CN101117639A - Method for acquiring disease-resistance expression of agrobacterium-mediated potato transgenic hrap - Google Patents

Abstract

The present invention discloses a method for attaining potato plates which are disease resistant by utilizing agrobacterium-mediated hrap gene. After the combined inoculation of the method and the bacteria allergic protein harpinPss, the allergic reaction caused by harpinPss can be promoted in the intercellular space of all inoculated plates, and the polymeride of harpinPss is dissociated, so the disease resisting effect is better, and the disease resistance of the potato plates can be promoted. The hrap gene is introduced in the potato plates through the agrobacterium-mediated, thus resulting that the potato plates have the ability to resist the late blight and the bacterial wilt. The present invention has the technical proposal that the pre-cultured explants are inoculated in the agrobacterium germ liquid to be soaked, explants after being soaked select the tube seedling which can root after two times of root selections by co-culturing, callus of induce, bud inducting and rootage cultivating and selecting, and the potted transformed plants are transferred after the mass propagation, the transformed plants select positive plants by the methods of the PCR detection and the Southern hybridization detection, and then the potato plates which are disease resistant can be obtained by the in-vivo inoculation resistance identification. The present invention improves the callus of inducing and the bud inducing of the explants, and enhances the agrobacterium-mediated ability. That the roots of the explants are selected by two times can eliminate the false positive plates in the transformation process , avoid the continuing cultivation to the false positive plates, save the labour force and decrease the manufacturing cost.

Description

Agrobacterium-mediated potato changes the hrap gene and obtains the method that disease resistance is expressed

Technical field

The present invention relates to utilize the agriculture bacillus mediated hrap gene of in potato, introducing to obtain the method that disease resistance is expressed.

Background technology

Potato is the world the 4th big important food crop, also be dish simultaneously, raise, processing and insutrial crop.The country of the potato of whole world plantation now has 148,300,000,000 tons of ultimate productions.International Potato Center (CIP) studies show that, worldwide the demand to potato will be expected to increase by 20% to the year two thousand twenty, surpasses the growth of paddy rice, wheat, corn; Developing country will be twice in 2000 to the demand of potato when the time comes.

China has the advantageous advantage of producing potato aspect geographic climate.About 4,700,000 hm of the long-term cultivated area of China potato ², ultimate production is above 70,000,000 tons.Output and area account for 1/5th and 1/4th of the whole world respectively, rank first in the world.After China entry into the World Trade Organization, the competitiveness in the international market of paddy rice, wheat, corn and Soybean and Other Crops weakens day by day, and potato demonstrates powerful advantage and development potentiality with characteristics such as its output height, cost are low.Yet, should see, though China is one of potato production big country, the ratio that the potato that is used to process accounts for ultimate production very low (being less than 5%), and the U.S., France, Holland account for about 60%-80%, the potato after the processing is than bright potato increment 10-30 doubly.The potato kind that is used to process now mainly is foreign kind, but the resistance against diseases of these kinds is very poor, especially anti-late blight ability; And also there is same problem in the higher special early maturing variety of other commodity value.The late blight of potato is one of destructive disease of restriction potato production, and every year is lost about 17,000,000,000 dollars, about about 1,000,000,000 dollars of China because of this disease in the whole world.International Potato Center (CIP) classifies the late blight of potato as preferential research project, has set up the research coorporative network, has strengthened the research to this disease.At first, the breeding scholar seeks disease-resistant gene from the wild provenance of potato.Yet potato is the autotetraploid crop, and with conventional breeding method improvement kind, reasons such as, process complexity long owing to dysgenesia, breeding cycle are difficult to direct utilization.The vertical resistance gene of wild species is because the variation of cause of disease microspecies loses resistance soon and easily simultaneously; The horizontal resistance gene is not enough to resist effectively the invasion and attack of late blight separately, and is subject to the influence of envrionment conditions and causes being very popular of late blight.Quality reduced after potato caught an illness, and potato shape is poor, causes commodity potato rate low, does not satisfy the demand in processing and market far away, has influenced economic benefit and sweet potato grower's enthusiasm simultaneously, has restricted the performance of production and working ability.

It is the biotechnology of coming along with DNA recombinant technology, gene genetic transformation technology and plant tissue culture technique development over nearly 20 years that plant gene transforms.In recent years, along with further investigation to plant disease-resistant reaction mechanism and pathogenic bacteria pathogenesis, antimycotic and Micobial Disease genetically engineered has obtained some breakthroughs, found some and the plant defense proteins associated factor, become the basic substance of developing the plant disease-resistant new resources at present by the genetic engineering means.In the breeding of potato disease-resistant gene engineering, also used the protein gene of relevant with these courses of disease (PR) in a large number, as LIU etc., ZHU etc. and Li Rugang etc. have reported that respectively tobacco Osmotin protein transgene potato plant can postpone late blight scab time of occurrence, reduce the frequency that infects of late blight, give the ability of the anti-late blight of blade.You have just waited report Lee, and the constitutive expression of HarpinEa gene and inducible expression are all given the ability of the anti-late blight of transgenic potato plant.Class monellin gene (tlp) that people such as golden red will separate from African stevia rebaudianum and closely linked antiweed bar gene thereof import in the potato with agrobacterium-mediated transformation, through the stripped resistance analysis of the free spore inoculating of late disease bacteria, transgenic line shows to the obvious restraining effect of late blight and to symptom delayed action.Raise the uncommon commentaries on classics nontoxic gene potato plant that just waits human avrD and elicitin gene to transform respectively and all have the resistance that significantly late disease bacteria is infected.

WEI in 1992 etc. are separated to the protein exciton harpin by the hrapN genes encoding first from pears fire epidemic disease Ou Wenshi germ (Erwinia amylovora) Ea321 hrap gene cluster.Harpins is that the hrap gene family of plant pathogenetic bacteria itself is coded in that the host go up to be caused a disease and the essential protein factor of induced hypersensitivity reaction on non-host, and anaphylaxis to be plant and cause of disease do in the process mutually, general manifestation and effective way that plant opposing cause of disease is contaminated.The allergic protein harpin of hrap (hypersensitiveresponse-assisting protein) and bacterium _PssAfter the combined inoculation, all can in the intercellular substance of inoculation plant, promote harpin _PssThe anaphylaxis that is caused, harpin dissociates _PssPolymer, reduce the virulence of bacterial pathogen, cause more serious supersensitivity necrocytosis.This anaphylaxis promotes the albumen of proteic sequence and known function or gene order all dissimilar, and sequence alignment finds to contain one section guiding by hrap, and it is secreted into the outer signal peptide of born of the same parents.Hrap can delay bacterial spot germ Xanthomonas campestris pv.vesicatoria in pimento breeding and alleviate its illness, also can delay the wild Xanthomonas campestris Xanthomonas of tomato campestris pv.vesicatoria in pimento breeding and alleviate its illness.People such as Ger import the hrap gene in the tobacco, find that there is resistance in transfer-gen plant to wildfire bacterium (Pseudomonas syringae pv.Tabaci) and soft rot Erwinia (Erwinia carotovora subsp.carotovora); Ajay etc. import Arabidopis thaliana by agriculture bacillus mediated with the hrap gene, and transfer-gen plant has also shown the effect of anti-soft rot Erwinia (Erwinia carotovora subsp.carotovora).But also do not have at present the hrap gene in potato conversion and the report aspect the disease resistance expression.

Summary of the invention

Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, provide a kind of agrobacterium-mediated potato that utilizes to change the method that the hrap gene obtains the disease resistance expression, this method is gone into the hrap gene in the potato plant by agriculture bacillus mediated, makes potato plant have the ability of anti-late blight and bacterial wilt.

In order to solve the problems of the technologies described above, the technical solution used in the present invention is: be inoculated in the bacterium liquid of the Agrobacterium LBA4404 that carries plasmid pBI hrap through pre-incubated explant and contaminate, explant after the dip-dye is through being total to cultivation, callus induction, inducing and sprout and the root culture selection, shift potted plant transformed plant behind the test tube seedling and propagating of selecting root to select to take root for twice, transformed plant detects with PCR and Southern hybridization detection method screening positive plant, identifies through the inoculation of live body disease resistance to obtain disease-resistant potato plant.

The above-mentioned Agrobacterium bacterium liquid that utilizes agrobacterium-mediated potato commentaries on classics hrap gene to obtain to use in the method for disease resistance expression is preparation like this: the Agrobacterium LBA4404 bacterium liquid that will carry plasmid pBI hrap is inoculated in the liquid LB substratum that adds 100mg/L Kan, 100mg/LSm and 100mg/L Rif, on shaking table 28 ℃, middling speed is cultured to OD ₆₀₀Value is 0.4～0.5.

Aforementionedly utilize agrobacterium-mediated potato to change the hrap gene to obtain used Agrobacterium LBA4404 in the method that disease resistance expresses, carry plasmid pBI hrap, contain neomycin phosphotransferase gene NPT II and anaphylaxis and promote protein gene hrap, the hrap upstream region of gene is a 35S promoter, and the downstream is the NOS terminator.

The aforementioned agrobacterium-mediated potato that utilizes changes the method that the hrap gene obtains the disease resistance expression, and the pre-cultivation of described explant is to get the potato tissue cultured seedling blade of cultivating 3～4 weeks, robust growth to cut off the edge, is cut into 0.40.5cm ²Fritter, face down is inoculated in the substratum, 22～25 ℃ of dark cultivations 2 days, the consisting of of described substratum: MS+2.0mg/L 6BA+0.2mg/LNAA+0.2mg/L 2,4-D+16g/L sucrose+6g/L agar.

The aforementioned agrobacterium-mediated potato that utilizes changes the method that the hrap gene obtains the disease resistance expression, following method is adopted in the dip-dye of described explant: the explant after pre-the cultivation is inoculated in the Agrobacterium bacterium liquid for preparing, and makes explant fully contact 8～20 minutes with Agrobacterium.

The aforementioned agrobacterium-mediated potato that utilizes changes the method that the hrap gene obtains the disease resistance expression, contaminating the back explant is to cultivate so altogether: take out explant from Agrobacterium bacterium liquid, blot with aseptic filter paper, be inoculated in the substratum, 22～25 ℃ of dark cultivations 2～3 days, described substratum consists of: MS+2.0mg/L 6-BA+0.2mg/L NAA+0.2mg/L 2,4-D+16g/L sucrose+6g/L agar.

The aforementioned agrobacterium-mediated potato that utilizes changes the method that the hrap gene obtains the disease resistance expression, the callus induction of described explant, induce and sprout and root culture adopts following step: the explant after will cultivating altogether inserts and consists of MS+2～2.5mg/L6BA+0.1～0.5mg/L NAA+30g/L sucrose+50mg/L Kan+500mg/L Cb, additional 0.6% agar, callus induction in the substratum of pH5.8; The back switching of 2～3 week of callus growth goes into to consist of in the BOA substratum of MS+2.5mg/L 6-BA+5.0mg/L GA3+30g/L sucrose+6g/L agar+50mg/L Kan+500mg/L Cb, be to induce under the condition of 12000LX to sprout at 22 ± 1 ℃, illumination every day 12 hours, intensity of illumination, 3～4 weeks were changed a BOA substratum; After treating that bud grows to 2～3 centimetres, bud downcut being inserted in the resistance screening substratum of MS+50mg/L Kan, is 12000LX at 22 ± 1 ℃, intensity of illumination, and periodicity of illumination is to cultivate under hour dark condition in 12 hours illumination/12 to take root.

The aforementioned agrobacterium-mediated potato that utilizes changes the method that the hrap gene obtains the disease resistance expression, described PCR detects: extract DNA from the transformed plant blade, with the transformed plant is template, the positive contrast of positive Agrobacterium bacterium liquid, the negative contrast of unconverted blade, carry out pcr amplification with hrap gene primer I:5 '-GTTGGAGTTGGAGGACGAGG-3 ' and hrap gene primer II:5 '-CGCGGATCCATGAAAATGAAGAACCTCTC-3 ', the screening positive plant.

The aforementioned agrobacterium-mediated potato that utilizes changes the method that the hrap gene obtains the disease resistance expression, described Southern hybridization detection method is: described Southern hybridization detection method is: extract DNA from the blade of the transformed plant of PCR test positive, cut the DNA of extraction with the BglII enzyme, after 1% agarose electrophoresis, change film, the screening positive plant.

The aforementioned agrobacterium-mediated potato that utilizes changes the method that the hrap gene obtains the disease resistance expression, specifically adopts following steps:

(1) bacterium liquid preparation: will preserve Agrobacterium LBA4404 bacterium liquid and be inoculated in the liquid LB substratum that adds 100mg/L Kan, 100mg/L Sm and 100mg/L Rif, on shaking table 28 ℃, middling speed is cultured to OD ₆₀₀Value is 0.4, gets Agrobacterium bacterium liquid, and is standby;

(2) the pre-cultivation: get the potato tissue cultured seedling blade of cultivating 4 weeks, robust growth, face down is inoculated in and consists of MS+2.0mg/L 6BA+0.2mg/L NAA+0.2mg/L 2, in the substratum of 4-D+16g/L sucrose+6g/L agar, 22 ℃ of dark cultivations 2 days;

(3) contaminate: the tissue cultured seedling blade inoculation after pre-the cultivation makes the tissue cultured seedling blade fully contact 8 minutes with Agrobacterium bacterium liquid in the bacterium liquid of the Agrobacterium LBA4404 that carries plasmid pBI hrap of step (1) preparation;

(4) cultivate altogether: take out the tissue cultured seedling blade, blot, be inoculated in the substratum in the step (2), 22 ℃ of dark cultivations 2 days with aseptic filter paper;

(5) callus induction, induce and sprout and root culture: tissue cultured seedling blade after will cultivating altogether inserts callus induction in the substratum that consists of MS+2.5mg/L 6BA+0.2mg/L NAA+30g/L sucrose+50mg/L Kan+500mg/L Cb, additional 0.6% agar, pH5.8; The back switching of 3 week of callus growth goes into to consist of in the BOA substratum of MS+2.5mg/L 6-BA+5.0mg/LGA3+30g/L sucrose+6g/L agar+50mg/L Kan+500mg/L Cb, be to induce under the condition of 12000LX to sprout at 22 ℃, illumination every day 12 hours, intensity of illumination, 4 weeks were changed a BOA substratum; After treating that bud grows to 3 centimetres, bud is downcut in the resistance screening substratum that inserts MS+50mg/L Kan, at 22 ℃, intensity of illumination is to cultivate under hour dark condition in 12 hours illumination/12 to take root for the 12000LX periodicity of illumination, shift behind the test tube seedling and propagating of selecting root to select can both to take root for twice potted plant must transformed plant;

(6) PCR detects: adopt the SDS micromethod to extract DNA from the transformed plant blade, with the transformed plant is template, the positive contrast of positive Agrobacterium bacterium liquid, the negative contrast of unconverted blade, carry out pcr amplification with hrap gene primer I:5 '-GTTGGAGTTGGAGGACGAGG-3 ' and hrap gene primer II:5 '-CGCGGATCCATGAAAATGAAGAACCTCTC-3 ', the screening positive plant; The PCR reaction system is 10 Bo uffer 2.5mL, 10mM dNTPs 0.5mL, 2.5U/mL Taq enzyme 0.5mL, 10mM gene primer I 0.5mL, 10mM gene primer II 0.5mL and template DNA 2mL, mends to 25mL with deionized water; The PCR reaction conditions: 94 ℃, 7min; 94 ℃, 50s; 53 ℃, 50s; 72 ℃, 1min; 30 circulations; 72 ℃, 7min; 4 ℃ of termination reactions;

(7) Southern detects: adopt the CTAB method from extract DNA through the blade of the transformed plant of PCR test positive, cut the DNA of extraction with the BglII enzyme, after 1% agarose electrophoresis, change film, screen positive plant;

(8) disease resistance is identified: the Southern positive plant is identified through the live body inoculation and is obtained disease-resistant potato plant.

The present inventor has done a large amount of experiments, immerged time, different varieties, different hormone concentration have been studied to the taking root of the influence of callus induction and bud differentiation, indefinite bud, transplanting condition etc., and transgenic potato plant has been carried out anti-kan screening, PCR detection, shouthern hybridization and disease resistance has identified that institute's experiment of doing is as follows in detail:

One, the regeneration of transfer-gen plant

1, for the examination material

1.1 vegetable material

The test plant material is available from the potato kind of vegetable or flower institute of the Chinese Academy of Agricultural Sciences " boolean class gram " and " Atlantic Ocean " tissue cultured seedling, and Canadian kind " Russet Burbank " tissue cultured seedling be so kind as to give of Nanjing agricultural university, three kinds are the susceptible late blight all.The substratum that successive propagation uses is additional 0.6% agar of MS minimum medium and 3% sucrose, and pH5.8 chooses robust growth, and the blade of seedling age about 4 weeks is explant.

1.2 reagent

DNTP Mixture gives birth to worker biotech firm available from Shanghai; Taq archaeal dna polymerase and Marker D2000 are available from Beijing TIANGEN company; Kan (sulphuric acid kanamycin), Sm (Vetstrep) and Cb (Pyocianil) sprout Pu Rui biotech firm available from Beijing section; Rif (Rifampin) capsule is available from Xinyi, Shanghai everything medicine company limited-liability company; 6-BA is available from the beautiful pearl east wind in Shanghai biotech firm; GA ₃Available from Biochemical Research institute of the Chinese Academy of Sciences; NAA is by Shanghai tetrad chemical plant packing; 2,4-D is available from China Medicine (Group) Shanghai Chemical Reagent Co.; Agarose is available from Britain Oxoid; Other conventional reagent is all selected homemade analytical reagent for use.

1.3hrap gene

Goal gene hrap is separated from the pimento that is caused allergic reaction by harpinPss by Taiwan's scholars Feng Tengyong researcher's laboratory to obtain, and is purchased again by Peking University woods loyal flat laboratory and builds and provide.The Agrobacterium that makes up is carried plasmid pBIhrap, contains neomycin phosphotransferase gene (NPTII) and anaphylaxis and promotes protein gene hrap, and the hrap upstream region of gene is a 35S promoter, and the downstream is the NOS terminator, as shown in Figure 1.The proteic molecular weight of hrap is 29kD, iso-electric point 8.6, and hrap gene cDNA total length 996bp, this gene are after Peking University rebuilds, about gene cDNA total length 750bp.

1.4 bacterium liquid preparation

Will carry plasmid pBI hrap Agrobacterium LBA4404 bacterium liquid be inoculated in the liquid LB substratum of additional 100mg/L Kan, 100mg/L Sm and 100mg/L Rif, on shaking table 28 ℃, middling speed is cultured to OD ₆₀₀Value is 0.4, gets Agrobacterium bacterium liquid.

2. method

The tissue cultured seedling blade of getting potato kind " boolean class gram ", " Russet Burbank " and " Atlantic Ocean " 4 weeks of cultivation, robust growth cuts off the edge, is cut into 0.4-0.5cm ²Fritter, face down is inoculated in and consists of MS+2.0mg/L6-BA+0.2mg/L NAA+0.2mg/L 2, in the substratum of 4-D+16g/L sucrose+6g/L agar, 22 ℃ of dark cultivations 2 days; Then with the tissue cultured seedling blade inoculation in the bacterium liquid of the Agrobacterium LBA4404 that carries plasmid pBI hrap, shake gently and make the tissue cultured seedling blade fully contact 8 minutes with Agrobacterium bacterium liquid; Take out the tissue cultured seedling blade, blot, be inoculated in and consist of MS+2.0mg/L 6-BA+0.2mg/L NAA+0.2mg/L 2, in the substratum of 4D+16g/L sucrose+6g/L agar, 22 ℃ of dark cultivations 2 days with aseptic filter paper; Then the tissue cultured seedling blade is inserted callus induction in the listed substratum of table 1; Callus is transferred after about 17 days and is gone into to consist of in the BOA substratum of MS+2.5mg/L 6-BA+5.0mg/L GA3+30g/L sucrose+6g/L agar+50mg/L Kan+500mg/L cb, 22 ℃, illumination every day 12 as a child, intensity of illumination is to induce under the condition of 12000LX to sprout, 4 weeks were changed a BOA substratum, cultivated 2-11 week " Invest, Then Investigate " callus of induce rate and bud differentiation rate.

Table 1 callus medium component

The substratum numbering	Hormone and content (mg/L)			Sucrose (g/L)	Kan (mg/L)	Cb (mg/L)	The substratum purposes
									Basal component	6-BA	NAA
AA	MS	2	0.1	30	50	500	Callus induction
								AB	MS	2	0.2	30	50	500	Callus induction
AC	MS
			2	0.3	30	50	500	Callus induction
AE	MS								2	0.5	30	50	500	Callus induction
		BA	MS	2.5	0.1	30	50	500							Callus induction
BB	MS								2.5	0.2	30	50	500	Callus induction
		BC	MS	2.5	0.3	30	50	500							Callus induction

Annotate: all additional 0.6% agar of various substratum, pH5.8.

3. experimental result

3.1 different substratum are to the influence of " Russet Burbank " kind callus of induce and bud differentiation

Relatively " Russet Burbank " kind callus induction and bud break up situation, and the result is as shown in table 2, and the callus of induce rate of different treatment, bud differentiation rate and every bud number have very big-difference.The callus of induce rate is handled the highest with BB and Bc, reach 100%; The bud differentiation rate then is that the AA processing is the highest, is 64.00%; What every bud number was maximum is that BA handles, about 4.33 buds, and sprouting minimum is that AB handles about 1.33 buds.This result shows, dependency is little between callus of induce rate, bud differentiation rate and the every bud number in our test.

The different substratum of table 2 are to the influence of " Russet Burbank " kind callus of induce and bud differentiation

Substratum	Inoculation piece number	Go out more piece number	Differentiation sprout tuber number	Differentiation bud number	Callus of induce rate (%)	Bud differentiation rate (%)	Every bud number
								AA	34	25	16	44	73.53	64.00	2.75
AB	21	19	9	12	90.48	47.37	1.33
								AC	31	28	5	13	90.32	17.86	2.60
BA	42	30	9	39	71.43	30.00	4.33
								BB	31	31	15	32	100.00	48.39	2.13
BC	20	20	4	10	100.00	20.00	2.50

Annotate: callus of induce rate (%)=(going out more piece number/inoculation piece number) * 100%; Bud differentiation rate (%)=(differentiation sprout tuber number/the piece number goes out to heal) * 100%; Every bud number (%)=differentiation bud number/differentiation sprout tuber number

3.2, NAA concentration is to the callus of induce rate of " boolean class gram " and the influence of bud differentiation rate

Be provided with 3 NAA concentration (0.1,0.3,0.5mg/L) in callus of induce substratum (table 1), relatively the callus induction rate of " boolean class gram " and bud differentiation rate are to the reaction of NAA concentration, result such as table 3.From table 3, " boolean class gram " callus of induce rate in three kinds of substratum is the highest in AA, minimum in AE.

Table 3 " boolean class gram " is to the reaction of NAA concentration

Substratum	Cultivated 16 days in the different HORMONE TREATMENT
					Inoculation piece number	Go out more piece number	Callus of induce rate (%)
AA	18	10	55.56
				AC	26	13	50.00
AE	33	14	42.42
				On average	26	12	46.15

Annotate: callus of induce rate=(going out more piece number/inoculation piece number) * 100%; Bud differentiation rate=(differentiation sprout tuber number/the piece number goes out to heal) * 100%

3.3, the taking root of indefinite bud

In the plant regeneration process, rooting of vitro seedling also is an important step.After treating that bud grows to 2-3cm, bud downcut being inserted in the resistance screening substratum of Ms+50mg/L Kan, is 12000LX at 22 ℃, intensity of illumination, and periodicity of illumination is to cultivate under hour dark condition in 12 hours illumination/12 to take root.Although whole transformation tissue culture process is to carry out in containing the substratum of Kan, we have carried out twice anti-Kan root choosing to regrowth.Compare with the contrast of non-transgenic indefinite bud, the transgenosis indefinite bud major part that inserts in the substratum that contains MS+50mg/L Kan can be taken root radical 5-6 bar; The non-transgenic indefinite bud then all can not be taken root to impinging upon in the identical substratum, have only a small amount of aerial root, and plant strain growth is short and small.This test obtains about 100 transgenic lines, more than 1000 transgenosis individual plant altogether.

Table 4 is twice comparison of selecting rooting rate of different varieties regeneration bud.The rooting rate of different varieties root choosing for the first time is about 65%～73%; All kinds rooting rate for the second time also are necessary all than slightly high for the first time so carry out twice choosing.

The rooting rate of twice choosing of table 4 transfer-gen plant

Kind	Root choosing for the first time			Root choosing for the second time
								Inoculation bud number	The bud number of taking root	Rooting rate %	Inoculation bud number	The bud number of taking root	Rooting rate %
Russet Burbank	84	62	73.81	48	36	75.00
							Boolean class gram	38	29	76.32	29	25	86.21
The Atlantic Ocean	79	61	77.22	61	57	93.44

Annotate: rooting rate=(the bud number of taking root/inoculation bud number) * 100%

3.4, the regenerative process of transgenic potato plant

To be inoculated on the callus inducing medium that contains 50mg/L Kan, 500mg/L Cb and hormone 6-BA and NAA through the leaf dish explant of cultivating altogether two days with Agrobacterium, cultivate about 2 weeks after, as seen the place has strumae at the leaf plate edge; Change it over to bud division culture medium BOA and induce the formation indefinite bud; When indefinite bud grows to 2-3cm when high, it is changed on the root media Ms+50mg/L Kan take root; After root being selected the part subculture test-tube plantlet flush away substratum of the good transformed plant of two secondary roots, plant in the nutrition pot, with vegetable mould: perlite (3: 1) is made matrix, does normal root water with 1: 1000 ground bacterium Lingshui Spring solution, irrigates matrix.Be put in shady and cool place, coating film is preserved moisture.Can move on to the place of open-air well lighted after about 1 week, regularly water, water one time of nutrition liquid (MS liquid nutrient medium) about 1 week.

Two, the detection of transformed plant

1, the PCR of transformed plant detects

1.1 method

According to the sequence of goal gene, design primer respectively, synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd:

Hrap gene bow I thing I:5 '-GTTGGAGTTGGAGGAcGAGG-3 '

Hrap gene primer II:5 '-CGCGGATCCATGAAAATGAAGAACCTCTC-3 '

Adopt the SDS micromethod to extract and transform the potato plant leaf DNA, expanding numerous single-strain blade with regeneration plant after transforming is template, the positive contrast of positive Agrobacterium bacterium liquid, the negative contrast of corresponding unconverted kind plant leaf DNA is carried out pcr amplification with gene primer I and gene primer II.The PCR reaction system is 10 Bo uffer 2.5mL, 10mM dNTPs 0.5mL, 2.5U/mLTaq enzyme 0.5mL, 10mM gene primer I 0.5mL, 10mM gene primer II 0.5mL and template DNA 2mL, mends to 25mL with deionized water; The PCR reaction conditions: 94 ℃, 7min; 94 ℃, 50s; 53 ℃, 50s; 72 ℃, 1min; 30 circulations; 72 ℃, 7min; 4 ℃ of termination reactions.

1.2 result

The transformation tissue culture plant of " boolean class gram ", " Russet Burbank ", " Atlantic Ocean " 3 kinds is expanded numerous transplanting, and the single-strain blade of getting the system of homophyletic not respectively extracts DNA and is PCR and detects, and the result is as shown in table 5.The strain of 3 kinds is that positive rate is 83.33%-100.00%, and the individual plant positive rate is 47.22%-65.28%.The strain of 3 kinds central " Atlantic Ocean " is that positive rate and individual plant positive rate are all the highest.In this test, expand numerous material that obtains from same strain system, some individual plant can not expand and specific band, as " boolean class gram " No. 1 strain is, has 15 strains to be positive in 22 individual plants of extraction, and 7 strains are negative; 10 strains show positive in 17 individual plants of " Atlantic Ocean " No. 2 strain system, and 7 strains are negative etc.

The electrophoresis result that Fig. 2 detects for part PCR.Positive individual plant and positive Agrobacterium bacterium liquid all can amplify the band about about 750bp, and unconverted plant can not amplify the purpose band.

Table 5 sampling individual plant number and positive individual plant number

Kind	Boolean class gram	Russet Burbank	The Atlantic Ocean
				The strain coefficient	16	12	12
Positive strain coefficient	15	10	12
				Positive strain is a rate	93.75％	83.33％	100.00％
Sampling individual plant number	157	36	72
				Positive individual plant number	96	17	47
Positive rate %	61.15％	47.22％	65.28％

2, Southern hybridization

2.1 method

Adopt the CTAB method from through the blade of the transformed plant of PCR test positive, extracting DNA, cut the DNA of extraction, after 1% agarose electrophoresis, change film, screen positive plant and get disease-resistant potato plant with the BglII enzyme.

2.2 result

The result of Fig. 3, Fig. 4 and Fig. 5 shows that " boolean class gram " detected 13 individual plants, wherein has 7 individual plant performances positive, and positive rate is 53.85%; " Atlantic Ocean " has detected 6 individual plants, wherein has 3 performances positive, and positive rate is 50.00%; " Russet Burbank " detected 7 individual plants, wherein has 2 individual plant performances positive, and positive rate is 28.57%; Show that goal gene has changed in the potato kind.

Three, the disease resistance of transfer-gen plant detects (entrusting plant protection institute of Yunnan Prov Agriculture University to carry out)

1, transgenic potato plant bacterial wilt resistance detection method

To on the TTC flat board, cultivate 24h through room temperature, 150r/m shaking table in the cultured Ralstonia solanacearum strain TB48 access TTC nutrient solution, stand-by; Cultured inoculation liquid adding is equipped with in the culturing bottle of MS substratum, makes the OD of substratum ₅₂₀Value is 0.200; Potato with different strains places culturing bottle then, observes in glasshouse; With time of occurring the wilting blade the earliest with whether whole strain is wilted as anti-sense standard.

2, transgenic potato plant late blight resistance detection method

Get being incubated on the rye tomato juice substratum of purifying for examination phytophthora infestans strain (ZY15, LSX18, XH05-5-4), about 10-15 days mycelia is covered with culture dish, adding a small amount of sterilized water and gently scraping the mycelia surface with little spatula falls into the water sporocyst, 270 order nets shine and filter, sporocyst number in the filtrate is measured with Hematocyte Counter, regulate filter liquor concentration, making the sporocyst number is 1000-2000/ml; To plant in seedling-growing container for each potato strain potato seed of examination or test-tube plantlet, every basin sowing 1 potato piece (or stem section) is put in the greenhouse, waters, applies fertilizer, and is used for inoculation when growing to the 6-9 leaf age.The sporangia suspension for preparing is sprayed on the potato plant, preserved moisture 24 hours, observe incidence.

3, detected result

Through Yunnan Prov Agriculture University transfer-gen plant is carried out disease resistance and detect (table 6), its result shows that the resistant strain rate of anti-late blight is 3.1-9.52%, and the anti-diseased plant rate of resistance to bacterial wilt is 50.00-77.78%.Transfer-gen plant has tangible resistance against diseases.

Show 6:3 the disease-resistant detection positive rate of kind

Kind		Anti-late blight detects			Resistance to bacterial wilt detects
										Detect number	Number positive	Positive rate (%)	Detect number	Number positive	Positive rate (%)
Boolean class gram	Strain system		8	4	50.00	6	3									50.00
								Individual plant	42	5	11.90	6	3	50.00
	Russet Burbank	Strain system		5	1	20.00	1								0	0.00
Individual plant								12	1	8.33	1	0	0.00
		The Atlantic Ocean	Strain system		9	1	11.11							8	6	75.00
Individual plant	27							1	3.7	9	7	77.78

Four, discuss

1 transgenic plant regeneration

At present as the explant material of gene transformation after deliberation very extensive, relate to each tissue, organ and the position of plant.In the genetic transformation system of potato, be that the conversion of explant is all succeedd with blade, stem section, stem tuber and test tube potato.Potato chips can directly form regeneration bud without the callus stage, and the test tube potato can obtain the regeneration bud of higher conversion on the suitable culture base.

The required hormone kind of potato different varieties callus of induce and bud differentiation is different with concentration, and hormone assembly and concentration suitable in the regeneration system substratum are to influence reproductive success whether key.

This experimental result shows, dependency is little between the callus of induce rate of potato leaf, bud differentiation rate and the every bud number, both has not been high kind of callus of induce rate or processing, and the bud differentiation rate is just high; Kind that the bud differentiation rate is the highest or processing, every bud number might not be just very high.

The evaluation of 2 transfer-gen plants

Though all be to contain on the substratum of Kan to carry out in whole regenerative process, we have still carried out twice anti-Kan to the regeneration bud that obtains and have selected to take root.The first time root choose and have 23%～50% the indefinite bud can not normally long root approximately, still having when square root selects 0～25% can not long root, this explanation jigs transformed plant still relatively effectively by twice, simultaneously in conversion process, also there is more false positive, can considers to select to press concentration to bring up to 75mg/L Kan.

The plant that can normally take root after the root choosing is extracted DNA carry out the PCR detection.The result shows that 3 kinds are changeed in 261 individual plants of hrap gene, and positive rate is 55.55%-68.11%, and the performance PCR positive over half (62.45%) is on average arranged.Lee elder generation equality report, the transfer-gen plant PCR positive rate that obtains with the HarpinEa gene transformation Atlantic Ocean is 55.14%, and Su Junfeng etc. import the Bt gene in the Atlantic research, and the PCR positive rate only is 2.96%.

The positive individual plant that changes the hrap gene of 26 PCR is carried out Southern hybridization, and the result has 12 strains positive, and the average positive rate of 3 kinds is 46.15%.The illustration purpose gene is incorporated in the regeneration plant.Except " Russet Burbank " has 1 strain is two copy hybridization signals, and all the other 11 strains are single copy hybridization signal.

Change hrap gene plant disease resistance qualification result, the resistant strain rate of " boolean class gram " anti-late blight is the highest in 3 kinds, and the resistance individual plant rate of Atlantic resistance to bacterial wilt is the highest.The individual plant that 86 of 3 kinds is changeed the hrap gene carries out the detection of anti-late blight live body inoculation disease resistance, has obtained 7 high anti-plant that anti-late blight is the 1-2 level, and average resistance individual plant rate is 8.14%.Transfer-gen plant has tangible resistance against diseases.

The disease resistance that the different individual plants that this test to each strain is carry out detects, find that the negative individual plant of small part PCR also has disease resistance, detect negative individual plant 1-2-32,1-4-8 as PCR and anti-late blight occurs, the high resistance to bacterial wilt of 3-18-2, the seed selection of this explanation resistance individual plant is necessary.It is because agriculture bacillus mediated gene transformation realizes by tissue culture procedures that the negative individual plant of small part PCR has disease resistance, in this process, because regeneration plant is to be formed by cell development, and individual cells is easy to take place heritable variation in inducing process, the variation plant of resistance wherein just might occur late blight is had.

Compared with prior art, the present invention has following advantage:

1, first anaphylaxis is promoted protein gene hrap to import potato, and obtained disease-resistant plant.

2, the method for agriculture bacillus mediated foreign gene provided by the invention has improved explant callus and inducing of sprouting, and has improved agrobacterium-mediated ability.

3, having set up with the different varieties potato leaf is the high-efficiency regeneration system of explant, has screened external introduced variety and all suitable shoot regeneration substratum BOA of domestic variety.

4, because the used hrap gene of experiment can improve effect relevant with the course of disease, anaphylaxis albumen harpinPss, reduce the virulence of bacterial pathogen, cause more serious supersensitivity necrocytosis, so the hrap gene mediated is gone into the potato transfer-gen plant (the late blight grade scale adopts international potato late blight-resistance grade scale, and inoculation bacterium liquid has been contained 11 internationally recognized R gene microspecies) that potato plant has obtained high anti-late blight (1-2 level) and high resistance to bacterial wilt by Agrobacterium.

Description of drawings

Fig. 1 is the used plasmid pBI of a present invention hrap collection of illustrative plates.

Fig. 2 is the part individual plant PCR detected result of changeing the hrap gene, among the figure over against photograph: positive Agrobacterium bacterium liquid pcr amplification product; Marker:D2000; 1-15: the different transgenosis individual plants of kind 1; 16-20: the different transgenosis individual plants of kind 2; 21-38: the different transgenosis individual plants of kind 3; Negative contrast 1: the unconverted plant of kind 1; Negative contrast 2: the unconverted plant of kind 2; Negative contrast 3: the unconverted plant of kind 3.

Fig. 3 is the Southern results of hybridization of Zhe-level Nie Tui transfer-gen plant, among the figure over against photograph: plasmid pBI hrap; Negative contrast: not genetically modified boolean class gram, 1-1,1-8, the band that 1-9 detects extremely a little less than, gene does not change into; 1-2,1-3,1-5 may be non-specific hybridizations a little less than being with, can not determine whether gene changes over to; 1-4,1-7,1-713,1-10,1-11,1-12,1-14-15 is defined as transgenic line.

Fig. 4 be " Atlantic Ocean " and the Southern results of hybridization of transformed plant, among the figure over against photograph: plasmid pBIhrap; Negative contrast: the Atlantic Ocean is transfer-gen plant (3) not; 3-5-12,3-18-1 do not have band, and gene does not change into; 3-24-1 can not determine whether gene changes over to a little less than being with; 3-3,3-17,3-19-1 are defined as transgenic line.

Fig. 5 is the Southern results of hybridization of " Russet Burbank " transformed plant, among the figure over against photograph: plasmid pBIhrap; 2-1,2-2 do not have band; A little less than 2-3,2-4,2-2-2 are with; 2-6,2-12 is defined as transgenic line.

Embodiment

Embodiment 1:

1, the conversion of explant and cultivation

(1) bacterium liquid preparation: the Agrobacterium LBA4404 bacterium liquid that will carry plasmid pBI hrap is inoculated in the liquid LB substratum of additional 100mg/L Kan, 100mg/L Sm and 100mg/L Rif, and on shaking table 28 ℃, middling speed is cultured to OD ₆₀₀Value is 0.4, gets Agrobacterium bacterium liquid, and is standby;

(2) the pre-cultivation: get " boolean class gram " tissue cultured seedling blade of cultivating 4 weeks, robust growth, be cut into 0.4cm ²Fritter, face down is inoculated in and consists of MS+2.0mg/L 6BA+0.2mg/L NAA+0.2mg/L 2, in the y2 substratum of 4D+16g/L sucrose+6g/L agar, 22 ℃ of dark cultivations 2 days;

(3) contaminate: " boolean class gram " the tissue cultured seedling blade inoculation after pre-the cultivation makes the tissue cultured seedling blade fully contact 8 minutes with Agrobacterium bacterium liquid in the bacterium liquid of the Agrobacterium LBA4404 that carries plasmid pBI hrap;

(4) cultivate altogether: take out the tissue cultured seedling blade, blot, be inoculated in the y2 substratum in the step (2), 22 ℃ of dark cultivations 2 days with aseptic filter paper;

(5) callus induction, induce and sprout and root culture: tissue cultured seedling blade after will cultivating altogether inserts callus induction in the substratum of callus substratum MS+2.5mg/L 6-BA+0.2mg/L NAA+30g/L sucrose+50mg/LKan+500mg/L Cb, additional 0.6% agar, pH5.8; The back switching of 2 week of callus growth goes into to consist of in the BOA substratum of MS+2.5mg/L 6BA+5.0mg/LGA3+30g/L sucrose+6g/L agar+50mg/L Kan+500mg/L Cb, be to induce under the condition of 12000LX to sprout at 22 ℃, illumination every day 12 hours, intensity of illumination, 4 weeks were changed a BOA substratum; After treating that bud grows to 3 centimetres, bud downcut being inserted in the resistance screening substratum of MS+50mg/L Kan, is 12000LX at 22 ℃, intensity of illumination, and periodicity of illumination is to cultivate under hour dark condition in 12 hours illumination/12 to take root, and root selects twice;

(6) will take root behind the part subculture test-tube plantlet flush away substratum of good transformed plant, and plant in the nutrition pot, with vegetable mould: perlite (3: 1) is made matrix, does normal root water with 1: 1000 ground bacterium Lingshui Spring solution, irrigates matrix.Be put in shady and cool place, coating film is preserved moisture.Can move on to the place of open-air well lighted after about 1 week, regularly water, water one time of nutrition liquid (MS liquid nutrient medium) about 1 week.

2, PCR detects: adopt the SDS micromethod to extract DNA from the transformed plant blade, with the transformed plant is template, the positive contrast of positive Agrobacterium bacterium liquid, the negative contrast of unconverted blade, carry out pcr amplification with two primer hrap gene primer I:5 '-GTTGGAGTTGGAGGACGAGG-3 ' and hrap gene primer II:5 '-CGCGGATCCATGAAAATGAAGAACCTCTC-3 ', the screening positive plant; The PCR reaction system is 10 Bo uffer 2.5mL, 10mM dNTPs 0.5mL, 2.5U/mL Taq enzyme 0.5mL, 10mM gene primer I 0.5mL, 10mM gene primer II 0.5mL and template DNA 2mL, mends to 25mL with deionized water; The PCR reaction conditions: 94 ℃, 7min; 94 ℃, 50s; 53 ℃, 50s; 72 ℃, 1min; 30 circulations; 72 ℃, 7min; 4 ℃ of termination reactions;

3, Southern detects: adopt the CTAB method from extract DNA through the blade of the transformed plant of PCR test positive, cut the DNA of extraction with the BglII enzyme, after 1% agarose electrophoresis, change film, screen positive plant

4, disease resistance is identified: identify through live body inoculation disease resistance to obtain disease-resistant potato plant.

Embodiment 2: the conversion of explant and cultivation: the Agrobacterium LBA4404 bacterium liquid that will preserve, carry plasmid pBI hrap is inoculated in the liquid LB substratum of additional 100mg/L Kan, 100mg/L Sm and 100mg/L Rif, on shaking table 28 ℃, middling speed is cultured to OD ₆₀₀Value is 0.5, gets Agrobacterium bacterium liquid, and is standby; " Atlantic Ocean " tissue cultured seedling blade of cultivating 3 weeks, robust growth is cut off the edge, be cut into 0.5cm ²Fritter, face down is inoculated in and consists of MS+2.0mg/L 6BA+0.2mg/L NAA+0.2mg/L 2, in the y2 substratum of 4D+16g/L sucrose+6g/L agar, 22 ℃ of dark cultivations 2 days; The tissue cultured seedling blade inoculation makes the tissue cultured seedling blade fully contact 10 minutes with Agrobacterium bacterium liquid in the bacterium liquid of the Agrobacterium LBA4404 that carries plasmid pBI hrap; Take out the tissue cultured seedling blade, blot, be inoculated in the y2 substratum, 22 ℃ of dark cultivations 2 days with aseptic filter paper; Tissue cultured seedling blade after cultivating is inserted callus induction in the substratum that consists of MS+2mg/L 6-BA+0.3mg/L NAA+30g/L sucrose+50mg/L Kan+500mg/L Cb, additional 0.6% agar, pH5.8; The back switching of 3 week of callus growth is gone in the BOA substratum, at 21 ℃, illumination every day 12 hours, intensity of illumination is to induce under the condition of 12000LX to sprout, and 4 weeks were changed a BOA substratum; After treating that bud grows to 2cm, bud downcut being inserted in the resistance screening substratum of MS+50mg/L Kan, is that 12000LX, periodicity of illumination are to cultivate under hour dark condition in 12 hours illumination/12 to take root at 22 ℃, intensity of illumination, and root selects twice; To take root behind the part subculture test-tube plantlet flush away substratum of good transformed plant, and plant in the nutrition pot, with vegetable mould: perlite (3: 1) is made matrix, does normal root water with 1: 1000 ground bacterium Lingshui Spring solution, irrigates matrix.Be put in shady and cool place, coating film is preserved moisture.Can move on to the place of open-air well lighted after about 1 week, regularly water, water one time of nutrition liquid (MS liquid nutrient medium) about 1 week.

Embodiment 3: the conversion of explant and cultivation: the Agrobacterium LBA4404 bacterium liquid that will preserve, carry plasmid pBI hrap is inoculated in the liquid LB substratum of additional 100mg/L Kan, 100mg/L Sm and 100mg/L Rif, on shaking table 28 ℃, middling speed is cultured to OD ₆₀₀Value is 0.5, gets Agrobacterium bacterium liquid, and is standby; Get " Russet Burbank " potato tissue cultured seedling blade of cultivating 4 weeks, robust growth and cut off the edge, be cut into 0.4cm ²Fritter, face down is inoculated in the y2 substratum, 23 ℃ of dark cultivations 2 days; The tissue cultured seedling blade inoculation makes the tissue cultured seedling blade fully contact 20 minutes with Agrobacterium bacterium liquid in the bacterium liquid of the Agrobacterium LBA4404 that carries plasmid pBI hrap; Take out the tissue cultured seedling blade, blot, be inoculated in the y2 substratum, 23 ℃ of dark cultivations 2 days with aseptic filter paper; Tissue cultured seedling blade after cultivating is inserted callus induction in the substratum that consists of MS+2mg/L 6-BA+0.5mg/L NAA+30g/L sucrose+50mg/L Kan+500mg/L Cb, additional 0.6% agar, pH5.8; The back switching of 2 week of callus growth is gone in the BOA substratum, at 23 ℃, illumination every day 12 hours, intensity of illumination is to induce under the condition of 12000LX to sprout, and 3 weeks were changed a BOA substratum; After treating that bud grows to 3 centimetres, bud downcut being inserted in the resistance screening substratum of MS+50mg/L Kan, is to cultivate under hour dark condition in 12 hours illumination/12 to take root for the 12000LX periodicity of illumination at 23 ℃, intensity of illumination, and root selects twice; To take root behind the part subculture test-tube plantlet flush away substratum of good transformed plant, and plant in the nutrition pot, with vegetable mould: perlite (3: 1) is made matrix, does normal root water with 1: 1000 ground bacterium Lingshui Spring solution, irrigates matrix.Be put in shady and cool place, coating film is preserved moisture.Can move on to the place of open-air well lighted after about 1 week, regularly water, water one time of nutrition liquid (MS liquid nutrient medium) about 1 week.

Claims (10)

1. an agrobacterium-mediated potato changes the method that the hrap gene obtains the disease resistance expression, it is characterized in that: be inoculated in the bacterium liquid of the Agrobacterium LBA4404 that carries plasmid pBI hrap through pre-incubated explant and contaminate, explant after the dip-dye is through being total to cultivation, callus induction, inducing and sprout and the root culture selection, shift potted plant transformed plant behind the test tube seedling and propagating of selecting root to select to take root for twice, transformed plant detects with PCR and Southern hybridization detection method screening positive plant, identifies through the inoculation of live body disease resistance to obtain disease-resistant potato plant.

2. agrobacterium-mediated potato changes the method that the hrap gene obtains the disease resistance expression according to claim 1, it is characterized in that: described Agrobacterium bacterium liquid is preparation like this: the Agrobacterium LBA4404 bacterium liquid that will carry plasmid pBI hrap is inoculated in the liquid LB substratum of additional 100mg/L Kan, 100mg/L Sm and 100mg/L Rif, on shaking table 28 ℃, it is 0.4～0.5 that middling speed is cultured to the OD600 value.

3. change the hrap gene as agrobacterium-mediated potato as described in the claim 2 and obtain the method that disease resistance is expressed, it is characterized in that: described Agrobacterium LBA4404, carry plasmid pBI hrap, contain neomycin phosphotransferase gene NPTII and anaphylaxis and promote protein gene hrap, the hrap upstream region of gene is a 35S promoter, and the downstream is the NOS terminator.

4. agrobacterium-mediated potato changes the method that the hrap gene obtains the disease resistance expression according to claim 1, it is characterized in that: the pre-cultivation of described explant is to get the potato tissue cultured seedling blade of cultivating 3～4 weeks, robust growth to cut off the edge, be cut into the fritter of 0.4-0.5cm2, face down is inoculated in the substratum, 22～25 ℃ of dark cultivations 2 days, consisting of of described substratum: MS+2.0mg/L 6-BA+0.2mg/L NAA+0.2mg/L 2,4-D+16g/L sucrose+6g/L agar.

5. agrobacterium-mediated potato changes the method that the hrap gene obtains the disease resistance expression according to claim 1, it is characterized in that: following method is adopted in the dip-dye of described explant: the explant after pre-the cultivation is inoculated in the Agrobacterium bacterium liquid for preparing, and makes explant fully contact 8～20 minutes with Agrobacterium.

6. agrobacterium-mediated potato changes the method that the hrap gene obtains the disease resistance expression according to claim 1, it is characterized in that: contaminating the back explant is to cultivate so altogether: take out explant from Agrobacterium bacterium liquid, blot with aseptic filter paper, be inoculated in the substratum, 22～25 ℃ of dark cultivations 2～3 days, described substratum consists of: MS+2.0mg/L6-BA+0.2mg/L NAA+0.2mg/L 2,4-D+16g/L sucrose+6g/L agar.

7. agrobacterium-mediated potato changes the method that the hrap gene obtains the disease resistance expression according to claim 1, it is characterized in that: the callus induction of described explant, induce and sprout and root culture adopts following step: the explant after will cultivating altogether inserts and consists of MS+2～2.5mg/L 6-BA+0.1～0.5mg/L NAA+30g/L sucrose+50mg/LKan+500mg/L Cb, additional 0.6% agar, callus induction in the substratum of pH5.8; The back switching of 2～3 week of callus growth goes into to consist of in the BOA substratum of MS+2.5mg/L 6-BA+5.0mg/L GA3+30g/L sucrose+6g/L agar+50mg/L Kan+500mg/L Cb, be to induce under the condition of 12000LX to sprout at 22 ± 1 ℃, illumination every day 12 hours, intensity of illumination, 3～4 weeks were changed a BOA substratum; After treating that bud grows to 2～3 centimetres, bud downcut being inserted in the resistance screening substratum of MS+50mg/L Kan, is 12000LX at 22 ± 1 ℃, intensity of illumination, and periodicity of illumination is to cultivate under hour dark condition in 12 hours illumination/12 to take root.

8. agrobacterium-mediated potato changes the method that the hrap gene obtains the disease resistance expression according to claim 1, it is characterized in that: described PCR detects and is: extract DNA from the transformed plant blade, with the transformed plant is template, the positive contrast of positive Agrobacterium bacterium liquid, the negative contrast of unconverted blade, carry out pcr amplification with hrap gene primer I:5 '-GTTGGAGTTGGAGGACGAGG-3 ' and hrap gene primer II:5 '-CGCGGATCCATGAAAATGAAGAACCTCTC-3 ', the screening positive plant.

9. agrobacterium-mediated potato changes the method that the hrap gene obtains the disease resistance expression according to claim 1, it is characterized in that: described Southern hybridization detection method is: extract DNA from the blade of the transformed plant of PCR test positive, cut the DNA of extraction with the BglII enzyme, after 1% agarose electrophoresis, change film, the screening positive plant.

As claim 1-9 arbitrary as described in agrobacterium-mediated potato change the hrap gene and obtain the method that disease resistance is expressed, it is characterized in that: described method is carried out as follows:

(1) bacterium liquid preparation: will preserve the liquid LB substratum that Agrobacterium LBA4404 bacterium liquid is inoculated in additional 100mg/L Kan, 100mg/L Sm and 100mg/L Rif, on shaking table 28 ℃, it is 0.4 that middling speed is cultured to the OD600 value, must Agrobacterium bacterium liquid, and standby;

(2) the pre-cultivation: get the potato tissue cultured seedling blade of cultivating 4 weeks, robust growth, face down is inoculated in and consists of MS+2.0mg/L 6-BA+0.2mg/L NAA+0.2mg/L 2, in the substratum of 4-D+16g/L sucrose+6g/L agar, 22 ℃ of dark cultivations 2 days;

(3) contaminate: the tissue cultured seedling blade inoculation after pre-the cultivation makes the tissue cultured seedling blade fully contact 8 minutes with Agrobacterium bacterium liquid in the bacterium liquid of the Agrobacterium LBA4404 that carries plasmid pBI hrap of step (1) preparation;

(4) cultivate altogether: take out the tissue cultured seedling blade, blot, be inoculated in the substratum in the step (2), 22 ℃ of dark cultivations 2 days with aseptic filter paper;

(5) callus induction, induce and sprout and root culture: tissue cultured seedling blade after will cultivating altogether inserts callus induction in the substratum that consists of MS+2.5mg/L 6-BA+0.2mg/L NAA+30g/L sucrose+50mg/L Kan+500mg/L Cb, additional 0.6% agar, pH5.8; The back switching of 3 week of callus growth goes into to consist of in the BOA substratum of MS+2.5mg/L 6-BA+5.0mg/LGA3+30g/L sucrose+6g/L agar+50mg/L Kan+500mg/L Cb, be to induce under the condition of 12000LX to sprout at 22 ℃, illumination every day 12 hours, intensity of illumination, 4 weeks were changed a BOA substratum; After treating that bud grows to 3 centimetres, bud is downcut in the resistance screening substratum that inserts MS+50mg/L Kan, at 22 ℃, intensity of illumination is to cultivate under hour dark condition in 12 hours illumination/12 to take root for the 12000LX periodicity of illumination, shift behind the test tube seedling and propagating of selecting root to select can both to take root for twice potted plant must transformed plant;

(6) PCR detects: adopt the SDS micromethod to extract DNA from the transformed plant blade, with the transformed plant is template, the positive contrast of positive Agrobacterium bacterium liquid, the negative contrast of unconverted blade, carry out pcr amplification with hrap gene primer I:5 '-GTTGGAGTTGGAGGACGAGG-3 ' and hrap gene primer II:5 '-CGCGGATCCATGAAAATGAAGAACCTCTC-3 ', the screening positive plant; The PCR reaction system is 10 Bo uffer 2.5mL, 10mM dNTPs 0.5mL, 2.5U/mL Taq enzyme 0.5mL, 10mM gene primer I 0.5mL, 10mM gene primer II 0.5mL and template DNA 2mL, mends to 25mL with deionized water; The PCR reaction conditions: 94 ℃, 7min; 94 ℃, 50s; 53 ℃, 50s; 72 ℃, 1min; 30 circulations; 72 ℃, 7min; 4 ℃ of termination reactions;

(7) Southern detects: adopt the CTAB method from extract DNA through the blade of the transformed plant of PCR test positive, cut the DNA of extraction with the BglII enzyme, after 1% agarose electrophoresis, change film, screen positive plant;

(8) disease resistance is identified: the Southern positive plant is identified through the live body inoculation and is obtained disease-resistant potato plant.

Images