CN101717785B - Method for obtaining high antiviral potato plant with gene engineering technology - Google Patents

Abstract

The invention discloses a method for obtaining a high-antiviral potato plant with a gene engineering technology, which comprises the steps of: cloning Tm-22 gene, building over-expression carrier thereof, infecting and transforming agrobacterium rhizogenes, and detecting the high-antiviral potato plant. According to the known disease-resistant mechanism of tomato disease-resistant gene Tm-22 and high resistance characteristic thereof to virus (such as TMV and ToM, and the like), the method uses the characteristic that the tomato and the potato has a nearer genetic relationship, adopts the gene engineering technology to transform the Tm-22 gene into the potato, and obtains the high-antiviral potato plant which has expressed the Tm-22 gene by means of screening and evaluating, thereby improving the resistance of the potato plant to normal virus. By transforming the potato by means of the plant gene, the method can prevent from environmental risk and food safety risk, is good for pushing the antiviral research of the potato into a new stage, and has energetic exploration for agricultural industry.

Description

Utilize genetic engineering technique to obtain the method for high antiviral potato plant

Technical field

The present invention relates to utilize genetic engineering technique to obtain the method for high antiviral potato plant.

Background technology

Yam is that output is only second to the fourth-largest food crop of wheat, corn and paddy rice in the world, and its unit surface yield of unit time is higher than the first three crop.Yam grain dish dual-purpose, nutritious, United Nations has affirmed fully the significant role that yam rises in guaranteeing world food safety and eradicating poverty, and is " international yam year " in definite 2008.But virus infection is the principal element that causes the yam deterioration of strains always, has a strong impact on yield of potato and quality.Traditional breeding mode can not satisfy the needs that yam produces, and can not fundamentally solve potato disease for a long time and poison problem.In recent years, develop perfect day by day with the transgenic technology system rapidly along with engineered, (especially antiviral) shown great potentiality to genetic engineering technique aspect the yam disease resistance improving, and will become the main means of the antiviral breeding of yam.

Because yam is the autotetraploid crop, it is very slow to go to improve the breed with conventional method, and generally breeding a kind needs 8～10 years at least, so virus disease just becomes in the potato raw delivery and feed infant kind a very stubborn problem.And the method for traditional control and control virus has stem apex detoxify tissue culture and pesticide application; The cultivation back was prone to limitation such as infective virus and qualitative variation again after wherein the stem apex detoxify tissue culture technique had its cost height, examined conditionality, detoxification; And the pesticide application serious environment pollution is poisoned problem so these two kinds of methods all can not effectively solve potato disease.

Hamilton has at first proposed the imagination of genetically engineered protection in early 1980s, in transgenic plant, expresses virus genome sequence and possibly be one of approach that defend against computer virus infects.So occurred the successfully anti-PVX of first example (potato virus X) transgenic Rhizoma Solani tuber osi in 1988, make that the engineering research of yam antiviral gene all make progress rapidly in the period of later 20 on breadth and depth, opened up new way for preventing and treating potato virus disease.

Although in yam antiviral gene engineering; Genetically modified technology and method have been carried out repeatedly improvement and perfect; The disease-resistant goal gene that changes over to is also towards diversified development, and experimental study has also been obtained a lot of gratifying achievements both at home and abroad, but exists some problems to need to be resolved hurrily:

1. in various experiment reports; The system of agrobacterium mediation converted yam is all relatively independent; At present, therefore also need the investigator to go further to optimize screening through experiment to most of yam kinds and the different explant transformation mode that generally is fit to of none also.

2. the high antiviral potato plant regeneration rate is lower and unstable, and transformation system standard and the method used in the different RRs are all inequality, and this just lets other investigators be difficult to compare and analyzes and estimate and use for reference.

3. take at present in the process of agrobacterium mediation converted yam, when importing goal gene, also imported antibiotics resistance gene mostly, this selective marker might be transferred in conversion process in other biological such as mikrobe or the potato plant body.

4. the heterologous gene that imports in now many experimental studies is that virogene is as the antiviral gene that transforms yam; Genes of these source viruses have has certain potentially dangerous property; Allos involucrum phenomenon like the CP gene; Change in the potato plant body of virogene and be prone to take place to produce new virus, cause the biological safety problem thereby virogene RNA produces sudden change or the like with the viral RNA reorganization.

There is limited evidence currently of has human non-viral gene (like plant gene) to transform yam.Fast development along with Protocols in Molecular Biology; People have have cloned and isolated some plant disease resistance genes; Especially with the very near solanaceous crops of yam sibship, as coming out to have identified Tm-1, Tm-2 and Tm-2 with anti-virus ability from separation of tomato karyomit(e) and clone ²Deng gene.Because homologous gene derives from the close kind of crop itself or close with it source, can get rid of environmental exposure and edible safety risk like this.The genetically modified yam of homology is accepted by the public more easily, with a breach that can become the transgenic industry most probably.

In the yam antiviral gene engineering research in future; Change the antiviral route of non-viral source gene, designated rna mediation over to; And take the complex gene strategy; To obtain to have the wide spectrum virus resistance and, will be current and development trend in the future to the high antiviral potato plant of environment and edible safety.

Summary of the invention

In view of this, the purpose of this invention is to provide a kind of method of utilizing genetic engineering technique to obtain high antiviral potato plant, according to known tomato disease-resistant gene Tm-2 ²Disease-resistant mechanism and to virus (like TMV, ToMV etc.) high anti-characteristic, utilize tomato and the nearer characteristics of yam sibship, the employing genetic engineering technique with Tm-2 ²Gene changes yam over to, and has expressed Tm-2 through screening, evaluation ²The high antiviral potato plant of gene improves the resistance of potato plant to common virus, avoids environmental exposure and edible safety risk simultaneously.

The method of utilizing genetic engineering technique to obtain high antiviral potato plant of the present invention comprises the steps:

(1) produces tomato disease-resistant gene Tm-2 ²

According to tomato disease-resistant gene Tm-2 ²Two pairs of Auele Specific Primers of sequences Design and add restriction enzyme digestion sites at upstream primer 5 ' end, extract tomato RNA then and carry out the RT-PCR amplification with two pairs of Auele Specific Primers respectively, obtain Tm-2 ²The amplified production of gene leading portion and back segment; With gained Tm-2 ²The extension amplification outcome of gene leading portion and back segment is gone into two pMD18-T carriers, carries out enzyme again and cuts, and enzyme is cut the purified back of product and connected, and gets total length Tm-2 ²Gene DNA sequence;

(2) structure of overexpression vector

Extract total length Tm-2 ²Fragment also is cloned in the pDH51 carrier that contains CaMV35S promotor and terminator, gets pDH51-Tm-2 ²Plasmid, enzyme is cut pDH51-Tm-2 ²Plasmid and with the gained fragment cloning to the pBIN19 carrier, overexpression binary vector pBIN19-Tm-2 ²

(3) Agrobacterium infects conversion

The yam explant that will pass through preparatory cultivation acquisition places and contains pBIN19-Tm-2 ²Dip-dye and warp are cultivated altogether, are induced and sprout and the selectivity root culture in the bacterium liquid of the LBA4404 Agrobacterium of binary vector, get transgenic potato plant;

(4) detection of transfer-gen plant

Adopt PCR, RT-PCR detection method screening positive plant from the gained transgenic potato plant, the gained positive plant is identified through the inoculation of live body disease resistance and is obtained the disease resistance high antiviral potato plant.Further, the said yam explant of step (3) is obtained by following steps:

A. potato detoxicating cuvette seedling single-unit section is inoculated on the substratum; Under the condition of 20 ± 2 ℃ of temperature, 16h/d photoperiod and 1000-2000lx intensity of illumination, cultivate then; Said substratum is the MS substratum; Component that comprises in the substratum and the respective components massfraction in substratum is respectively: sucrose 3%, agar 0.6-0.8%, and regulating pH is 5.8;

B. treat that detoxification test tube plantlet grows to behind 6-7 the blade stem section that the potato detoxicating cuvette seedling of robust growth is cut into 1 leaf, 1 joint, with said stem section be seeded to induce in the solid medium and under 20 ± 2 ℃ full dark condition inducing culture to obtain the test tube potato;

C. said test tube potato section gets the yam explant;

Further, the said solid medium of inducing of step b is the MS substratum, induces the component and the massfraction of respective components in inducing solid medium that comprise in the solid medium to be respectively: sucrose 8-10%, and regulating pH is 5.8;

Further, the said solid medium of inducing also comprises agar and 6-BA, and the massfraction of agar in inducing solid medium is 0.6%, and the mass concentration of 6-BA in inducing solid medium is 2mg/L;

Further, the said pBIN19-Tm-2 that contains of step (3) ²The bacterium liquid of the LBA4404 Agrobacterium of binary vector makes as follows:

1. will contain pBIN19-Tm-2 ²The LBA4404 Agrobacterium of binary vector be inoculated in the YEB solid medium and under 28 ± 2 ℃ of conditions dark culturing 2-3d, culture;

2. join the gained culture in the YEB liquid nutrient medium according to 0.01: 1 volume ratio and be cultured to OD ₆₀₀For behind the 1.8-2.0 under the 4000-6000rpm condition room temperature centrifugal and with the resuspended thalline of fresh YEB liquid nutrient medium, bacteria suspension;

3. the gained bacteria suspension with the resuspended thalline of MS salts solution, must contain pBIN19-Tm-2 in the centrifugal back of 4000-6000rpm room temperature ²The bacterium liquid of the LBA4404 Agrobacterium of binary vector.

Component that said YEB solid medium comprises and the concentration of respective components in the YEB solid medium are respectively: massfraction is 1.5% agar, the Kan of 50mg/L, the Sm of 500mg/L and the Rif of 50mg/L, and regulating pH is 7.0;

Component that said YEB liquid nutrient medium comprises and the concentration of respective components in the YEB liquid nutrient medium are respectively: the Rif of the Kan of 50mg/L, the Sm of 500mg/L and 50mg/L, and regulating the pH value is 7.0;

The pH of said MS salts solution is 5.8;

Further, the said pBIN19-Tm-2 that contains ²The LBA4404 Agrobacterium bacterium liquid immerged time of binary vector is 8-10min; The said cultivation altogether is with in the bud inducing culture, altogether cultivating 2-3d 20 ± 2 ℃ of following dark through the yam explant of LBA4404 Agrobacterium bacterium liquid dip-dye; Said bud inducing culture is the MS substratum; Component that comprises in the bud inducing culture and the respective components concentration in the bud inducing culture is respectively: massfraction is that 3% sucrose, massfraction are 0.8% agar, the IAA of 1mg/L, the ZT of 2mg/L, the GA of 0.2mg/L and the BA of 0.5mg/L, and regulating the pH value is 5.8;

Further; Said induce to sprout be operating as: the yam explant that will pass through common cultivation forwards resistance to and selects in the substratum; And cultivation produces and forms new plant until resistant buds under the condition of 20-25 ℃ of illumination 16h, 18-20 ℃ dark 8h and 1000-2000lx light intensity; It is the MS substratum that said resistance is selected substratum; Component and the concentration of respective components in selecting the resistance substratum that resistance is selected to comprise in the substratum are respectively: massfraction is that 3% sucrose, massfraction are 0.8% agar, the IAA of 1mg/L, the ZT of 2mg/L, the GA of 0.2mg/L, the 6-BA of 0.5mg/L, the CarB of 500mg/L and the Kan of 65-80mg/L, and regulating the pH value is 5.8;

Said selectivity root culture is operating as: will be through inducing the new plant segment that forms after sprouting; Gained stem section being placed selects root media under the condition of 20-25 ℃ of illumination 16h, 18-20 ℃ dark 8h and 1000-2000lx light intensity, to cultivate and the screening of taking root again; Said selection root media is the MS substratum; Component and the concentration of respective components in selecting root media selecting to comprise in the root media are respectively: massfraction is that 3% sucrose, massfraction are 0.8% agar, the CarB of 200mg/L and the Kan of 50mg/L, and regulating the pH value is 5.8;

Further, said PCR of step (4) and RT-PCR detection method comprise the steps:

I is according to overexpression binary vector pBIN19-Tm-2 ²NPT II reporter gene sequences Design Auele Specific Primer (F1, R1), its sequence is following:

F1：5’-CTATGACTGGGCACAACAGACAAT-3’

R1：5’-CAATATCACGGGTAGCCAACG-3’；

II is with Tm-2 ²Compare with non-transgenic potato gene group, search Tm-2 ²In contain with the lower segment area of non-transgenic potato gene group homology, design primer in view of the above, its sequence is following:

To-F：5′-CGCGCGTGTTATAGAGATTATGGAC-3′

To-R：5′-ATAACCATTTGCCTCGCCCG-3′

III PCR detects

Extract the genomic dna of gained transgenic potato plant and non-transgenic potato plant respectively; Be template with the DNA that extracts then; Carry out pcr amplification with said Auele Specific Primer (F1, R1) (To-F, To-R); After obtaining object tape, connect pMD18-T carrier and order-checking respectively, screening positive plant;

IV RT-PCR detects

Respectively the geneome RNA of extraction step III gained positive plant and wild-type potato plant carries out the RT-PCR amplification with said Auele Specific Primer (To-F, To-R), obtain object tape after, connect the pMD18-T carrier and also check order screening positive plant;

Further; The said Auele Specific Primer of step (1) is two pairs; Be respectively: (TF2, TR2) and (T22F, T22R), 5 ' end of T22F primer adds restriction enzyme digestion sites (the underscore position is the digestion with restriction enzyme site), its sequence separately is following:

TF2：5’-GCATGTTAAGGGCAAGAGAA-3’

TR2：5’-GCGACAACTTGAAATTCTTCC-3’

T22F：5’-GGATCCTTGGCCGTGGACTCCATCT-3’

T22R：5’-GTCGACCACTACTACACTCACGTTGC-3’。

The Chinese of each English name is explained as follows among the present invention: 6-BA is that 6-benzyl aminopurine, Kan are that kantlex, LSm are that Streptomycin sulphate, Rif are that Rifampin, IAA are that indolylacetic acid, ZT are that zein, CarB are that Pyocianil, CaMV 35S are the cauliflower mosaic virus 35S promoter.

The invention has the beneficial effects as follows:

The method of utilizing genetic engineering technique to obtain high antiviral potato plant of the present invention has following advantage:

1, according to known tomato disease-resistant gene Tm-2 ²Disease-resistant mechanism and function thereof, and Tm-2 ²Transform the plant of Solanaceae tobacco and to the report of the high anti-characteristic of virus (like TMV, ToMV etc.) tool of infringement plant of Solanaceae, utilize tomato and the nearer characteristics of yam sibship, the employing genetic engineering technique is with Tm-2 ²Gene changes yam over to, and screens, identifies and expressed Tm-2 ²The high antiviral potato plant of gene is analyzed its resistance to harm yam common virus, obtains having the potato plant than the strong virus resistance, and antiviral spectrum is wide simultaneously.

2, the Tm-2 on the very near tomato karyomit(e) of utilization of the present invention and yam sibship ²The gene transformation yam has got rid of due to illness virus gene RNA reorganization or sudden change etc. to environment and the edible risk of bringing, and makes for the disease-resistant research of yam and trying to explore, and helping provides reference for external source disease-resistant gene successful expression on potato plant.

3, the inventive method has improved the transformation efficiency that obtains the test tube potato.

Description of drawings

Fig. 1 and Fig. 2 are Tm-2 ²Clone's schema of gene;

Fig. 3 and Fig. 4 are the total RNA of tomato gained Tm-2 after the RT-PCR amplification ²The agarose gel electrophoresis figure of two sections sequence amplification products before and after the gene;

Fig. 5 is the structure schema of overexpression binary vector;

Fig. 6 is pBIN19-Tm-2 ²Plasmid PCR detected result figure;

Fig. 7 cuts pBIN19-Tm-2 for enzyme ²Carrier products therefrom PCR detected result figure;

Fig. 8 farming bar engineering bacteria is contaminated yam explant operational flowchart;

Fig. 9 and Figure 10 are the PCR product agarose gel electrophoresis figure of No. 3 transfer-gen plants of Hubei Province yam and wild-type potato plant genomic dna, and 1 is the contrast of wild-type yam strain system among Fig. 9, and 2 is No. 3 transgenic lines of Hubei Province yam; 1-4 is No. 3 transgenic lines of Hubei Province yam among Figure 10, and 5-6 is the contrast of wild-type yam strain system);

Figure 11 is the RT-PCR product agarose gel electrophoresis figure that detects the geneome RNA of gained No. 3 transgenic positive plant of Hubei Province yam and wild-type potato plant through PCR; Wherein, 1-6 is No. 3 transgenic positive strains of a Hubei Province yam system, and 7-12 is the contrast of wild-type yam strain system.

Embodiment

Below will combine accompanying drawing that the preferred embodiments of the present invention are carried out detailed description.Should be appreciated that preferred embodiment has been merely explanation the present invention, rather than in order to limit protection scope of the present invention.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example; J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press; 2002) described in condition, or the condition of advising according to manufacturer.

Supply the examination material: tomato KBV strain spire, No. 3 detoxic seedlings of Hubei Province yam (virus-free seed potato breeding center, Chongqing City provides).

Plasmid: -T Easy Vector (Promega); PDH51 (carrying CaMV35S promotor and terminator), pBIN19 etc. are preserved by this laboratory.

Bacterial strain: e. coli jm109 is preserved by this laboratory

Reagent: TaKaRa RNA PCR Kit (AMV) Ver.3.0 test kit, 5-Full RACE CoreSet test kit, restriction enzyme, Taq enzyme etc. are available from Dalian TaKaRa company; It is the TIANGEN Company products that ultra-thin/plain agar sugar gel DNA reclaims test kit (centrifugal column type); X-gal and IPTG match hundred victory companies available from Beijing; All the other reagent are import or homemade AR.

1, Tm-2 ²The clone of gene, clone's flow process such as Fig. 1 and Fig. 2;

1.1 design of primers

According to the antiviral resistant gene Tm-2 of the tomato of having reported ²(AF536201) two pairs of Auele Specific Primers of sequences Design are respectively: (TF2, TR2) and (T22F, T22R), the T22F primer 5 ' end add BamHI restriction enzyme site (underscore position), the sequence of three pairs of Auele Specific Primers is following:

①TF2：5’-GCATGTTAAGGGCAAGAGAA-3’

②TR2：5’-GCGACAACTTGAAATTCTTCC-3’

③T22F：5’-GGATCCTTGGCCGTGGACTCCATCT-3’

④T22R：5’-GTCGACCACTACTACACTCACGTTGC-3’

1.2RT-PCR

1. be template with the total RNA of KBV strain tomato spire, carry out the synthetic of the first chain cDNA according to TaKaRa RNA PCR Kit (AMV) Ver.3.0 operation instructions;

2. be template with cDNA first chain, carry out pcr amplification with Auele Specific Primer (T22F, TR2) and (TF2, T22R) respectively, the PCR product adopts agarose gel electrophoresis to identify, getting big or small is the amplified fragments of 1321bp and 1502bp, respectively like Fig. 3 and Fig. 4.

Reaction system such as table 1:

Table 1

ddH 2O	16.5μl
		10×PCR?Buffer	2.5μl
MgCl 2	2.5μl
		dNTP(20μM)	1.0μl
F	0.5μl
		R	0.5μl
The cDNA template	1.0μl
		Taq?DNA?polymerase	0.5μl
Total	25.0μl

Pcr amplification loop parameter to (T22F, TR2) and (TF2, T22R) is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 2min, totally 35 circulations; 72 extend 3min eventually, finish pcr amplification.

3. the recovery of PCR product and purifying: adopt ultra-thin/plain agar sugar gel DNA to reclaim the operation of test kit (centrifugal column type) (TIANGEN Company products) by specification.

4. the conversion of recombinant plasmid and evaluation; Operation as follows; With step 3. the gained purified product be connected with

-T Easy Vector; Carry out transformation experiment with escherichia coli jm109 competent cell; Blue hickie screening positive clone is used M13 primer (the prompt base in the English Weihe River (Shanghai) trade Co., Ltd product) to carry out single bacterium colony PCR then and is detected, and positive recombinant is identified by the order-checking of the prompt base in the English Weihe River (Shanghai) trade Co., Ltd.

1.3 total length Tm-2 ²The splicing of gene fragment

The amplified fragments that sequence verification is correct is respectively 1321bp and 1502bp, and wherein the cDNA sequence of 1321bp adds its 5 ' end through RACE and then two amplified fragments is connected into the PstI restriction enzyme site

-T Easy Vector carrier is designated as pMD18-Tm-2 ², carry out transformation experiment with escherichia coli jm109 competent cell, blue hickie screening positive clone.

2, the structure of overexpression binary vector

2.1 concrete operations

The BamHI enzyme is cut pMD18-Tm-2 ²Plasmid and the empty carrier pDH51 that contains CaMV35S promotor and terminator are with gained total length Tm-2 ²Fragment imports among the pDH51 and obtains transition plasmid vector pDH51-Tm-2 ², and then with EcoRI digested plasmid carrier pDH51-Tm-2 ²(the gained fragment comprises CaMV35S promotor, Tm-2 ²Fragment and CaMV35S terminator) and be cloned into expression vector pBIN19, obtain overexpression binary vector pBIN19-Tm-2 ², its flow process such as Fig. 5.

2.2 result's checking

The EcoRI enzyme is cut pBIN19-Tm-2 ²The carrier result occur about 11000bp with 3400bp about 2 electrophoresis bands, like Fig. 7; With the M13 universal primer with pBIN19-Tm-2 ²Plasmid is that template is carried out pcr amplification, and the electrophoresis band about 3400bp appears in electrophoresis result, like Fig. 6, proves that the result is consistent with theory.

3, Agrobacterium infects conversion

The transfer of this step inserts the potato haulm section of inducing culture and directly carries out dark processing without illumination cultivation; Make in the short period of time and can induce the test tube potato; Maximum because of the stem tuber wound area, so infection ability is strong, and stem tuber is easy to operate; Can directly induce into seedling without the callus process, on the suitable culture base, make the test tube potato obtain the transformation tissue culture bud of upper frequency.Operating process such as Fig. 8, operation steps is following:

3.1 yam explant preparation

1. the shoot proliferation of potato virus-free plantlet

Substratum adopts the MS substratum; Component that comprises in the substratum and the respective components massfraction in substratum is respectively: sucrose 3% and agar 0.6%; Adjustment pH is 5.8 before the medium sterilization, after the sterilization substratum is sub-packed in the group training vial by 20-25ml; Potato detoxicating cuvette seedling single-unit section with said two kinds is inoculated on the substratum respectively; Place the light temperature incubator of 20 ± 2 ℃ of temperature, 16h/d illumination condition and 1000lx intensity of illumination to cultivate; Subculture once around the basis seedling was every; In this step, the massfraction of agar is that 0.6-0.8%, intensity of illumination can all can for 1000-2000lx in the MS substratum.

2. the test tube potato induces

When treating that detoxification test tube plantlet grows to about 6-7 blade; Robust growth, the consistent potato detoxicating cuvette seedling in source are cut into the stem section of 1 leaf, 1 joint; Under the aseptic condition stem section of each kind is seeded to respectively and is equipped with in the petridish that 25ml induces solid medium, every petridish 10-15 stem section places postvaccinal petridish under 20 ± 2 ℃ of full dark conditions and directly to induce the knot potato; Get the test tube potato; The said solid medium of inducing is the MS substratum, induces the component and the concentration of respective components in inducing solid medium that comprise in the solid medium to be respectively: massfraction is that 10% sucrose, massfraction are 0.6% agar, and regulating pH is 5.8; This step is described induces in the solid medium; The sucrose massfraction all can at 8-10%; Also can add 6-BA simultaneously, the concentration 2mg/L of 6-BA in inducing solid medium gets final product, and table 2 adds test tube potato inductivity difference behind 6-BA and the KT separately for inducing solid medium;

Table 2 is respectively to adding the difference of test tube potato inductivity behind hormone 6-BA and the KT in the substratum

3. treat that said two kinds test tube potato that induces after two weeks of growing is cut into the thin slice about 0.5cm, is required yam explant.

3.2 the conversion of agriculture bacillus mediated test tube potato

3.2.1 contain pBIN19-Tm-2 ²The preparation of the LBA4404 Agrobacterium bacterium liquid of binary vector

1. will contain pBIN19-Tm-2 ²The LBA4404 Agrobacterium of binary vector be inoculated in the YEB solid medium and under 28 ± 2 ℃ of conditions dark culturing 2d, culture, the said dark culturing of this step all can between 2-3d;

2. picking list bacterium colony is after the 20mlYEB liquid nutrient medium is cultivated 2d, according to 0.01: 1 volume ratio the gained culture joined in the YEB liquid nutrient medium and spends the night enlarged culturing to OD ₆₀₀Be 1.8-2.0, room temperature is centrifugal and use the resuspended thalline of fresh YEB liquid nutrient medium once more under the 6000rpm condition then, bacteria suspension, in this step, centrifugal rotational speed is that 4000-6000rpm all can;

3. the gained bacteria suspension with the resuspended thalline of MS salts solution, must contain pBIN19-Tm-2 in the centrifugal back of 4000rpm room temperature ²The LBA4404 Agrobacterium bacterium liquid of binary vector, in this step, centrifugal rotational speed is that 4000-6000rpm all can;

Component that said YEB solid medium comprises and the concentration of respective components in the YEB solid medium are respectively: massfraction is 1.5% agar, the Kan of 50mg/L, the Sm of 500mg/L and the Rif of 50mg/L, and regulating pH is 7.0;

Component that said YEB liquid nutrient medium comprises and the concentration of respective components in the YEB liquid nutrient medium are respectively: the Rif of the Kan of 50mg/L, the Sm of 500mg/L and 50mg/L, and regulating the pH value is 7.0;

The pH of said MS salts solution is 5.8.

Because of the concentration of Agrobacterium at first influences its thalline adhering on vegetable cell; The big more adhesive rate of bacterial concentration is high more within the specific limits; Transformation efficiency is big more; Can be but surpassed after the certain limit because the toxic action of bacterium or grow too fastly etc. makes explant receive excessively injury and to cause brownization of explant otch to be rotted serious of Agrobacterium, and is unfavorable for common cultivations back removal Agrobacterium; Lower bacterial concentration causes T-DNA can not be integrated into the vegetable cell genome effectively, reduces changing effect greatly; The Agrobacterium bacterial concentration of gained of the present invention is suitable, can effectively guarantee to contain Tm-2 ²The transformation efficiency of gene plasmid.

3.2.2 the dip-dye of yam explant

The yam explant of gained two each and every one kinds is dipped in contains pBIN19-Tm-2 ²10min in the LBA4404 Agrobacterium bacterium liquid of binary vector; For immerged time, if too short, fully on the adherent cell, influence transforms Agrobacterium; Time is oversize, and the wound of explant is understood brownization and Agrobacterium is seriously polluted and be difficult to control in subsequent experimental, and in this step, soak time all can fully ensure the dip-dye effect between 8-10min.

3.2.3 cultivate altogether

Yam explant after contaminating is placed the bud inducing culture and cultivates 2d altogether in 20 ± 2 ℃ of following dark; Said bud inducing culture is the MS substratum; Component that comprises in the bud inducing culture and the respective components concentration in the bud inducing culture is respectively: massfraction is that 3% sucrose, massfraction are 0.8% agar, the IAA of 1mg/L, the ZT of 2mg/L, the GA of 0.2mg/L and the BA of 0.5mg/L, and regulating the pH value is 5.8; Cultivate altogether is the important factor that influence transforms; Overlong time can make explant pollute dead because of Agrobacterium; Too short meeting of time causes agroinfection and DNA to transform insufficient; Thereby the reduction transformation efficiency also can increase the appearance of false transformant simultaneously, and the suitable time of cultivating altogether among the present invention is 2-3d.

Sprout 3.2.4 induce

Yam explant after will cultivating altogether respectively forwards resistance to and selects in the substratum; And under the condition of 25 ± 2 ℃ of illumination 16h, 18 ± 2 ℃ of dark 8h and 1000lx light intensity, be cultured to resistant buds and produce and form new plant; It is the MS substratum that said resistance is selected substratum; Component and the concentration of respective components in selecting the resistance substratum that resistance is selected to comprise in the substratum are respectively: massfraction is that 3% sucrose, massfraction are 0.8% agar, the IAA of 1mg/L, the ZT of 2mg/L, the GA of 0.2mg/L, the 6-BA of 0.5mg/L, the CarB of 500mg/L and the Kan of 75mg/L, and regulating the pH value is 5.8; In this step, 20-25 ℃ of illumination 16h, 18-20 ℃ dark 8h and 1000-2000lx light intensity are the suitable culture condition of gained yam explant of the present invention, and the false positive plant generally can occur not growing or poor growth phenomenon, only positive plant normal growth.

Hormone is one of principal element that influences regeneration frequency; The combination of different hormone level and hormone is different with the influence of sprouting to inducing the different explants callus, because potato set is very sensitive to kantlex (Kan), the low pressure of selecting of the concentration of Kan is not enough; Be prone to produce the non-transgenic plant; Otherwise then be unfavorable for the bud differentiation, it is 75mg/L that the resistance of present embodiment is selected the concentration of Kan in the substratum, and the safe level of Kan in resistance selection substratum is between the 65-80mg/L among the present invention.

3.2.5 selectivity root culture

Will be through inducing the new plant segment that forms after sprouting; Again gained stem section is placed and select the root media cultivation and screening of taking root under the condition of 25 ± 2 ℃ of illumination 16h, 18 ± 2 ℃ of dark 8h and 2000lx light intensity; Get transgenic potato plant; Said selection root media is the MS substratum; Component and the concentration of respective components in selecting root media selecting to comprise in the root media are respectively: massfraction is that 3% sucrose, massfraction are 0.8% agar, the CarB of 200mg/L and the Kan of 50mg/L; Regulating the pH value is 5.8, adopt selection root media according to the invention to carry out the selectivity root culture after, its rooting rate can reach more than 60%.

Transform in the yam at agriculture bacillus mediated foreign gene; Except that above factor; Transformation efficiency also is subject to the influence of yam kind; Secondly this enforcement experimental data shows that the inventive method is preferably applied in the conversion of No. 3 kinds of Hubei Province yam, also applicable to the gene transformation of (like No. 27, river potato) of the toxicity-removing white potato of other kind.

4, the detection of gained transfer-gen plant

4.1 design primer

A is according to present embodiment gained overexpression binary vector pBIN19-Tm-2 ²Last NPT II gene order designs a pair of Auele Specific Primer (F1, R1), and its sequence is following:

F1：5’-CTATGACTGGGCACAACAGACAAT-3’

R1：5’-CA?ATATCACGGGTAGCCAACG-3’

B carries out the gene order contrast, according to Tm-2 ²In contain with the lower zone of non-transgenic potato gene group homology, the design primer:

To-F：5′-CGCGCGTGTTATAGAGATTATGGAC-3′

To-R：5′-ATAACCATTTGCCTCGCCCG-3′

4.2PCR detect

Get step 3.2.5 gained transgenic potato plant and expand numerous transplanting; Get the genomic dna of system of homophyletic not and wild-type potato plant respectively; Be template with the DNA that extracts then, carry out the pcr amplification detection, screen and obtain positive plant with Auele Specific Primer (F1, R1) and (To-F, the To-R) of above-mentioned design; The reaction system such as the table 1 of pcr amplification, the loop parameter of amplification is:

Pcr amplification loop parameter to (F1, R1) is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 40s, totally 35 circulations; 72 extend 10min eventually, finish pcr amplification.

Pcr amplification loop parameter to (To-F, To-R) is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ are extended 40s, totally 35 circulations; 72 extend 10min eventually, finish pcr amplification.

Fig. 9 and Figure 10 are the PCR product agarose gel electrophoresis figure of No. 3 transfer-gen plants of Hubei Province yam and wild-type potato plant genomic dna, and the gained target stripe connects the pMD18-T carrier, and be correct through order-checking proof conversion results.(1 is the contrast of wild-type yam strain system among Fig. 9, and 2 is No. 3 transgenic lines of Hubei Province yam; 1-4 is No. 3 transgenic lines of Hubei Province yam among Figure 10, and 5-6 is the contrast of wild-type yam strain system)

4.3RT-PCR detect Tm-2 ²Expression in potato plant

No. 3 transgenic positive plant of Hubei Province yam that screening obtains in the extraction step 4.2 respectively and the geneome RNA of wild-type potato plant, (To-F, To-R) carries out the RT-PCR augmentation detection with Auele Specific Primer, further screening positive plant.Amplification system and parameter to Auele Specific Primer (To-F, To-R) are identical with step 4.2; Figure 11 is the RT-PCR product agarose gel electrophoresis figure that detects the geneome RNA of gained No. 3 transgenic positive plant of Hubei Province yam and wild-type potato plant through PCR; After obtaining object tape; Connect pMD18-T carrier and order-checking with connecting liquid respectively, prove that the result is correct.(among Figure 11,1-6 is No. 3 transgenic positive strains of a Hubei Province yam system, and 7-12 is the contrast of wild-type yam strain system)

4.4 virus infection

4.4.1 vegetable material, malicious source

Vegetable material: No. 3 transfer-gen plants of gained Hubei Province yam, wild-type yam

The poison source: TOMV (ToMV) and tobacco mosaic virus(TMV) (TMV) are provided by Zhejiang University; Potato virus X (PVX) and marmor upsilon (PVY) are provided by Hua Zhong Agriculture University yam laboratory and Hubei Province's yam Engineering Technical Research Centre.

4.4.2 operation steps

1. the preparation of virus inoculation: the malicious source of TOMV (ToMV) tobacco mosaic virus(TMV) (TMV) is seeded on the Nicotiana glutinosa earlier; Collect morbidity Nicotiana glutinosa blade after 20 days; According to the sick leaf of 1g add 20ml phosphoric acid buffer (0.01mol/L, pH7.0) back homogenate in mortar is filtered with double gauze then; Collect filtrate for later use, potato virus X (PVX) and marmor upsilon (PVY) through test tube continue be commissioned to train foster;

2. inoculation method: the blade that rubs gently back and forth, use the distilled water flushing blade face then;

4.4.3 experimental result

Observe through virus infection, get rid of the external environment factor affecting, the transgenic line virus resistance result such as the following table that obtain.

Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although with reference to preferred embodiment the present invention is specified, those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement technical scheme of the present invention; And not breaking away from the aim and the scope of technical scheme of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.

Sequence table

< 110>University Of Chongqing

< 120>utilize genetic engineering technique to obtain the method for high antiviral potato plant

<160>8

<210>1

<211>20

<212>DNA

< 213>artificial sequence

<220>

<223>TF2

<400>1

gcatgttaag?ggcaagagaa 20

<210>2

<211>21

<212>DNA

< 213>artificial sequence

<220>

<223>TR2

<400>2

gcgacaactt?gaaattcttc?c 21

<210>3

<211>25

<212>DNA

< 213>artificial sequence

<220>

<223>T22F

<400>3

ggatccttgg?ccgtggactc?catct 25

<210>4

<211>26

<212>DNA

< 213>artificial sequence

<220>

<223>T22R

<400>4

gtcgaccact?actacactca?cgttgc 26

<210>5

<211>24

<212>DNA

< 213>artificial sequence

<220>

<223>F1

<400>5

ctatgactgg?gcacaacaga?caat 24

<210>6

<211>21

<212>DNA

< 213>artificial sequence

<220>

<223>R1

<400>6

caatatcacg?ggtagccaac?g 21

<210>7

<211>25

<212>DNA

< 213>artificial sequence

<220>

<223>To-F

<400>7

cgcgcgtgtt?atagagatta?tggac 25

<210>8

<211>20

<212>DNA

< 213>artificial sequence

<220>

<223>To-R

<400>8

ataaccattt?gcctcgcccg 20

Claims (3)

1. a method of utilizing genetic engineering technique to obtain high antiviral potato plant is characterized in that: comprise the steps:

(1) produces tomato disease-resistant gene Tm-2 ²

According to tomato disease-resistant gene Tm-2 ²Two pairs of Auele Specific Primers of sequences Design and add restriction enzyme digestion sites at upstream primer 5 ' end, extract tomato RNA then and carry out the RT-PCR amplification with two pairs of Auele Specific Primers respectively, obtain Tm-2 ²The amplified production of gene leading portion and back segment; With gained Tm-2 ²The amplified production of gene leading portion and back segment is cloned into two pMD18-T carriers respectively, carries out enzyme again and cuts, and enzyme is cut the purified back of product and connected, and gets Tm-2 ²Full length gene cDNA sequence;

(2) structure of overexpression vector

Extract total length Tm-2 ²Fragment also is cloned in the pDH51 carrier that contains CaMV35S promotor and terminator, gets pDH51-Tm-2 ²Plasmid, enzyme is cut pDH51-Tm-2 ²Plasmid and with the gained fragment cloning to the pBIN19 carrier, overexpression binary vector pBIN19-Tm-2 ²

(3) Agrobacterium infects conversion

The yam explant that will pass through preparatory cultivation acquisition places and contains pBIN19-Tm-2 ²Dip-dye and warp are cultivated altogether, are induced and sprout and the selectivity root culture in the bacterium liquid of the LBA4404 Agrobacterium of binary vector, get transgenic potato plant;

Said yam explant is obtained by following steps:

A. potato detoxicating cuvette seedling single-unit section is inoculated on the substratum; Under the condition of 20 ± 2 ℃ of temperature, 16h/d photoperiod and 1000-2000lx intensity of illumination, cultivate then; Said substratum is the MS substratum; Component that comprises in the substratum and the respective components massfraction in substratum is respectively: sucrose 3%, agar 0.6-0.8%, and regulating pH is 5.8; Said potato detoxicating cuvette seedling is the detoxification test tube plantlet of No. 27, Hubei Province yam No. 3 or river potato;

B. treat that detoxification test tube plantlet grows to behind 6-7 the blade stem section that the potato detoxicating cuvette seedling of robust growth is cut into 1 leaf, 1 joint, with said stem section be seeded to induce in the solid medium and under 20 ± 2 ℃ full dark condition inducing culture to obtain the test tube potato; The said solid medium of inducing is the MS substratum; Induce the component and the concentration of respective components in inducing solid medium that comprise in the solid medium to be respectively: massfraction is the sucrose of 8-10%; Massfraction is 0.6% agar, the 6-BA of 2mg/L, and regulating pH is 5.8;

C. said test tube potato section gets the yam explant;

The said pBIN19-Tm-2 that contains ²The bacterium liquid of the LBA4404 Agrobacterium of binary vector makes as follows:

1. will contain pBIN19-Tm-2 ²The LBA4404 Agrobacterium of binary vector be inoculated in the YEB solid medium and under 28 ± 2 ℃ of conditions dark culturing 2-3d, culture; Component that said YEB solid medium comprises and the concentration of respective components in the YEB solid medium are respectively: massfraction is 1.5% agar, the Kan of 50mg/L, the Sm of 500mg/L and the Rif of 50mg/L, and regulating pH is 7.0;

2. join the gained culture in the YEB liquid nutrient medium according to 0.01: 1 volume ratio and be cultured to OD ₆₀₀For behind the 1.8-2.0 under the 4000-6000rpm condition room temperature centrifugal and with the resuspended thalline of fresh YEB liquid nutrient medium, bacteria suspension; Component that said YEB liquid nutrient medium comprises and the concentration of respective components in the YEB liquid nutrient medium are respectively: the Rif of the Kan of 50mg/L, the Sm of 500mg/L and 50mg/L, and regulating the pH value is 7.0;

3. the gained bacteria suspension with the resuspended thalline of MS salts solution, must contain pBIN19-Tm-2 in the centrifugal back of 4000-6000rpm room temperature ²The bacterium liquid of the LBA4404 Agrobacterium of binary vector; The pH of said MS salts solution is 5.8;

The said pBIN19-Tm-2 that contains ²The LBA4404 Agrobacterium bacterium liquid immerged time of binary vector is 8-10min; The said cultivation altogether is with in the bud inducing culture, altogether cultivating 2-3d 20 ± 2 ℃ of following dark through the yam explant of LBA4404 Agrobacterium bacterium liquid dip-dye; Said bud inducing culture is the MS substratum; Component that comprises in the bud inducing culture and the respective components concentration in the bud inducing culture is respectively: massfraction is that 3% sucrose, massfraction are 0.8% agar, the IAA of 1mg/L, the ZT of 2mg/L, the GA of 0.2mg/L and the BA of 0.5mg/L, and regulating the pH value is 5.8;

Said induce to sprout be operating as: the yam explant that will pass through common cultivation forwards resistance to and selects in the substratum; And cultivation produces and forms new plant until resistant buds under the condition of 20-25 ℃ of illumination 16h, 18-20 ℃ dark 8h and 1000-2000lx light intensity; It is the MS substratum that said resistance is selected substratum; Component and the concentration of respective components in selecting the resistance substratum that resistance is selected to comprise in the substratum are respectively: massfraction is that 3% sucrose, massfraction are 0.8% agar, the IAA of 1mg/L, the ZT of 2mg/L, the GA of 0.2mg/L, the 6-BA of 0.5mg/L, the CarB of 500mg/L and the Kan of 65-80mg/L, and regulating the pH value is 5.8;

Said selectivity root culture is operating as: will be through inducing the new plant segment that forms after sprouting; Gained stem section being placed selects root media under the condition of 20-25 ℃ of illumination 16h, 18-20 ℃ dark 8h and 1000-2000lx light intensity, to cultivate and the screening of taking root again; Said selection root media is the MS substratum; Component and the concentration of respective components in selecting root media selecting to comprise in the root media are respectively: massfraction is that 3% sucrose, massfraction are 0.8% agar, the CarB of 200mg/L and the Kan of 50mg/L, and regulating the pH value is 5.8;

(4) detection of transfer-gen plant

Adopt PCR, RT-PCR detection method screening positive plant from the gained transgenic potato plant, the gained positive plant is identified through the inoculation of live body disease resistance, is obtained high antiviral potato plant.

2. the method for utilizing genetic engineering technique to obtain high antiviral potato plant according to claim 1, it is characterized in that: said PCR of step (4) and RT-PCR detection method comprise the steps:

I is according to overexpression binary vector pBIN19-Tm-2 ²NPT II reporter gene sequences Design Auele Specific Primer F1, R1, its sequence is following:

F1：5’-CTATGACTGGGCACAACAGACAAT-3’

R1：5’-CAATATCACGGGTAGCCAACG-3’；

II is with Tm-2 ²Compare with non-transgenic potato gene group, search Tm-2 ²In contain with the lower segment area of non-transgenic potato gene group homology, design primer in view of the above, its sequence is following:

To-F：5′-CGCGCGTGTTATAGAGATTATGGAC-3′

To-R：5′-ATAACCATTTGCCTCGCCCG-3′

III PCR detects

Extract the genomic dna of gained transgenic potato plant and non-transgenic potato plant respectively; Be template with the DNA that extracts then; Carry out pcr amplification with said Auele Specific Primer (F1, R1) and (To-F, To-R); After obtaining object tape, connect pMD18-T carrier and order-checking respectively, screening positive plant;

IV RT-PCR detects

Respectively the geneome RNA of extraction step III gained positive plant and non-transgenic potato plant carries out the RT-PCR amplification with said Auele Specific Primer To-F, To-R, obtain object tape after, connect the pMD18-T carrier and also check order screening positive plant.

3. the method for utilizing genetic engineering technique to obtain high antiviral potato plant according to claim 1, it is characterized in that: the said Auele Specific Primer of step (1) is two pairs, is respectively: T22F, TR2 and TF2, T22R, its sequence separately is following:

TF2：5’-GCATGTTAAGGGCAAGAGAA-3’

TR2：5’-GCGACAACTTGAAATTCTTCC-3’

T22F：5’-GGATCCTTGGCCGTGGACTCCATCT-3’

T22R：5’-GTCGACCACTACTACACTCACGTTGC-3’。

Images