation verticillium wilt Phenotype after bacterium " V991 "；(B) disease refers to statistical conditions to transgenic arabidopsis inoculation Huang wife pathogenic bacteria " V991 " afterwards；

Figure 13 is in germination period and sprouting later stageGbABR1Transgenic arabidopsis are insensitive to salt stress, Mannitol, ABA, (A) salt WT and transgenic arabidopsis germination rate statistics under treatment conditions；Under normal condition and treatment with mannitol under the conditions of WT andGbABR1Turn The germination rate of gene arabidopsiss seed;Under normal condition and ABA treatment conditions under WT andGbABR1Transgenic arabidopsis seed Germination rate；(B) under the conditions of salt treatment WT andGbABR1Transgenic arabidopsis growth phenotype；Under normal condition and treatment with mannitol Under the conditions of WT andGbABR1The phenotype of transgenic arabidopsis seed.Under normal condition and ABA treatment conditions under WT andGbABR1Turn The phenotype of gene arabidopsiss seed.

Specific embodiment

Explanation is further explained to the application with reference to embodiment.Before specific embodiment is introduced, with regard to following realities Partial material situation briefly introduction in applying example is described as follows.

Cotton material includes：Gossypium hirsutum L. sense verticillium wilt kind Ji cotton 11, by the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute Zhu He Qin researcher provides；

The new sea 15 of resisting verticillium sea island cotton, is provided by the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute Du Xiongming researcher；

Verticillium wilt bacterial strain：Cotton verticillium wilt High pathogenicity fungus strain V991, by Plant Protection institute, Chinese Academy of Agricultral Sciences's letter osmanthus Good researcher provides；

Arabidopsiss material：Wildtype Arabidopsis thaliana seed (Columbia-0), by the brave class of He'nan University's plant adverse circumstance laboratory Anguo Topic group is provided, and condition of culture is：21 DEG C, 16 h illumination, 8 h are dark；

Primer sequence：There is provided by Hua Da science and technology synthesis；

Other biological material includes：

Strain Escherichia coli DH5 α, Agrobacterium strain GV3101, TA cloning vehicle pMD18-T Vector, BP reaction entry vectors PDONOR221, gene overexpression vector PK7WG2.0, gene Subcellular Localization carrier PK7FWG2.0, VIGS interference technique carrier TRV1 and TRV2；Commercialization strain or plasmid are, are no longer illustrated one by one；

Experiment reagent：

Plasmid extraction kit, oligo (dT) 18, RNAase inhibitor, dNTP, pMD18-T Vector, T4-DNA ligase, Restriction endonucleaseBamH I andKpn I、Ex TaqEnzyme, PCR primer QIAquick Gel Extraction Kit, PCR kit for fluorescence quantitative etc. are TaKaRa Products；

Quantitative fluorescent PCR personality board is Labwares Products；

RNA extracts kits are purchased from Aidlab Biotech（Beijing, China）Company；

Reverse transcription M-MLV is Promega Products；

Used medium and solution have：LB liquid（Solid）Culture medium, YEP liquid（Solid）Culture medium, 0.6 MS culture medium（MS 0.443 g, 3 g of sucrose, 100 mL of water, 0.6 g of agar powder, pH 5.8 ~ 6.0）, Cha Shi（Czapek）Culture medium, PDA cultures Base, 50 × TAE Buffer（Na₂EDTA∙2H₂37.2 g of O, 57.1 mL of glacial acetic acid, NaOH adjust pH=8.3, add water and are settled to 1 L）Deng by the preparation of this area customary preparation methods；

The reagents such as antibiotic that other do not describe, hormoness are reagent commonly used in the art, repeat no more.

Embodiment 1

On the basis of early-stage Study, inventor is had found after being infected by verticillium wilt, Cotton GossypiiGbABR1The expression of gene there occurs bright Aobvious change, thus, inventor is with sea island cotton（New sea 15）Based on, it is rightGbABR1Gene has carried out labor, detailed process It is briefly discussed below.

First, sea island cottonGbABR1The acquisition of gene

First, plant cotton, extracts the RNA of seedling Cotton Gossypii, and carries out reverse transcription acquisition cDNA；

RNA extraction steps refer to EASYspin Plus plant RNA rapid extraction test kits, and reverse transcription step is as follows：

Total RNA, 1000/RNA concentration；

Oligo dT18 (10 μM), 1 μ L；

Random primer, 1 μ L；

ddH₂O (RNase-free) polishings are to 13 μ L；

In PCR instrument, 65 DEG C of 5 min of operation are placed on rapidly 2 min on ice；

Following reagent is added in above-mentioned reaction system：

5 × RT Buffer, 4 μ L；

50U/ μ L RNasin, 1 μ L；

10 mM dNTPs, 1 μ L；

200U/L M-MLV, 1 μ L；

This program is run in PCR instrument：42 DEG C, 60 min；85 DEG C, 5min.

With cDNA as template, design primer and carry out the amplification of gene, design of primers is as follows：

GbABR1F:5 '-CGCCAAGGATCATTGATGTATC-3 ',

GbABR1R: 5’-CGGTGACTTTATTTAGTGGTTGG-3’；

PCR amplification system：

Taq enzyme (2.5 U/ L）, 10 μ L；

Primer F（GbABR1F）, 0.2 μ L；

Primer R（GbABR1R）, 0.2 μ L；

CDNA, 1 μ L；

ddH₂O, 8.6 μ L；

PCR amplification programs, 95 DEG C, 5min；95 DEG C, 30s, 54 DEG C, 30s, 72 DEG C, 1min20s, 30 circulations；72 DEG C, 10min。

Electrophoresis detection is carried out to PCR electrophoresis products, as a result as shown in Figure 1.It can be seen that geneGbABR1Size It is for 1113 bp, consistent with expected resultss.

2nd,GbABR1The underlying biological informaticss information analysiss of gene

Pcr amplification product in recycling step one, with agarose gel test kit (Tiangeng purification kit) after purification, with PMD18-T carriers connect, and then carry out the detection of bacterium solution PCR, and positive plasmid is further sequenced.

（1）GbABR1The Tertiary structure predictions of albumen and analysis

Preliminary analyses are thought, in the applicationGbABR1Gene and arabidopsissAtERF1The DNA binding domain of gene is more similar, because This withAtERF1(PDB ID：1gccA) as comparison model, by http://www.sbg.bio.ic.ac.uk/~ Phyre2/html/page.cgi id=index coupleGbABR1Protein steric structure be analyzed, as a result as shown in Figure 2.

Fig. 2 is analyzed and be can be seen thatAtERF1、GbABR1Composition it is essentially identical, by 1 α spiral and 3 β Fold composition.

（2）To speculatingGbABR1The sequence analysis of coded aminoacid

It is rightGbABR1The aminoacid sequence that gene is derived carries out Blastp homology searches, and carries out sequence using DNAMAN softwares Compare, as a result as shown in Figure 3.

Analysis shows：GbABR1It is very much like in certain sector sequence with the aminoacid sequence of arabidopsiss some homologous proteins, All contain AP2 domains.Contain two conservative sequential elements containing AP2/EREBP domain:YRG and RAYD, also WLG are protected The sequential element kept, and aminoacid sequence meets the feature of typical AP2/ERF transcription factor.

（3）GeneGbABR1Sequence alignment and phylogenetic analysis

Due in arabidopsiss and Oryza sativa L. AP2/ERF families research ratio it is more complete, so have chosen allusion quotation in arabidopsiss and Oryza sativa L. The AP2/ERF classes transcription factor of type carries out homologous evolution and compares, to geneGbABR1Construct homologous cladogram.As a result such as 5 institutes Show.

Phylogenetic analysis are carried out to the AP2 albumen of the albumen and other species of this gene using MEGA5.0 softwares, is analyzed Show：

By in cladogram as can be seen that geneGbABR1Belong to the ERF classes (figure in AP2/ERF transcription factor on evolutionary relationship 4) ；

WillGbABR1Phylogenetic analysis are carried out with ERF classes B1 in AP2 transcription factor in arabidopsiss～B6 families, is as a result shown, base CauseGbABR1Belong to ERF class B4 subtribes (Fig. 5)；GbABR1The aminoacid phase of the arabidopsiss AT5G64750 nearest with homology It is 31.62% like rate.

GbABR1Gene promoter sequence is analyzed

With the online websites of PlantCare, cis acting element is predicted, it is as a result as shown in the table.

Table 1, predictionGbABR1Promoter cis element：

。

Above-mentioned promoter cis element is analyzed, it can be seen thatGbABR1Contain TC-rich in the upstream of gene Repeats, the element play an important role responding the aspect such as degeneration-resistant, disease-resistant.Meanwhile,GbABR1Gene promoter cis element In containing the ARE motif for responding and adjusting hypoxia or oxygen free condition.Additionally, the genoid containing with light, phytohormone Response related elements.These results indicate thatGbABR1Expression pattern may be regulated and controled by many factors, participate in Cotton Gossypii to biology Adverse circumstance, the response of abiotic stress and the regulation and control in hormone signal path.

3rd,GbABR1Subcellular Localization of the albumen in cell

First, by the Gateway technologies of homologous recombination, with reference to existing vector construction and the method for identification, willGbABR1ORF The PCR primer of section (not containing termination codon) is connected in destination carrier pK7WGF2.0, obtains fusion expression vector p35S-GFP::GbABR1。

Secondly, above-mentioned identification correct carrier is converted into Agrobacterium, positive colony is verified with bacterium solution PCR（The result such as Fig. 6 It is shown）.

Finally, with reference to existing Agrobacterium infection method, by above-mentioned identification correct Agrobacterium infiltration injection tobacco leaf；2- After 3 days, a part of blade is cut, after tabletting, GFP green fluorescences are detected with laser confocal microscope.As a result it is as shown in Figure 7.

Testing result shows that the GFP albumen of p35S-GFP carriers conversion has stronger table in each position in tobacco cell Reach；GFP::GbABR1 is expressed on nucleus, and this result is tentatively illustratedGbABR1Nucleus may be positioned at.

Embodiment 2

For gene in previous embodiment 1GbABR1On the basis of fundamental characteristics understands, inventor further exists to this gene The tissue expression pattern in the case of nature in cotton tissue and tissue expression situation is made a concrete analysis of after being coerced, phase Close experiment to be briefly discussed below.

First, (wild type) gene in the case of natureGbABR1Tissue expression pattern

New extra large 15 root, stem, the tissue of leaf are taken respectively, are extracted RNA, and are obtained cDNA using reverse transcription reagent box, using as follows Quantification PCR primer carries out qPCR amplifications：

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

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

Reference gene selects ubiquitin protein 7 (Ubiquitin7, UB7), and primer sequence is as follows：

UBQ7F:5 '-GAAGGCATTCCACCTGACCAAC-3 ',

UBQ7R: 5’-CTTGACCTTCTTCTTCTTGTGCTTG-3’。

QRT-PCR is in 7500 Real-time PCR sequence detection systems of ABI and software (Applied Biosy Stems, USA) on carry out.The design of 10 L reaction systems is as follows：

cDNA（Suitably can dilute）, 4.4 L；

10 × SYBR Green PCR Master Mix, 5 L；

Primer, 0.2 L（Positive and negative primer is respectively 0.1 L）；

ROX II, 0.2 L；

PCR programs are：95 DEG C of 1 min of degeneration, then (95 DEG C of 5 s of degeneration, 60 DEG C of renaturation extend 40 s) for 40 circulations.

After 40 circulations, the specificity of amplified production is detected with solubility curve analysis.Each reaction includes at least three Repeat.Variable concentrations are diluted to single template, the Ct values of each dilute sample are mapped by the log values of template extension rate Detection primer amplification efficiency.

Testing result is as shown in Figure 8.Analysis shows：GbABR1Expression is higher in root, relatively low in leaf.This One result showsGbABR1Expression with certain histological difference.

2nd, in the case of biotic (verticillium wilt pathogen infects)GbABR1Expression pattern

Inventor had found in early stage basic research,GbABR1Before and after Cotton Gossypii inoculation verticillium wilt pathogen, expression there occurs significantly change Change, in order to further analyze the expression pattern after the gene is infected by verticillium wilt pathogen in Cotton Gossypii, inventor is further with RT- PCR and qRT-PCR technologies are analyzed to the gene, and detailed process is as follows.

Choosing two leaves disease-resistant variety sea island cotton wholeheartedly new extra large 15 carries out hindering root inoculation, inoculation verticillium wilt pathogen V991 used Conidium liquid concentration is 1 × 10⁷Spores/mL, with water process as control.Respectively inoculation 1 hour, 12 hours, it is 24 little When, 48 hours, 120 hours equi-time points choose cotton root system, and respectively 0 h, 1 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h and 72 h equi-time points take sample blade, after extracting RNA and reverse transcription goes out cDNA and carries out expression pattern analysis.

When RT-PCR is analyzed, analysisGbABR1During gene, design of primers is as follows：

F:5 '-TCCTTATCGTCGTCATCGCCTG-3 ',

R: 5’-CTCCAATACCGCCACCTTGACTAC-3’；

Reaction system design is as follows：

Taq enzyme (2.5 U/ L）, 10 μ L；

Primer F, 0.2 μ L；

Primer R, 0.2 μ L；

CDNA, 5.0 μ L；

ddH₂O, 4.6 μ L；

PCR amplification programs：95 DEG C, 5min；94 DEG C, 30s, 58 DEG C, 30s, 72 DEG C, 30s, 30 circulations；72 DEG C, 10min.

With ubiquitin protein 7 (Ubiquitin7, UB7) used as reference gene, design primer carries out parallel PCR reactions（It is related Experimentation with reference to it is aforementioned " nature in the case of (wild type) geneGbABR1Tissue expression pattern " partial content）.

Sample is carried out after homogeneity analysis, pcr amplification product carries out electrophoretic separation detection on 1% agarose gel.

QRT-PCR courses of reaction with reference to it is aforementioned " nature in the case of (wild type) geneGbABR1Tissue expression pattern " portion Divide content.

Experimental result as shown in Figure 9 A, is analyzed to which and can be seen that：The result of RT-PCR shows, V991 conidium After liquid and water process,GbABR1Expression 1 hour after cotton root system is inoculated with pathogen, 12 hours, 24 hours, it is 48 little When, all show as within 120 hours raise.The result of qRT-PCR shows,GbABR1Expression 12 hours in cotton leaf, 24 Hour, all show as within 36 hours expression rise, and variation tendency closely similar (Fig. 9 B).

The above results show, after new sea 15 is infected by verticillium wilt pathogen, geneGbABR1Expression be varied from, explanationGbABR1Participate in this biotic process.

Embodiment 3

Further, inventor utilizes VIGS technique constructions VIGS interference vectors, with to geneGbABR1Carry out silence.Pass through Character mutation situation when Cotton Gossypii is infected to verticillium wilt pathogen after observation gene silencing, as a result further proves, geneGbABR1With Resistance to verticillium wilt height correlation, related experiment situation are briefly discussed below.

（1）Build VIGS interference vector pTRV::GbABR1

In geneGbABR1Non-conservative section design primer：

F:5 '-CGCGGATCCCCCAATCATTCACC-3 ',

R: 5’- CGGGGTACCGGCGATGACGAC-3’；

Designing design principle is：Forward primer adds enzymeKpnThe restriction enzyme site and protection base of I, downstream primer add enzymeBamH The restriction enzyme site and protection base of I；

PCR amplifications target sequence (about 200 bp)；

Then with empty carrier pTRV2 after reclaim the product of PCR, enzyme is used respectivelyBamH I and enzymeKpn Bars of the I in 37 °C of water-baths Double digestion is carried out under part；

Enzyme action system is：

10 × K Buffer, 5 μ L；

BamH I andKpnI, each 2.5 μ L；

DNA fragmentation, 30 μ L（During double digestion pTRV2 plasmids, consumption is 20 μ L)；

DdH2O, 10 μ L（During double digestion pTRV2 plasmids, ddH₂O adds 20 μ L),

Digestion products carry out 1% agarose gel electrophoresiies；Aim sequence is reclaimed, and is attached using T4 DNA ligases, built VIGS interference vector pTRV::GbABR1.

（2）Gene in Ji cotton 11 and Xin Hai 15GbABR1After silence, Cotton Gossypii is to verticillium wilt pathogen response condition

By pTRV1, pTRV2 and pTRV2::GbABR1 is converted in Agrobacterium respectively, difference shake-flask culture to OD₆₀₀Reach 0.6 ~ 0.8 or so；

By pTRV1 infect liquid respectively with pTRV2, pTRV2::GbABR1's infects liquid according to volume ratio 1:1 ratio uniform is mixed Close（When liquid is infected in preparation, centrifugation first obtains thalline, then resuspension, and during resuspension, suspension formulation is：AS, 100 mmol/L、200 μL；MES, 1 mol/L, 1000 μ L；MgCl₂, 1 mol/L, 1000 μ L；ddH₂O adds to 100mL）.

Injection bacterium solution（Infect liquid）When, to be injected using 2 mL syringes, detailed process is：

It is open and flat in cotyledon, when also not growing true leaf, an inclined-plane is gently drawn at the back side of Cotton Seeding Cotyledon, bacterium solution is slow Slowly squeeze in cotyledon, cotyledon is all filled with as far as possible；

Lucifuge 24 hours afterwards, whne Cotton Seedling-Growth to two leaves wholeheartedly when, taking blade, to extract RNA (preferably new long true Leaf), RT-PCR is then carried out, whether detection target gene is lowered expression.

The method for infecting Cotton Gossypii is the method using root inoculation is hindered, with verticillium wilt pathogen V991 spore liquid (1 × 10⁷ Conidia/mL) infect Cotton Gossypii.The situation of cotton verticillium wilt morbidity is counted when starting and flavescence, wilting occur to true leaf.

Reference《Verticillium dahliae quarantine detection and identification》Middle method, counts the disease index (standard No. of Cotton Gossypii:GB/ T28084-2011)：

Disease index=∑ (sick level strain number × represent numerical value) × 100/ investigates the representative numerical value of total strain number × morbidity most heavy duty.

As a result show, the disease of the cotton plants of gene silencing refer to higher than matched group cotton plants 20% or so (Figure 10 A, B) 。

Morphologic observation shows that the yellowing leaf of gene silencing group cotton plants, the blade for dropping are all than the Cotton Gossypii of matched group Seriously (Figure 10 A, B).

Summary result is can be seen that either in susceptible variety Ji cotton 11 or disease-resistant variety new extra large 15, silenceGbABR1After gene, when being infected by verticillium wilt pathogen, the sickness rate and disease of Cotton Gossypii refer to and significantly improve Cotton Gossypii, explanationGbABR1Base The silence of cause reduces resistance of the Cotton Gossypii to verticillium wilt, i.e. Cotton Gossypii is more easy to susceptible.

Embodiment 4

Further, above-mentioned rightGbABR1On the basis of gene function tentatively understands, its arabidopsis thaliana transformation is built by inventor Overexpression plant, and the resistance to verticillium wilt to overexpression plant and other resistances have carried out preliminary assessment, specific experiment process It is briefly discussed below.

（One）BuildGbABR1Overexpression vector

In geneGbABR1ORF two ends, plus adapter-primer attBl and attB2, primer sequence specific design is as follows for design：

F:5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCTCATGTATCAGTTGATGATG-3 ',

R: 5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTTAGTGGTTGGAAT-3’；

PCR is expanded, and PCR primer is carried out BP first and is reacted to recombinate on carrier pDONR221, is then carried out LR and is reacted with homologous Recombinate on carrier pK2GW7.0；

In BP courses of reaction, the design of 2.5 L reaction systems is as follows：

TE8.0,1.0 μ L；

BP enzymes, 0.5 μ L；

PDONR221,0.5 μ L；

PCR primer, 0.5 μ L；

2 ~ 3 hours are reacted under 25 DEG C of water-baths；

In LR courses of reaction, the design of 2.5 L reaction systems is as follows：

TE8.0,1.0 μ L；

LR enzymes, 0.5 μ L；

PK2GW7.0,0.5 μ L；

Middle interstitial granules（I.e. aforementioned BP product）, 0.5 μ L；

2 ~ 3 hours are reacted under 25 DEG C of water-baths.

By above-mentioned homologous recombination Gateway technologies, the PCR primer of the ORF sections of gene GbABR1 is connected to into target In carrier pK7WG2.0, final structure obtains overexpression vector pK7WG2.0-GbABR1 (qualification result is as shown in Figure 11 A).

（Two）The homozygous screening of transgenic arabidopsis and acquisition

200 μ L Agrobacterium competent cells are taken, competent cell is placed in after dissolving naturally on ice, add the above-mentioned steps of 4 ~ 5 μ L Suddenly overexpression vector pK7WG2.0-GbABR1 constructed in (), after gently mixing, stands 30 minutes on ice；

Then 70 s of quick-freezing in liquid nitrogen, then it is put in rapidly 37 DEG C of water-baths 5 ~ 6 minutes, place 2 minutes then on ice；

Add 1 mL YEP liquid mediums, 28 DEG C, 180 rpm shaken cultivation 4 ~ 5 hours；

Under room temperature, 4000 rpm centrifugation 4-5 minutes, discard part YEP liquid mediums, and bacterium solution is gone in YEP resistant panels Even coating, after drying, is inverted culture 2 ~ 3 days for 28 DEG C in incubator.

Picking positive bacterial plaque is added to 5 mL containing rifampicin and Spe（Spectinomycin）YEP liquid mediums in, 28 DEG C, After 220 rpm shaken cultivation, take 1 ~ 2 ml bacterium solutions and add to 200 mL resistances（Containing rifampicin and Spe）In YEP liquid mediums, mistake Night is cultivated to OD₆₀₀For 1.2 ~ 1.6；

Bacterium solution is transferred in 50 mL collection tubes, is centrifuged 20 minutes in 4000 rpm, collects thalline simultaneously is added thereto to infect liquid (5% sucrose, 0.5%MS), piping and druming are mixed, and adjust OD₆₀₀Value so as between 0.8 ~ 1.0, calculates the volume for infecting liquid for adding. It is eventually adding transformation of Arabidopsis thaliana auxiliary reagent L-77,300 μ L/L.

Prepared the container that liquid pours a wide aperture into is infected containing overexpression vector pK7WG2.0-GbABR1 by above-mentioned In, then by it is having bloomed, and cut off other immaturity Fruit pods arabidopsiss spica immersion it is above-mentioned infect in liquid, take after 30 seconds Go out；

After all infecting, the Seedling of arabidopsiss is kept flat, avoid light place is covered with preservative film, recover normal and cultivate after 24 hours.

Transformation efficiency is infected to improve, after seven days, can carry out twice transformation.

After plant maturation, the arabidopsiss seed in T1 generations is collected.

By dried T1 for seed, with 0.1% mercuric chloride soaking disinfection 4 to 5 minutes, then arrived with aseptic water washing 4 It is 5 times, front to need twice to get express developed.The seed program request after cleaning will be sterilized with sterilized big pipette tips containing kanamycin and On 0.6% MS solid mediums of cephamycin, the shrouding after the surface of the seed moisture is dried up.Culture dish is put in refrigerator, 4 DEG C Vernalization 2 to 3 days.Culture dish is placed in into culturing room (150 mmolm of intensity of illumination again^-2s^-1, light dark be 16 h/8 h, temperature 18 ~ -22 DEG C, relative humidity is cultivated in being about 80%).Continue culture 1-2 it is all after, select can normally long root, grow positive seedling Carry out individual plant culture sowing；Ripe sowing of culture.Until screen to T3 generations obtaining homozygous plants.

（Three）In arabidopsiss transfer-gen plantGbABR1Expression conditions

GbABR1The arabidopsiss T1 of overexpression does not have before bolting for plant, extracts the RNA of the leaf tissue of 3-4 piece arabidopsiss.

The result of qRT-PCR shows：L3, L9, L11 in overexpression strain（L3, L9, L11 are numbered for strain, are contained without special Justice）Expression higher (Figure 11 B).Therefore subsequent experimental has carried out some with the third generation seed of two strains of L9, L11 Correlational study.

The arabidopsiss and wildtype Arabidopsis thaliana that be sheerly the two are transplanted in little alms bowl, observe plant phenotype, find super table Up to arabidopsiss than wildtype Arabidopsis thaliana the height of plant, bolting sooner or later, the many-side such as blossom and bear fruit have some differences, turn base Have that bolting is early, the early, plant that blooms is short and small than wildtype Arabidopsis thaliana because of arabidopsiss, it is precocious the features such as (Figure 11 C).Illustrate intending south Overexpression gene in mustardGbABR1In the case of normal growth, the development to arabidopsiss has certain effect.

（Four）The resistance to verticillium wilt identification of transgenic Arabidopsis plants

The growth seedling of 8-12 days is removed from culture dish, the tip of a root removes about 1mm, accomplish identical injury root as far as possible.Seedling Root be dipped into 1 × 10⁶5-6 minutes are processed in the V991 bacterium solutions of conidia/mL, seedling kind is returned in earthen bowl after process.Connect Investigation or so bacterium two weeks after arabidopsiss incidence.

The arabidopsiss of contrast wild type,GbABR1Overexpression arabidopsiss more resisting verticillium bacterium V991, overexpression arabidopsiss strain The blade of system turn yellow less, growing way it is good.Wild-type Arabidopsis plants death is more.Concrete outcome is as shown in figure 12.

Summary result can be seen thatGbABR1After gene overexpression, transfer-gen plant refers to than the disease of WT lines It is substantially low, i.e.GbABR1After gene overexpression, arabidopsiss can be obviously improved to verticillium wilt pathogen resistance.

（Five）Resistance of the transgenic Arabidopsis plants to abiotic factor salt

By the seed of wildtype Arabidopsis thaliana and the seed of L11, L9 strain, program request respectively is being not added with NaCl and added with 120 mM On the solid medium of 1% MS of NaCl, after 4 DEG C of vernalization treatments 2-3 days, grow in being put into culturing room, count germination rate.

As a result as shown in figure 13, analysis shows：

On the solid medium of 1%MS, L11, L9 strain does not have too big difference compared with wildtype Arabidopsis thaliana, on germination rate Not, all sprout and only need to 1-2 days；

Have on the solid medium of 1%MS of NaCl, overexpression strain L11, L9 is substantially fast than wild type in the germination rate of seed； At germination and growth to the 2nd, 3,4 days it is observed that difference between germination rate, wherein the various materials when growing into the 3rd day The difference of germination rate is the most notable.The germination rate of overexpression strain L11 is about 30-50%, the germination rate 10% of wild type；

Have on the solid medium of 1%MS of NaCl, germination and growth 10 or so, it is larger that overexpression strain L11, L9 shows as bizet, Root is longer, and lateral root is more, cotyledon greening it is many, overall growing way is better than wild type.

Summary phenotypic results and germination rate statistical result, it is believed that overexpression material is insensitive to high-salt stress； That is,GbABR1After gene overexpression, the salt tolerant resistance of plant can be improved.

（Six）Transgenic Arabidopsis plants are to abiotic factor arid（Osmotic stress）Resistance

By the seed of wildtype Arabidopsis thaliana and the seed of L11, L9 strain, program request respectively is being not added with Mannitol and added with 240 mM The solid medium of the 1%MS of Mannitol, after 4 DEG C of vernalization treatments 2-3 days, grows in being put into culturing room, counts germination rate.

As a result as shown in figure 13, analysis shows：

On the solid medium of 1%MS, L11, L9 strain does not have too big difference compared with wildtype Arabidopsis thaliana, on germination rate Not, all sprout and only need to 1-2 days.But on the solid medium of the 1%MS containing Mannitol, overexpression strain L11, L9 It is substantially fast than wild type in the germination rate of seed；At germination and growth to the 3rd, 4,6 days it is observed that difference between germination rate It is different, wherein the difference of various material germination rates is the most notable when growing into the 4th day.The germination rate of overexpression strain L11 is about 55%, the germination rate 20% of wild type.

On the solid medium of the 1%MS containing Mannitol, germination and growth 10 or so, the performance of overexpression strain L11, L9 Larger for bizet, root is longer, and growing way is better than wild type, is more tolerant of hyperosmotic stress than wild type.

（Seven）Transgenic Arabidopsis plants are to ABA signal pathway dependencies

By the seed of wildtype Arabidopsis thaliana and the seed of overexpression strain L11, L9, program request is in 1%MS and added with 1M ABA respectively 1%MS solid medium, after 4 DEG C of vernalization treatments 2-3 days, be put in culturing room and grow, count germination rate.As a result such as Figure 13 It is shown, analysis shows：

On the solid medium of normal 1%MS, L11, L9 strain does not have not too compared with wildtype Arabidopsis thaliana on germination rate Big difference, all sprouts and only needs to 1-2 days.But on the solid medium of the 1%MS containing 1uM ABA, overexpression strain It is L11,1 L9 substantially fast than wild type in the germination rate of seed；At germination and growth to the 2nd, 3 days it is observed that germination rate Between difference, wherein the difference of various material germination rates is the most notable when growing into the 3rd day.Overexpression strain L11 is sprouted The rate of sending out is about 40%, the germination rate 20% of wild type；

Grow 10 days on normal MS, the phenotype of different materials is not different；But train in the solid of the 1%MS containing 1uM ABA On foster base, the growing way of overexpression strain L11, L9 is substantially better than wild type, shows as that main root is longer, and bizet is larger, cotyledon greening It is many；The result shows that overexpression material is insensitive to ABA during the sprouting of seed and early stage growth of seedling.

Therefore may determine that：GbABR1Sprout with involved in plant in early stage seedling life growth course to Exogenous ABA in seed Response, in this physiological process with negative regulation effect.

Claims (3)

1. Cotton GossypiiGbABR1Application of the gene in resisting verticillium, it is characterised in that describedGbABR1Gene, GenBank numberings For KP259809, its ORF has 1113bp, and sequential analysis of protein shows, which contains a typical highly conserved AP2/EREBP Domain；The gene belongs to ERF class B4 members, AP2 family members in category sea island cotton；

Subcellular Localization result shows that the gene mapping is in nucleus；

The gene has expression in the root of Cotton Gossypii, stem, leaf, after Cotton Gossypii is infected by verticillium wilt pathogen, the gene expression amount It is significantly improved.

2. Cotton Gossypii as claimed in claim 1GbABR1Application of the gene in resisting verticillium, it is characterised in that by VISG technologies WillGbABR1After gene silencing, the susceptible verticillium wilt of Cotton Gossypii.

3. Cotton Gossypii is utilizedGbABR1The method of gene constructed high resistance to verticillium wilt new variety of plant, it is characterised in that willGbABR1Base Because carrying out overexpression, the verticillium wilt disease resistance of the transfer-gen plant after overexpression is strengthened.