CN105543268B - Plant is improved to the method for resistance to verticillium wilt using verticillium wilt pathogen VdP4-ATPase gene - Google Patents

Abstract

The present invention relates to the VdP4-ATPase genes of coding verticillium wilt pathogen P class ATPase to improve plant to the purposes in resistance to verticillium wilt, and plant is improved to the method for resistance to verticillium wilt, the plant is improved to the resistance of verticillium wilt and expression encodes the VdP4-ATPase gene of verticillium wilt pathogen P class ATPase in target plant body.The present invention improves the resistance of plant using the removing toxic substances to pathogen toxin, does not have the characteristics of pathogen microspecies resistance.Therefore, the characteristics of can be widely applied to the resistance of raising various pathogenic bacteria biological strain, small species specificity resistance be not present.

Description

Plant is improved to resistance to verticillium wilt using verticillium wilt pathogen VdP4-ATPase gene Method

Technical field

The invention belongs to plant genetic engineering fields.Specifically, relating to the use of technique for gene engineering improves plant disease-resistant The method of property.

Technical background

Verticillium wilt seriously affects the yield and quality of crop, and the whole world is every year because loss caused by verticillium wilt reaches multi-million dollar (Pegg et al.,2002).Report that potato, under normal conditions can be with because infection verticillium wilt annual output reduces 50% or more Cause 10-15% the underproduction (Powelson et al., 1993；Rowe et al., 1987,2002)；Romaine lettuce growing area, Huang wither Loss caused by sick is very easy to reach 100% (Subbarao et al., 1997)；Levinet (2003) etc. the study found that Olive can achieve 70% or more because of the underproduction caused by verticillium wilt；Verticillium wilt is also that most cottons for planting cotton country are mainly sick Evil, for example, Australia, Brazil, Bulgaria, China, Greece, Peru, Turkey, Uganda, the U.S. and Uzbekistan this Smooth equal countries, can cause the underproduction (Bolek et al., 2005) of 30% or more cotton, and the serious regional or time of falling ill subtracts Production can achieve 100%.Verticillium wilt is a kind of Major Diseases for restricting Cotton in China production, at present the morbidity face in each cotton region in China 50% or more of the long-pending Zhan Zhimian gross area, annual ten thousand tons of gined cotton 7.5-10 of loss, hundred million yuan of (Xiao Song of direct economic loss 16-20 China etc., 2006).Increase year by year in Cotton in China verticillium wilt grave illness plot, harm aggravates year by year, it has also become current to realize that cotton is high It produces, the key constraints of stable yields.Verticillium wilt not only causes a large amount of underproduction of crop and product quality to reduce, what pathogenic bacteria generated Toxin can also endanger the health of the mankind.

Verticillium wilt is mainly by big beautiful branch bacterium Verticillium dahliae Kleb and verticilliumalbo-atrum Verticillium alboatrum infects a kind of caused soil-borne vascular bundle disease.Once in pathogen invaded plants body, just There is no effectively preventing method.Host range is wide for verticillium wilt pathogen, can infect more than 200 kinds including all dicotyledons Plant, pathogen can be survived in the soil 20 years or so (Klosterman et al., 2009) in the form of Microsclerotia, be removed Kawchuk in 2001 etc. is obtained other than 1 disease-resistant gene Ve for Solanum verticillium wilt pathogen from clone in tomato, almost without gram It is grand to arrive the disease-resistant gene from other plant, show the gene only to the part of verticillium wilt pathogen the Mechanism Study of Ve disease-resistant gene Microspecies are resistant (Fradin et al., 2009).The research of early stage thinks that after plant infection verticillium wilt, conduit is by spore The blocking such as jelly that son, mycelia and pathogen are formed by plant cell causes wilt downright bad (Agrios, 2005), but It is that subsequent studies have shown that catheter blockage is not the main reason for verticillium wilt is wilted, with the continuous deepening of research, people recognize The toxin for knowing verticillium wilt pathogen generation is (Fradin et al., 2006) an important factor for leading to plant wilt.There are also some Person thinks that the blocking of vascular bundle is also based on the inductive effect (Chen Xusheng etc., 1998) of toxin.

It was verified that being the unique cost-effective approach for preventing and treating verticillium wilt harm using disease-resistant variety.Traditional breeding method side Although method can be using crop itself or the resistant gene breeding resistant variety of relationship kind, there is available resistant variety moneys The disadvantages of source is few, and the breeding time is long, and cost is big；Application chemical agent is not only difficult to effectively prevent verticillium wilt but also pollution environment. It understands in depth with the continuous development of plant gene engineering technology and to what plant and pathogen interacted so that by external source Resistant gene imports plant to improve disease resistance as an effective way.It can be broken in traditional breeding method by transgenic technology Inter-species not affinity phenomenon, eliminate crossing barrier, greatly widened the source of resistant gene and applied (Grover and Gowthama,2003)。

Plant pair can be improved in the intracorporal growth of plant and breeding using external source disease-resistant gene Product inhibiton verticillium wilt pathogen The resistance of verticillium wilt, on the other hand, the pathogenesis based on Amber box policy improves removing toxic substances of the plant to Amber box policy Ability, and plant is improved to one of the effective ways of resistance to verticillium wilt.But number researcher big absolutely mainly utilizes former side Method achievees the purpose that improve plant resistance to environment stress, there is not yet the toxin successfully generated using the expression product removing toxic substances pathogen of foreign gene To improve the report to resistance to verticillium wilt.

Summary of the invention

It is an object of the present invention to provide the VdP4-ATPase genes of coding verticillium wilt pathogen P class ATPase to improve To the purposes in resistance to verticillium wilt, the VdP4-ATPase gene integration by that will encode verticillium wilt pathogen P class ATPase enters plant Target plant constructs genetically modified plants, and the VdP4-ATPase gene is made to express in plant and improve plant and wither to Huang The resistance of disease.

It is another object of the present invention to provide a kind of raising plants to the method for resistance to verticillium wilt, by planting in target Expression alien gene in object and improve the plant to the resistance of verticillium wilt, the foreign gene is coding verticillium wilt pathogen P class The VdP4-ATPase gene of ATPase.

Specifically, the present invention is by the verticillium wilt pathogen VdP4-ATPase base containing nucleotide sequence shown in SEQ ID NO.9 Because being integrated into plant, realizes the constitutive expression of these genes, improve genetically modified plants to the removing toxic substances energy of Amber box policy Power, and then obtain the genetically modified plants of resisting verticillium.

More specifically, method of the invention, includes the following steps:

VdP4-ATPase gene integration is entered into target plant building genetically modified plants, and makes the VdP4-ATPase Gene is expressed in plant.

Preferably, the method, includes the following steps:

1) building contains the recombinant plant expression vector from verticillium wilt pathogen VdP4-ATPase gene；

2) recombinant plant expression vector is imported in target plant, so that VdP4-ATPase gene is in target plant Middle constitutive expression；

3) genetically modified plants with the resisting verticillium improved are obtained.

Preferably, the nucleotide sequence of the VdP4-ATPase gene is as shown in SEQ ID NO.9.

The target plant that method of the invention can be adapted for is preferably tomato, tobacco or cotton.

In the method for the present invention, the recombinant plant expression vector in step 1) has structure as shown in Figure 4.

It is also another object of the present invention to provide a kind of preparation methods of genetically modified plants with resistance to verticillium wilt, including Following steps:

I) the VdP4-ATPase gene of coding verticillium wilt pathogen P class ATPase is obtained, and is operably inserted plant table Up in carrier, plant expression vector is constructed；

Ii host) is converted with the plant expression vector that step i) is obtained, obtains transformant；

Iii plant) is converted with the transformant that step ii) is obtained, obtains genetically modified plants.

The present invention improves plant to the method for resistance to verticillium wilt using verticillium wilt pathogen VdP4-ATPase gene, specifically includes Following step:

1) it obtains verticillium wilt pathogen VdP4-ATPase gene: SpeI and SmaI restriction enzyme site design primer is introduced, then with Huang The germ cDNA that withers is that template carries out PCR amplification, and amplified production is the VdP4-ATPase gene order for adding restriction enzyme site；

2) plant expression vector of constitutive expression VdP4-ATPase gene: the VdP4-ATPase that amplification is obtained is constructed Gene order is connected into plant expression vector pLGN-35S-Nos, is constructed a new plant expression vector, is named as pLGN- 35S-VdP4-ATPase；

3) genetic transformation of plant: utilize agrobacterium tumefaciens-mediated transformation, by above-mentioned steps 2) obtain pLGN-35S- VdP4-ATPase plant expression vector is integrated into Plant Genome, realizes group of the VdP4-ATPase gene in genetically modified plants Molding expression improves genetically modified plants to the resistance of verticillium wilt；

4) acquisition of the VdP4-ATPase transgenic plant of resisting verticillium: the genetically modified plants that step 3) is obtained are into one Step bred, Molecular Identification and anti-disease enzyme, obtains the VdP4-ATPase transgenic plant improved to resistance to verticillium wilt.

Further, the step of pLGN-35S-VdP4-ATPase plant expression vector construction of constitutive expression includes: to obtain After the cDNA for obtaining verticillium wilt pathogen, design upstream primer: 5 '-GACTAGTATGGCTGGACGACCCACGGG (SpeI) -3 ', downstream Primer 5 '-CCCCGGGTCAGGTTCC CTGTCCTTGTG (SmaI) -3 ' carries out PCR amplification, amplified production and pLGN-35S- Nos vector plasmid carries out SpeI and SmaI double digestion respectively, and the large fragment after digestion is separately recovered, and recycles ligase that will return The section that takes up is attached, and is constructed a new plant expression vector, is named as pLGN-35S-VdP4-ATPase, to realize Constitutive expression of the VdP4-ATPase gene in genetically modified plants.

Plant of the present invention is mainly tobacco, tomato and cotton.

Abovementioned steps 2) described in building constitutive expression verticillium wilt pathogen VdP4-ATPase gene plant expression vector side Method is the conventional method of this field, and the carrier used is the conventional carrier of plant genetic engineering, and composition type expression promoter is flower Cauliflower mosaic virus CaMV35S promoter can also reach same purpose using other promoters with composing type characteristic.

Abovementioned steps 3) described in Agrobacterium tumefaciens mediated methods for plant transformation, wherein plant expression vector is transferred to agriculture The method of bacillus is electrotransformation, and plant expression vector is integrated into the method for tobacco, tomato and cotton as Agrobacterium tumefaciems Jie Inducing defecation by enema and suppository.

Abovementioned steps 4) described in Molecular Identification be identification technology used in the fields such as molecular biology, genetic engineering, such as The technologies such as GUS histochemical stain, PCR amplification identification.

Abovementioned steps 4) described in anti-disease enzyme refer to that seedling plants are to the resistance of verticillium wilt in phjytotron or greenhouse Identification, transgene tobacco and tomato, which are all made of, fills the inoculation that bacterium solution method carries out pathogen, and cotton is then inoculated with using leaching root method.

For raising plant provided by the present invention to the method for resistance to verticillium wilt, being will be from the VdP4- of verticillium wilt pathogen ATPase gene of MP is transferred to respectively in tobacco, tomato and cotton cells, realizes composing type table of these genes in transgenic plant It reaches, the toxin generated in plant using the P4 class ATP enzyme removing toxic substances verticillium wilt pathogen of foreign gene coding improves genetically modified plants To the detoxification ability of Amber box policy, and then genetically modified plants are improved to the disease resistance of verticillium wilt.

Cyclosporin A (CsA) is a kind of lipophilic hydrophobicity cyclic peptide being made of 11 amino acid, and present invention incorporates CsA With the similitude of Amber box policy part-structure and the characteristic of verticillium wilt pathogen pathogenesis, creatively propose using yellow Wither germ VdP4-ATPase gene constitutive expression in genetically modified plants, improves genetically modified plants to Amber box policy Detoxification ability, and then genetically modified plants are improved to the strategy of the resistance of verticillium wilt.

The present invention is mainly to improve disease resistance of plant using detoxification ability of the transgene product to the yellow verticillium toxin that withers, more into one Step is also in an identical manner protection model of the invention using the detoxification ability of transgene product raising plant against fungal toxin It encloses.Plant expression vector involved in the present invention, transformed cells etc. are also within the scope of the present invention simultaneously.

The VdP4-ATPase transgene tobacco obtained using the method for the present invention, T₀It is connect for seedling using root filling bacterium solution method is hurt The high virulence L2-1 bacterial strain 30d of kind, disease index 29.35, compared with wild type control 46.34.It is inoculated with 30d, 46 VdP4-ATPase Still there are 14 transformants apparent illness do not occur in transformant.VdP4-ATPase transgene cotton T₁For seedling, leaching is utilized Root inocalation method is inoculated with V991 defoliation verticillium wilt bacterial strain 20d, and non-transgenic control disease index has reached 82.24, there are two The disease index of VdP4-ATPase strain is lower than 10, respectively 6.25 and 8.33.The transgene tomato T obtained using the present invention₀ The high virulence L2-1 bacterial strain of bacterium solution method inoculation is filled using root is hurt for seedling plants, the disease index of non-transgenic tomato reaches 78.33, The disease index of VdP4-ATPase transgene tomato is 40.83, reduces by 37.5 compared with wild type control.Result of study shows to utilize Transgene cotton, tobacco and the tomato that the method for the present invention obtains can significantly mention genetically modified plants to the disease resistance of verticillium wilt.It says It is bright, it is provided by the invention to improve plant to the method significant effect of resistance to verticillium wilt.This method be applicable not only to cotton, tobacco and Tomato, all resistances that can be improved using this method by the plant that verticillium wilt pathogen infects to verticillium wilt.

Method provided by the invention mainly improves the resistance of plant using the removing toxic substances to pathogen toxin, because without having The characteristics of pathogen microspecies resistance.Therefore, the resistance that can be widely applied to improve various pathogenic bacteria biological strain, is not present The characteristics of small species specificity resistance.

Detailed description of the invention

Fig. 1 is sensitivity Detection interpretation of result of the phytopathogen to CsA

1: Chinese cabbage blackspot bacterium；2: verticillium wilt pathogen；3: sporulation；Cyclosporin A (CsA) concentration is 0 μ g/mL, 10 μ g/ ML, 50 μ g/mL and 100 μ g/mL.The CsA of 5 μ L various concentrations is added in each hole, is cultivated one week or so under the conditions of 26 DEG C.

Fig. 2 is pLGN-35S-Nos plant expression vector construction flow chart

LF-LF: recombination enzyme recognition site (LoxpRT) synthesizes for full genome；35S-MCS-Nos comes from pBIN AR Carrier (GenBank:AB752377.1), is obtained by PCR；AphA2 of II gene of NPT on bacterial transposon Tn5；

Gus gene is reporter gene, comes from Escherichia coli.

Fig. 3 is pLGN-35S-VdP4-ATPase carrier digestion verification result

M:DNA molecular weight standard Marker15；VdP:pLGN-35S-VdP4-ATPase vector plasmid obtaining after digestion The VdP4-ATPase target fragment obtained.

Fig. 4 is pLGN-35S-VdP4-ATPase plant expression vector figure

The table of II fusion of GUS::NPT comprising 2 × 35S starting in pLGN-35S-VdP4-ATPase carrier Up to frame, the expression cassette of the VdP4-ATPase gene of 35S starting.

Fig. 5 is VdP4-ATPase transgene tobacco GUS histochemical stain analysis

WT: the non-transgenic tobacco of wild type；VdP:VdP4-ATPase transgene tobacco.

Fig. 6 is that the PCR amplification of VdP4-ATPase transgene tobacco GUS positive plant identifies partial results

Bp: base-pair；M:DNA standard molecular weight DL2000；1: Wild-type non-transgenic negative control；2: plasmid positive pair According to；3-17:GUS stained positive transgenic tobacco plant.The PCR of VdP:VdP4-ATPase transgene tobacco GUS positive plant expands Increase result.

Fig. 7 is inoculation verticillium wilt pathogen 30d, the phenotype of VdP4-ATPase transgenic tobacco plant

WT: the non-transgenic tobacco of wild type；VdP:VdP4-ATPase transgene tobacco.

Fig. 8 is that the expression of foreign gene and sick grade compare in VdP4-ATPase transgenic line

WT: wild type control；VdP-1, VdP-4 ... VdP-42:VdP4-ATPase transgene tobacco strain.

Fig. 9 is VdP4-ATPase transgene cotton GUS histochemical stain analysis

WT: the non-transgenic cotton of wild type；VdP:VdP4-ATPase transgene cotton.

Figure 10 is that the PCR amplification of VdP4-ATPase transgene cotton GUS positive plant identifies partial results

Bp: base-pair；M:DNA standard molecular weight DL2000；1: Wild-type non-transgenic negative control；2: plasmid positive pair According to；3-17:GUS positive transgenic tobacco plant.VdP:VdP4-ATPase transgene cotton.

Figure 11 is inoculation verticillium wilt pathogen 20d, VdP4-ATPase transgene cotton T₁Sick grade, disease incidence (%) for strain And disease index

Null: the nontransgenic plants control separated in transgenic line；B1,B10,B50,B53,B56,B57,B58, The independent transformant of B6, B60 and B61:BbP4-ATPase transgene cotton.VdP7,VdP8,VdP10,VdP15,VdP16, The independent transformant of VdP23, VdP24, VdP33, VdP48 and VdP56:VdP4-ATPase transgene cotton.

Figure 12 is inoculation verticillium wilt pathogen 20d, VdP4-ATPase transgene cotton T₁For the phenotype of seedling

WT: the non-transgenic cotton of wild type；VdP:VdP4-ATPase transgene cotton.

Figure 13 is the expression of VdP4-ATPase transgene cotton foreign gene

Null: the nontransgenic plants control separated in transgenic line；VdP7,VdP8,VdP10,VdP15,VdP16, The independent transformant of VdP23, VdP24, VdP33, VdP48 and VdP56:VdP4-ATPase transgene cotton.

Figure 14 is VdP4-ATPase transgene tomato GUS histochemical stain analysis

WT: wild type control；VdP:VdP4-ATPase transgene tomato.

Figure 15 is that the PCR amplification of VdP4-ATPase transgene tomato GUS positive plant identifies partial results

Bp: base-pair；M:DNA standard molecular weight；1: Wild-type non-transgenic negative control；2: plasmid positive control；3- The tomato plant of 17:GUS stained positive reaction.

Figure 16 is inoculation verticillium wilt pathogen 30d, the phenotype of plant

WT: wild type control；VdP:VdP4-ATPase transgene tomato.

Specific embodiment

Present invention will be described in further detail below with reference to the accompanying drawings, but following explanation does not limit the present invention It is fixed.

What the medicine and reagent in embodiment of the present invention was not specifically described is domestic conventional chemical reagent, material side What method was not specifically described refers to " Molecular Cloning:A Laboratory guide " (Sambrook and Russell, 2001).

1, the proposition of inventive technique core strategy

Sensibility of 1.1 verticillium wilt pathogens to CsA

Analysis finds the amino acid sequence and beauveria bassiana of P class ATPase gene of MP from verticillium wilt pathogen coding The amino acid sequence similarity of BbP4-ATPase gene coding is up to 78%.Therefore, we are by the genoid from verticillium wilt pathogen It is named as VdP4-ATPase.

Early-stage study the result shows that, the BbP4-ATPase gene disruption mutant from beauveria bassiana is more quick to CsA Sense.BbP4-ATPase is a kind of memebrane protein, participates in intracellular vesicle transport, so it is presumed that, CsA may be transported with vesica Defeated mode is transported to a certain privileged site and is decomposed or utilized (such as vacuole), to improve beauveria bassiana to CsA's Tolerance, so speculating that the VdP4-ATPase from verticillium wilt pathogen may have similar function with BbP4-ATPase.We Result of study be also shown that relative to other plant pathogenic fungis (such as Chinese cabbage blackspot bacterium, sporulation etc.), verticillium wilt pathogen pair CsA has stronger tolerance, and verticillium wilt pathogen itself can generate toxin, and the toxin generated may growth to its own Certain influence is generated, so, VdP4-ATPase may play the role of removing toxic substances.

In order to verify verticillium wilt pathogen to the sensibility of CsA, with defoliation verticillium wilt pathogen V991 (Verticillium Dahliae, scientia Agricultura Sinica research institute Plant Protection Institute, simple Gui Liang researcher give), Chinese cabbage blackspot bacterium (Alternaria brassicae, this laboratory early period save), sporulation (Alternaria solani, this laboratory Early period saves) verticillium wilt pathogen V991, Chinese cabbage blackspot bacterium, sporulation are inoculated in PDA (potato solid as research object Culture medium) in culture, cultivate 14 days under the conditions of 26 DEG C, take appropriate each bacterial strain conidium in 0.05%Tween-80, preparation Concentration is 2 × 10⁶The conidial suspension of a/mL takes each bacterial strain conidial suspension of 400 μ L in 60mL PDA culture medium In (being cooled to 45 DEG C or so), after mixing, take 20mL culture medium and conidial mixture in the culture dish that diameter is 90mm In (each bacterial strain does 3 repetitions), after culture medium solidifies completely, with diameter be 5mm punch punch, in each Kong Zhongjia Enter the CsA of 5 μ L various concentrations, is cultivated one week or so under the conditions of 26 DEG C, concrete outcome is as shown in Figure 1.Fig. 1's the result shows that opposite In Chinese cabbage blackspot bacterium and sporulation, verticillium wilt pathogen has stronger tolerance to CsA.

The it is proposed of 1.2 inventive technique core strategies

The studies above shows verticillium wilt pathogen relative to plant pathogenic fungis such as Chinese cabbage blackspot bacterium and sporulations to CsA With compared with strong tolerance, meanwhile, CsA is a kind of lipophilic hydrophobicity cyclic peptide being made of 11 amino acid, and structure may be with Huang Wither pathogenic toxin certain pathogenic components structure it is similar, therefore, in conjunction with verticillium wilt pathogen pathogenic mechanism, the present invention is creatively mentioned The core strategy of the invention out, " using the P class ATP enzyme of verticillium wilt pathogen come the Amber box policy that detoxifies in transgenic plant, into And transgenic plant is improved to the resistance of verticillium wilt ".

2, the clone of VdP4-ATPase gene

The extraction of 2.1 verticillium wilt pathogen RNA and the synthesis of cDNA

Take appropriate verticillium wilt pathogen wild-type strain V991 mycelia in 50mL PDB (potato fluid nutrient medium) culture medium, 200rpm, 26 DEG C shaken cultivation 4 days, filter, collect mycelia, using liquid nitrogen grinding method, by the EASYspin of Aidlab company RNA rapidly extracting kit operation instructions extract the total serum IgE of verticillium wilt pathogen, and the RNA of acquisition is detected using 1% agarose electrophoresis Quality.Recycle the PrimeSc- of TaKaRA companyThe synthesis of RT reagent Kit with gDNA Eraser kit CDNA operates stringent by specification and carries out.

The clone of 2.2VdP4-ATPase gene

The verticillium wilt pathogen cDNA obtained using embodiment 2.1 is template, with VdP4-ATPase upstream region of gene

Primer 5 '-GACTAGTATGGCTGGACGACCCACGGG(SpeI)

-3’/5’-CCCCGGGTCAGGTTCCCTGTCCTTGTG (SmaI) -3 ' is primer, utilizes KOD-Plus-Neo (TOYOBO) enzyme carries out PCR amplification, recycles amplified production, obtains the VdP4-ATPase piece for having SpeI/SmaI restriction enzyme site Section.

PCR amplification system are as follows: 10 × PCR Buffer, 5 μ L, 2mM dNTPs, 5 μ L, 25mM MgSO₄3 μ L, about 10 μM Each 2 μ L of primer is swum, 41 μ L (1U/ μ L) of μ L, KOD-Plus-Neo of template (50 μ g/ μ L) adds water to 50 μ L, amplification program 94 ℃2min；98 DEG C of 10s, 68 DEG C of 4min 30s are recycled 40 times.

3, the building of pLGN-35S-VdP4-ATPase plant expression vector and the acquisition of Agrobacterium-mediated Transformation

The acquisition of 3.1pLGN-35S-Nos plant expression vector

PLGN-35S-Nos is a binary plant expression vector of this laboratory from pCambia2300 transformation.Its T-DNA section (region between RB and LB) is substituted for the Reporter gene GUS and label base of constitutive promoter CaMV35S-P control It is respectively added to a LoxpFRT recombination enzyme recognition site because of the track fusion box of NPTII, and at this expression cassette both ends, And another by the expression cassette of CaMV35S-P control, detailed process is as shown in Figure 2.

The building of 3.2pLGN-35S-VdP4-ATPase plant expression vector

Double digestion is carried out with SpeI and SmaI after the segment that the amplification of above-mentioned embodiment 2.2 obtains is recycled, and recycles enzyme It is sliced section, while carrying out double digestion pLGN-35S-Nos plasmid with SpeI and SmaI, and recycle the large fragment after digestion, it is then sharp With the VdP4-ATPase segment and pLGN-35S-Nos segment of DNA ligase connection recycling, connection product converts Escherichia coli DH5 α, screening positive clone simultaneously carry out digestion verification (as shown in Figure 3), the results showed that, successfully by VdP4-ATPase gene piece Section is connected into pLGN-35S-Nos carrier.Sequencing analysis, the BbP4- that sequence verification obtains are carried out according to carrier sequence design primer ATPase gene of MP sequence is shown in sequence 9 (SEQ ID NO.9), -80 DEG C of preservation pLGN-35S-VdP4-ATPase plant expression vectors Positive colony is spare, and carrier has structure shown in Fig. 4.

The acquisition of the recombinational agrobacterium of 3.3pLGN-35S-VdP4-ATPase plant expression vector

Using electrotransformation, the pLGN-35S-VdP4-ATPase plant expression vector that step 3.2 obtains is transferred to agriculture bar Bacterium LBA4404 competent cell carries out resistance screening using antibiotic-screening marker gene, obtains positive colony, then extract agriculture Bacillus plasmids simultaneously carry out double digestion verifying (verification result is identical as Fig. 3 digestion verification result) with SmaI and SpeI, are contained The recombinational agrobacterium of pLGN-35S-VdP4-ATPase plant expression vector.

Escherichia coli or the Agrobacterium plasmid of the conversion of pLGN-35S-VdP4-ATPase carrier are extracted, SpeI and SmaI are carried out Electrophoresis is carried out using 1% Ago-Gel after double digestion, obtains the target fragment of about 4128bp.

4, the Molecular Identification of transgenic plant

The GUS histochemical stain of 4.1 transgenic plants is identified

Referring to the method for (1987) such as Jefferson, hole device is cut reconstituted tobacco, tomato or cotton with the aperture 9mm and is planted Strain young leaflet tablet sets GUS dyeing liquor (500mg/L X-Gluc, 0.1mol/L K₃Fe(CN)₆, 0.1mol/L K₄Fe(CN)₆, 1% Triton X-100 (v/v), 0.01mol/L Na₂The 0.1mol/L phosphate buffer of EDTA, pH7.0) in, 37 DEG C of heat preservation 2h. After dyeing, 75% ethanol decolorization, every 4h replaces a destainer, until the color of uncolored part is taken off completely.Blade does not all have The regrown material for having blue to occur is nontransgenic plants, and dyeing blue is transgenic plant.

The PCR amplification of 4.2VdP4-ATPase transgenic plant is identified

Tobacco, tomato and the cotton plants that all regenerated GUS histochemical stains are positive, take its seedling leaves About 0.1g, operation sequence to specifications are mentioned using the plant genes group DNA rapidly extracting kit of Aidlab company DNA is taken, is expanded with the quality of 1% agarose gel electrophoresis detection DNA then using the DNA of extraction as template after obtaining DNA The segment of VdP4-ATPase gene, to detect whether regeneration plant all incorporates VdP4-ATPase gene.

VdP4-ATPase gene PCR amplimer are as follows: upstream primer: 5 '-TATTCCTCTCGTT GCCGTGC-3 '；Under Swim primer: 5 '-CAATGTCTGCGGGAAAAGGC-3 '.

25 μ L amplification systems include: 10 × LA PCR Buffer, 2.5 μ L；25mmol/L MgCL₂, 2.5 μ L； 2.5mmol/L dNTP Mixture, 2 μ L；Template DNA, 1 μ L (about 10ng)；5 μm of ol/L upstream primers, 1 μ L；Under 5 μm of ol/L Swim primer, 1 μ L；LA Taq enzyme, 0.2 μ L；ddH₂O, 14.8 μ L.

PCR thermal cycler parameters: 94 DEG C, 5min；94 DEG C, 30s, 57 DEG C, 30s, 72 DEG C, 30s, expand 30 circulations；72 DEG C, 10min.Last amplified production is detected with 1% agarose gel electrophoresis.

5, the acquisition of anti-disease enzyme pathogenic bacteria and plant disease grade SS

The acquisition of pathogenic bacteria of 5.1 anti-disease enzymes

PDA solid medium activation defoliation verticillium wilt pathogen V991 or High pathogenicity L2-1 bacterial strain (Agricultural University Of Hebei, Professor Ma Zhiying give), a little mycelium inoculation of picking enters in CZB (Cha Shi fluid nutrient medium), 180rpm, 26 DEG C of shaken cultivations 7d, then be inoculated in the ratio of 10% (bacterium solution/CZB culture medium) into CZB culture medium, 180rpm, 26 DEG C of shaken cultivation 7d, with four layers Sterile gauze filters off mycelia and impurity in bacteria-removing liquid, and deionized water adjustment verticillium wilt pathogen spore concentration reaches 10⁷A/ml, The spore suspension is the pathogenic bacteria inoculation liquid of anti-disease enzyme inoculation.

The sick grade SS of 5.2 transgenic plants

Referring to 5 grades of sick grade statistical methods of the Young Cotton seedling diseases grade of Bhat and Subbarao (1999), cotton, tobacco are counted With the sick grade of tomato plant.

0 grade: plant appearance is without illness；

1 grade: 1/3 or less plant leaf shows illness；

2 grades: plant leaf 1/3-2/3 shows illness；

3 grades: 2/3 or more plant leaf shows illness；

4 grades: plant leaf all shows illness.

5.3 disease incidence and disease index calculation formula

6, gene transcript expression detects

The extraction of 6.1RNA and the synthesis of cDNA

Using plant RNA rapidly extracting kit (Aidlab product), plant RNA is extracted, all operations are stringent illustratively Book carries out, and the RNA of acquisition detects quality using 1% agarose electrophoresis.It recyclesRT reagent Kit With gDNA Eraser kit (TaKaRa product) synthesizes cDNA, then using cDNA as template amplification target gene, plants in RNA The removal of object genomic DNA and the stringent by specification of the synthesis of cDNA carry out.

The detection of 6.2 gene transcript expressions

Utilize the expression of transgenosis in Real-time PCR method detection plant.To uniform cDNA concentration, cotton Using GhHIS3 gene as internal standard, tobacco is then using 18S gene as internal standard.

20 μ L reaction systems include: 1 μ L of cDNA, each 1 μ L, 2 × iQ SYBR Green Supermix of upstream and downstream primer 10 μ L supply 20 μ L with the distilled water of no RNA enzyme.

Amplification program are as follows: 95 DEG C of pre- amplification 3min；94 DEG C of 10s, 56 DEG C of 30s, 72 DEG C of 30s, coamplification 40 circulations.Amplification The relative expression quantity of Gene Study software analysis gene is utilized after the completion.

7, the acquisition of the genetic transformation of tobacco and transgenic plant

7.1 tobacco genetic transformation culture mediums

MSB:MS inorganic salts (Murashige and Skoog, 1962)+B5 organic (Gamborg etc., 1968).

Seed germination medium: MSB+30g/L sucrose is added 2.0g/L Gelrite and is solidified, pH6.0.

Co-culture medium: MSB+30g/L sucrose+0.5mg/L IAA (heteroauxin)+2.0mg/L 6-BA (6- benzyl ammonia Base purine), 2.0g/L Gelrite is added and is solidified, pH5.4.

Calli induction media: MSB+30g/L sucrose+0.5mg/L IAA+2.0mg/L 6-BA+400mg/L Cef (head P0-357)+100mg/L Km (kanamycins), 2.0g/L Gelrite is added and is solidified, pH5.8.

Young shoot induced medium: MSB+30g/L sucrose+2.0mg/L 6-BA, 200mg/L Cef+100mg/L Km is added 2.0g/L Gelrite is solidified, pH5.8.

Root media: MSB+30g/L sucrose+200mg/L Cef is added 2.5g/L Gelrite and is solidified, pH5.8。

The acquisition of 7.2 tobacco genetic transformation explants

Mature tobacco seed subnumber grain is taken, 75% alcohol sterilizing 1min, quickly incline alcohol；1% liquor natrii hypochloritis Sterilize 15min, and during which failure of oscillation is not swung, to reach adequately disinfected purpose；Incline sodium chlorate solution, is rinsed using aseptic double-distilled water Seed 7-8 times.120rpm under aseptic condition, shaken at room temperature culture to seed germination, then it is transferred to seed germination medium, 16h light According to culture under the conditions of/8h dark culture to 4-6 piece true leaf.Full-grown true leaf is taken, uses scalpel under aseptic condition on super-clean bench The leaf dish that piece is cut into the Jie side 3-5mm is used as the explant of conversion.

The preparation of 7.3 During Agrobacterium liquid

The agrobacterium strains scribing line culture of the carrier containing LGN-35S-VdP4-ATPase of -80 DEG C of preservations obtains single bacterium respectively It falls, then picking single colonie, is inoculated with (5g/L in the YEB culture medium of additional 50mg/L Km and 125mg/L Sm (streptomycin sulphate) Sucrose, 1g/L bacto-yeast extract, 10g/L bacterium tryptone, 0.5g/L MgSO₄·7H₂O, pH7.0), 28 DEG C, 200rmp shaken cultivation stay overnight, until take bacterium solution to be centrifuged when bacterium solution OD600 value reaches 0.8, thallus addition same volume MSB liquid Body culture medium is resuspended, and re-suspension liquid is the During Agrobacterium liquid of conversion.

The genetic transformation and plant regeneration of 7.4 tobaccos

The leaf dish explant that step 7.2 obtains in disseminating 30min in During Agrobacterium liquid prepared by step 7.3, hypsokinesis Bacterium solution is gone, the tobacco explant infected through Agrobacterium is seeded in co-culture medium, 26 DEG C of dark culture 2d, then after substitution Calli induction media, the subsequent substitution bud inducement cultivation base of 14d, is further cultured for about 14d, cuts regenerated green bud after substitution culture of rootage It cultivates in base, in root media to 2-3 leaf seedling, cleans the agar around root, be transplanted into the nutritive cube for containing turfy soil, And it is placed in greenhouse and is cultivated.

The acquisition of the Molecular Identification and transgenic plant of 7.5 transgene tobaccos

GUS histochemical stain is carried out to regenerated tobacco plant respectively according to the method for embodiment 4 and PCR amplification is reflected It is fixed.The plant that all tobacco leaf GUS histochemical stains can obtain blue as shown in Figure 5 (right side) is transgenic plant.Through GUS histochemical stain identification obtains 46 VdP4-ATPase transgenic lines altogether.In order to further determine GUS systematism It learns and whether all incorporates VdP4-ATPase gene in the plant that dyeing identification is positive, all GUS stained positive reactions Regeneration plant extract blade total DNA after, then using DNA as template, the piece of VdP4-ATPase gene is expanded with sequence 3 and sequence 4 Section, the results show that all VdP4-ATPase transgenosis reconstituted tobacco GUS positive reaction plant, which can expand, obtains VdP4- The target fragment (Fig. 6) of ATPase gene of MP about 200bp, illustrate to incorporate in GUS stained positive plant BbP4-ATPase or VdP4-ATPase gene.It is identified through GUS histochemical stain and PCR amplification, obtains 38 BbP4-ATPase transgenosis altogether Independent transformation of tobacco, independent transformation of 46 VdP4-ATPase transgene tobaccos, all these transformants are completely used for Anti-disease enzyme analysis.

8, resistance of the transgene tobacco to verticillium wilt

6-10 piece leaf is grown in the transgenic tobacco plant greenhouse obtained through 7 genetic transformation of embodiment and Molecular Identification When, it carries out hurting root processing at from plant about 1-2cm using knife blade, then pours L2- prepared by embodiment 7.1 for every plant 1 inoculation liquid 30mL (hurt root fill bacterium solution method) sets culture in 22 DEG C (night) -26 DEG C (daytime) of artificial climate room.30d is inoculated with, By the sick grade of the canonical statistics transgenic plant of embodiment 7.2.The results show that 46 independent VdP-ATPase transformants Average disease grade is 1.17, disease index 29.25.And the average disease of Wild-type non-transgenic control (regenerated WT lines) Grade is 3.03, and disease index is 75.65 (tables 1), and compared with non-transgenic control, the state of an illness of VdP-ATPase transgenic line refers to Number reduces 46.34.

It is inoculated with 30d, not only sick grade and disease index are significantly lower than Wild-type non-transgenic to VdP4-ATPase transgene tobacco It compares, there are 14 transformants there is no apparent illness in 46 VdP4-ATPase transformants, rather than non-transgenic control lines are whole It is serious to wilt, leaf chlorosis (Fig. 7).It randomly selects 5 transformants of not falling ill and carries out expanding propagation using axillary bud, then recycle Identical mode is inoculated with verticillium wilt pathogen, carries out anti-disease enzyme, is inoculated with 30d as the result is shown, these transformants are not shown obviously yet Illness, further confirm obtain disease-resistant transgenic tobacco the resistance of verticillium wilt is significantly improved.The result shows that utilizing VdP4-ATPase gene can significantly improve transgene tobacco to the disease resistance of verticillium wilt.

In order to further clarify the disease-resistant horizontal relationship improved between transgene expression level of transgenic line, randomly select 20VdP4-ATPase transgene tobacco is research object, extracts transgenic tobacco plant blade in the method for embodiment 6.1 RNA, and cDNA is synthesized by the method for embodiment 6.1, then using cDNA as template amplification VdP4-ATPase genetic fragment, then Quantitative pcr amplification is carried out by the method for embodiment 6.2, using 18S rRNA gene as internal standard.The results show that all genes of walking around VdP4-ATPase gene can carry out effective transcriptional expression, and the disease of the higher transgenic plant of transcriptional expression level in plant Grade is lower, i.e., for resistance with the transcriptional expression level of transgenosis there is certain correlation, the transcriptional expression of transgenosis is horizontal Higher, transgenic plant is better to the resistance of verticillium wilt.The expression of transgenic line and sick grade are shown in Fig. 8.

Using transgene tobacco seedling leaves as material, the RNA of transgenic plant is extracted, and reverse transcription synthesizes cDNA, with sequence Column 3 and sequence 4 are primer amplification VdP4-ATPase gene, are interior with the 18S rRNA gene of tobacco for homogenization cDNA concentration Gene is marked, and is that primer is expanded with sequence 5 and sequence 6, Gene Study software analysis gene is utilized after the completion of amplification Relative expression quantity.

Table 1 is inoculated with verticillium wilt pathogen 30d, the sick grade and disease index of VdP4-ATPase transgene tobacco

WT: WT lines；VdP4-ATPase:VdP4-ATPase transgenic tobacco plant.

46 VdP4-ATPase transgene tobacco transformants are outstanding all of root filling bacterium solution method inoculation verticillium wilt pathogen spore is hurt Supernatant liquid (10⁷A spore/mL), it is inoculated with 30d, the sick grade of each transformant is counted by 0-4 grades of 5 grade standards, is then calculated separately The average sick grade and disease index of all transformants and control.Non-transgenic control all serious morbidities, average disease grade reach 3.03, disease index has reached 75.69, and the average sick grade of VdP4-ATPase transgenic line is 1.17, and disease index is 29.35。

9, the genetic transformation of cotton:

9.1 Cotton Transformation culture mediums

Minimal medium: MSB (MS inorganic salts+B5 is organic)；

Seed germination medium: 1/2MSB+20g/L sucrose+6g/L agar, tap water are prepared, natural pH；

Co-culture medium: MSB+0.5mg/L IAA+0.1mg/L KT (6-Furfurylaminopurine)+30g/L glucose+100 μm ol/L acetosyringone+2.0g/L Gelrite, pH5.4；

Screen de- bacterium culture medium: MSB+0.5mg/L IAA+0.1mg/L KT+75mg/L Km+500mg/L cef+30g/L Glucose+2.0g/L Gelrite, pH5.8；

Calli induction media: MSB+0.5mg/L IAA+0.1mg/L KT+75mg/L Km+200mg/L cef+30g/L Glucose+2.0g/L Gelrite, pH5.8；

Embryo callus subculture induced medium: MSB+0.1mg/L KT+30g/L glucose+2.0g/L Gelrite, pH5.8；

Liquid suspension culture base: MSB+1.91g/L potassium nitrate+0.1mg/L KT+30g/L glucose, pH5.8；

Body embryo maturation medium: MSB+15g/L sucrose+15g/L glucose+0.1mg/L KT+2.5g/L Gelrite, pH6.0；

Seedling culture medium: SH+0.4g/L activated carbon+20g/L sucrose, pH6.0.(Schenk&Hildebrandt, 1972)

The acquisition of 9.2 conversion explants

Upland cotton seed decladding, 0.1% mercuric chloride of seed benevolence sterilizing 10min are inoculated in kind after aseptic water rinses 5-6 times Sub- germination medium, 28 DEG C of dark culture 5-7d.Sterile hypocotyl is cut into the dissection of 3-5mm long, as conversion explant.

The 9.3 conversions preparation of During Agrobacterium liquid

Conversion is with the preparation of Agrobacterium with embodiment 7.3, and after the Agrobacterium bacterium solution centrifugation of culture, thallus is androgynous with addition Long-pending co-cultivation fluid nutrient medium is resuspended, and re-suspension liquid is conversion During Agrobacterium liquid.

The induction of the genetic transformation and embryo callus subculture of 9.4 Cotton Hypocotyls

Explant disseminates 20min, bacterium solution of inclining with During Agrobacterium liquid, then sucks explant excess surface with aseptic filter paper Bacterium solution, the hypocotyl segment after dip dyeing is inoculated in co-culture medium, and it is de- to be seeded to screening by 26 DEG C of dark culture 2d for hypocotyl Bacterium culture medium, the subsequent calli induction media progress callus for substituting into additional kanamycins (Km) and cephalosporin (cef) of 20d Induction, interval 20d subculture is primary, and the subsequent substitution embryo callus subculture induced medium of 60d carries out liquid suspension after obtaining embryo callus subculture Culture, to obtain the consistent embryo callus subculture of raised growth.

The induction and seedling culture of 9.5 body embryos

The embryo callus subculture of liquid suspension culture, the filtering of 30 mesh stainless steel mesh, the embryo callus subculture under sieving connect evenly dispersedly Kind enters body embryo maturation medium, and about 15d generates a large amount of body embryo, by it after substituting into SH culture medium, promotes the further seedling of body embryo. The regrowth of 3-4 leaf is transplanted into greenhouse and is bred.

9.6VdP4-ATPase acquisition and the Molecular of transgene cotton

Using regenerated cotton leaf as material, GUS histochemistry is carried out to regeneration plant respectively by the method for embodiment 4 Dyeing and PCR amplification identification.The plant that GUS histochemical stain can obtain blue (right side) shown in Fig. 9 is transgenic plant, To be that a strain is calculated from the regeneration plant of an explant, obtained through GUS histochemical stain identification VdP4-ATPase transgene cotton is 10 GUS positive strains, is 111 plants of transgenic plants.To further determine that the GUS positive Whether VdP4-ATPase gene all is incorporated in plant, further transgenic plant is carried out by the method for embodiment 4.2 PCR amplification identification, the results show that the plant that all GUS histochemical stains are positive can amplify VdP4-ATPase The target of (about 200bp) gene is specifically with (Figure 10).In an identical manner to T₁Molecular Identification is carried out for seedling, through Molecular Identification Transgenic plant T₁Disease-resistant analysis is used to for seedling.

Using the DNA of transgene cotton blade as template, with composition sequence 3 and sequence 4 for primer amplification VdP4-ATPase base The segment of cause, the target that the plant of all GUS stained positive reactions can amplify VdP4-ATPase (about 200bp) gene are special Different band.

10, resistance of the transgene cotton to verticillium wilt

Breed T in greenhouse₀Seed benevolence is stayed in seed decladding for plant harvest, Aquaponic is carried out after 28 DEG C of sproutings, until cotyledon is flat Expansion begins true leaf occur, and the V991 defoliation verticillium wilt pathogen obtained using leaching root inocalation method inoculation embodiment 7.1, root infects 2d Transplanting is joined the army in feeding alms bowl afterwards, and then 22 DEG C (night) -26 DEG C (daytime), 16h illumination is cultivated under 8h dark condition, after inoculation between The sick grade for counting a plant by the sick grade standard of embodiment 5.2 every 5d is inoculated with 20d, non-turn separated in transgenic line Gene plant (Null) is all fallen ill, and disease index reaches 82.24, the disease of 10 transgenosis VdP4-ATPase strains of acquisition Feelings index is also below non-transgenic control, wherein VdP8, VdP10, VdP15, VdP16, VdP24, VdP33 and VdP56 etc. 7 The disease incidence of strain is lower than 50%, respectively 28.57%, 50.00%, 50.00%, 33.33%, 50.00%, 46.25% and 25.00%, disease index is respectively 14.29,12.50,12.50,8.33,25.00,21.15 and 6.25, VdP16 therein and The disease index of VdP56 strain rate is respectively 8.33 and 6.25, is lower than 10, has reached disease-resistant level.Compared using average sick grade Compared with the results show that inoculation 20d, the average sick grade of non-transgenic control is 3.29, to non-defoliation verticillium wilt V991 strains expressed To be susceptible, the sick grade of this 7 strains of VdP8, VdP10, VdP15, VdP16, VdP24, VdP33 and VdP56 is respectively 0.57, 0.50,0.50,0.33,1.0,0.85 and 0.25 (Figure 11) equally shows as verticillium wilt V991 bacterial strain anti-or resistance to.

All there is the typical illness of verticillium wilt because verticillium wilt pathogen infects blade in inoculation 20, non-transgenic control plant, on The discoloration of portion's leaf chlorosis, partial blade necrosis even fall off, and VdP4-ATPase transgene cotton true leaf leaf color is normal, and plant is raw Long normal, only there is slight illness in individual plant cotyledons, have a little scab (Figure 12), hence it is evident that improve to verticillium wilt Resistance.

In order to further illustrate the relationship between transgene expression level and disease resistance, detected using Real-time PCR outer The transcriptional expression of source gene is horizontal.Using transgenic line as material, each strain takes three sample extraction RNA, and reverse transcription is closed It is uniform with the sequence 3 of synthesis and sequence 4 for primer amplification VdP4-ATPase gene then using cDNA as template at cDNA Change cDNA concentration, using GhHIS3 gene as internal standard gene, amplimer sequence is sequence 7 and sequence 8.(Figure 13) as the result is shown, In all VdP4-ATPase transgenic plants VdP4-ATPase gene also can effective transcriptional expression, with the transgenic line state of an illness Index compares, the results show that the height of transgene expression level affects the resistance level of transgenic line, transgene expression When horizontal higher, the disease resistance of transgenic line is more preferable.

It is inoculated with defoliation verticillium wilt pathogen V991 bacterial strain using leaching root inocalation method, is inoculated with 20d, by 0-5 grades of sick grade standard statistics Sick grade, and disease incidence, disease index are calculated, and calculate the average sick grade of each strain.The results show that the hair of transgenic line Sick rate, disease index and average sick grade are all significantly lower than non-transgenic control, show that VdP4-ATPase transgene cotton withers to Huang The resistance of disease significantly improves.

11, the genetic transformation of tomato

11.1 tomato genetic transformation culture mediums

Minimal medium: MSB0 (organic+30g/L sucrose of the inorganic+B5 of MS, pH5.8).The fine jade of solid medium addition 6g/L Rouge (Murashige and Skoog, 1962；Gamborg etc., 1968)；

Co-culture medium MSB1:MSB0+2.0mg/L 6-BA+0.2mg/L IAA+100uM AS (acetosyringone)+ 6g/L agar, pH5.4；

Screening and culturing medium MSB2:MSB1+500mg/L cb (carbenicillin)+100mg/L Km+6g/L agar, pH5.8；

Subculture medium MSB3:MSB0+200mg/Lcb+100mg/L Km+6g/L agar, pH5.8；

Root media MSB4:MSB0+0.5mg/L IAA+200mg/L Cef+50mg/L Km+6g/L agar, pH6.0.

The acquisition of 11.2 tomato genetic transformation explants

1% liquor natrii hypochloritis of Micro-Tom tomato seeds sterilizing 10-15min, then aseptic water rinses 5-6 times, Be seeded on MSB0 culture medium, 25 DEG C, 16h illumination/8h dark photoperiod in sprouting 7d or so, open and flat sterile of robust growth Seedling cotyledon cuts both ends, and the part at middle part about 2/3 gives over to the explant of Agrobacterium tumefaciens-mediated Transformation.

The 11.3 conversions preparation of During Agrobacterium liquid

Conversion is with the preparation of Agrobacterium with embodiment 7.3, and after the Agrobacterium bacterium solution centrifugation of culture, thallus is androgynous with addition Long-pending MSB0 fluid nutrient medium is resuspended, and re-suspension liquid is conversion During Agrobacterium liquid.

11.4 tomato genetic transformation

Crown gall agriculture bar is utilized using the cotyledon for growing about 7d aseptic seedling as explant with reference to the method for (2004) Cortina etc. Bacterium mediated method carries out genetic transformation.

Bacterium solution is gone in the dip dyeing explant 10min hypsokinesis of During Agrobacterium liquid, and sterile blotting paper sucks explant excess surface bacterium Then liquid is inoculated with into the co-culture medium MSB1,25 DEG C of dark co-cultivation 2d for being covered with one layer of aseptic filter paper.After the completion of co-cultivation, Explant is inoculated in screening and culturing medium MSB2 and carries out differentiation culture, 25 DEG C, 16h illumination/8h dark photoperiod culture 2 In week, then by explant after substituting into the generation of MSB3 culture medium callus induction, subculture is primary every 2 weeks.It, will after generating Km resistance young shoot Young shoot, which is cut, to be inoculated with into MSB4 root media, and Km resistance regeneration plant is obtained.The vegetative seedling of root long 3-5cm, is transplanted into temperature Room grows seedling.

The acquisition of the Molecular Identification and transgenic plant of 11.5 transgene tomatos

GUS histochemical stain is carried out to regenerated tomato plant respectively according to the method for embodiment 6 and PCR amplification is reflected It is fixed.The plant that all tomato leaf GUS histochemical stains can obtain blue as shown in figure 14 (right side) is transgenic plant, warp GUS histochemical stain identification obtains 30 plants of VdP4-ATPase Transgenic Tomato Plants altogether, in order to further determine GUS group VdP4-ATPase gene whether is all incorporated in the plant that weave chemistry dyeing identification is positive, all regeneration plants extract Blade total DNA, then be primer with composition sequence 3 and sequence 4 using DNA as template, expand the segment of VdP4-ATPase gene, knot Fruit shows that all GUS positive reaction plant can expand the target for obtaining VdP4-ATPase (about 200bp) gene specifically with (figure 15).PCR amplification result illustrates, incorporates VdP4-ATPase base in the VdP4-ATPase plant of GUS stained positive reaction Cause.It is identified through GUS histochemical stain and PCR amplification, obtains 30 VdP4-ATPase transgenic plants altogether, it is all these Transformant T₀Anti-disease enzyme analysis is completely used for for plant.

12, resistance of the transgene tomato to verticillium wilt

The Transgenic Tomato Plants obtained through 11 genetic transformation of embodiment and Molecular Identification grow to 4-6 piece in greenhouse Ye Shi carries out hurting root processing at from plant about 1-2cm using knife blade, and every plant is symmetrically hurt root then every plant of pouring implementation L2-1 inoculation liquid 15mL prepared by example 5.1 sets in the culturing room on 22 DEG C (night) -26 DEG C (daytime) and cultivates.It is inoculated with 20d, is pressed The sick grade of the canonical statistics transgenic plant of embodiment 7.2.

Anti-disease enzyme as the result is shown (table 2), is inoculated with 30d, the average sick grade of wild type control (regenerated WT lines) It is 3.13, disease index 78.33.The average sick grade of 30 VdP4-ATPase transformants is 1.63, disease index 40.83, Compared with non-transgenic control, the disease index of transgenic line reduces 37.50.It is inoculated with 30d, 30 VdP4-ATPase turn There is the sick grade of 18 transformants to be not above 1 in beggar, the transgenic plant that mentioning property improves is that the blade of bottommost occurs slightly Illness, rather than the sick grade of non-transgenic control lines is all 3 or more, and the blade of middle and upper part has begun chlorosis and turns yellow (figure 16)。

Anti-disease enzyme the result shows that, transgene tomato can be significantly improved using VdP4-ATPase gene, verticillium wilt is resisted Characteristic of disease.

Table 2 is inoculated with verticillium wilt pathogen 30d, the sick grade and disease index of transgene tomato

WT: wild type control；VdP:VdP4-ATPase transgene tomato.

All plant fill bacterium solution method inoculation verticillium wilt pathogen spore suspension (10 using root is hurt⁷A spore/mL), it is inoculated with 30d, The sick grade that each transformant is counted by 0-4 grades of 5 grade standards, then calculate separately all transformants and control average sick grade and Disease index.Non-transgenic control is all fallen ill, and average disease grade is 3.13, and disease index has reached 78.33, VdP4-ATPase The sick grade and disease index of transgene tomato are remarkably decreased.

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Claims (6)

1. encoding the VdP4-ATPase gene of verticillium wilt pathogen P class ATPase is improving plant to the purposes in resistance to verticillium wilt, In by the way that VdP4-ATPase gene integration is entered target plant construct genetically modified plants, and make the VdP4-ATPase base Plant is improved because expressing in plant to the resistance of verticillium wilt, wherein the nucleotide sequence of the VdP4-ATPase gene is such as Shown in SEQ ID NO.9, the target plant is tomato, tobacco or cotton.

2. a kind of improve plant to the method for resistance to verticillium wilt, wherein improving by the expression alien gene in target plant body For the plant to the resistance of verticillium wilt, the foreign gene is the VdP4-ATPase gene for encoding verticillium wilt pathogen P class ATPase, Wherein for the nucleotide sequence of the VdP4-ATPase gene as shown in SEQ ID NO.9, the target plant is tomato, tobacco Or cotton.

3. method as claimed in claim 2, includes the following steps:

The VdP4-ATPase gene integration for encoding verticillium wilt pathogen P class ATPase is entered into target plant building genetically modified plants, and So that the VdP4-ATPase gene is expressed in plant.

4. method according to claim 2 includes the following steps:

1) recombinant plant expression vector of the building containing the VdP4-ATPase gene from coding verticillium wilt pathogen P class ATPase；

2) recombinant plant expression vector is imported in target plant, so that VdP4-ATPase gene group in target plant Molding expression；

3) genetically modified plants with the resisting verticillium improved are obtained.

5. method as claimed in claim 4, wherein recombinant plant expression vector described in step 1) has knot as shown in Figure 4 Structure.

6. a kind of preparation method of the genetically modified plants with resistance to verticillium wilt, comprising the following steps:

I) the VdP4-ATPase gene of coding verticillium wilt pathogen P class ATPase, the coding verticillium wilt pathogen P class ATPase are obtained The nucleotide sequence of VdP4-ATPase gene be operably inserted plant table as shown in SEQ ID NO.9, and by the gene Up in carrier, plant expression vector is constructed；

Ii host) is converted with the plant expression vector that step i) is obtained, obtains transformant；

Iii plant) is converted with the transformant that step ii) is obtained, obtains genetically modified plants；

Wherein the genetically modified plants are tomato, tobacco or cotton.