CN1995362B - Gibberella fujikuroi electroporation genetic transformation method - Google Patents
Gibberella fujikuroi electroporation genetic transformation method Download PDFInfo
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Abstract
The invention discloses a genetic conversing method of exogenous gene for Gibberalla fujikuroi through electroporation technique, which is characterized by the following: utilizing cell wall degraded enzyme of fungi to dispose the cell wall; obtaining the protoplast to purify; blending purified protoplast and fitful exogenous gene/DNA solution; proceeding electric shock effect; making protoplasm adsorb the exogenous DNA.
Description
(1) technical field
The present invention relates to the electroporation genetic transformation technology of gibberella Gibberella fujikuroi, be applicable to fields such as the genetic engineering of gibberella and gibberella molecular biology research.
(2) background technology
Gibberella fujikuroi (Gibberellafujikuroi) is the generation bacterium of Plant hormones regulators,gibberellins, causes bakanae disease because of excessively producing Plant hormones regulators,gibberellins on paddy rice, and this disease all has generation in each paddy rice producing region of China, produces to paddy rice and causes serious threat.Plant hormones regulators,gibberellins is a plant growth regulators, and important effect is arranged in growth and development of plants, has very widely to use on crops such as fruit, vegetables, paddy rice.Plant hormones regulators,gibberellins can also be used for aspects such as beer production industry, clinical medicine.
Gibberella has become one of filamentous fungus that has good DEVELOPMENT PROSPECT, and at present a lot of researchs are being attempted by engineered way improvement bacterial classification.Simultaneously, gibberella also attracts great attention to the pathogenic molecular biology of paddy rice and the research of growth.But the research and development of genetic transforming method are the prerequisites of gibberella being implemented genetically engineered and molecular biology research.
The genetic transformation of filamentous fungus mainly contains the spore conversion method, PEG mediation protoplast transformation method of Lithium Acetate mediation, agriculture bacillus mediated conversion method and electroporation conversion method at present.In gibberella (G.fujikuroi), have only preceding 2 kinds of method for transformation that application is arranged at present---spore conversion method, PEG mediation protoplast transformation method, the not favourable as yet report that transforms with electroporation.The spore conversion method only limits to can spore-bearing gibberella bacterial strain, and PEG mediation protoplast transformation method is used more, but step of converting is many, and efficient is not high.The electroporation transformation technology has very high efficient in the genetic transformation of microorganism etc., and simple, has been widely used in researchs such as genetically engineered and molecular biology.World-renowned company such as Bio-rad, Phamacia has developed electroporation apparatus.
The present invention has utilized electroporation technology that gibberella fujikuroi (G.fujikuroi) has been implemented the foreign gene conversion first, find suitable conversion parameter and optimized the combination of parameter, more existing other method of Transformation Program is simpler, improved transformation efficiency, this method can be used for genetic engineering and molecular biology research and the application of gibberella.
(3) summary of the invention
The object of the invention provides a kind of genetic transforming method that utilizes electroporation (electroporation) technology gibberella fujikuroi (G.fujikuroi) to be carried out foreign gene, and find the higher conversion parameters combination of transformation efficiency, can be used for fields such as gibberella genetic engineering and molecular biology research, with the weak point of the genetic transforming method that solves other.
The present invention reaches the technical scheme that goal of the invention adopts: utilize fungall cell wall degrading enzyme that the cell walls of gibberella is handled, obtain protoplastis and carry out purifying, the protoplastis of purifying and an amount of foreign gene/thymus nucleic acid (DNA) aqueous solution mix, utilize electroporation apparatus to the mixed solution processing of shocking by electricity then, the protoplastis that electric shock is handled can receive foreign DNA, thereby has realized the genetic transformation of foreign DNA to gibberella.
The electroporation genetic transforming method of described gibberella fujikuroi Gibberella fujikuroi carries out as follows:
1) the gibberella fujikuroi protoplastis is suspended with 1M Sorbitol Solution USP A, with the microscopy counting, the protoplastis concentration of adjusting protoplasma body fluid is 10
7-10
8/ mL;
2) to be dissolved in aseptic deionized water to concentration be 5～10 μ g/ul to the foreign DNA that is used to transform, and adds 10～15 μ gDNA in every 100ul protoplasma body fluid, mixing, and ice bath 30～40min becomes mixed solution;
3) in the electric shock cup of precooling, add step 2) the gained mixed solution, behind ice bath 5～10min, shock by electricity with electroporation apparatus;
4) adding concentration in the electric shock back electric shock cup is the Sorbitol Solution USP B of 1M, ice bath 30min～1h, coat the solid regenerated culture medium flat plate of MYG after the suspension, behind 28 ℃ of cultivation 12h, on regenerated plate, cover the soft agar MYG regeneration culture medium that contains with the corresponding microbiotic final concentration 50 μ g/mL of described foreign DNA, be cultured to transformant at 28 ℃ and occur; Contain 10mM Tris-Cl, 50mM CaCl in the described Sorbitol Solution USP B solution
2, and pH7.5; The solid regenerated substratum final concentration of described MYG is: maltose 5g/L; Yeast extract paste 5g/L; Glucose 10g/L; Agar powder 15g/L; Sucrose 0.5mol/L; PH6.5; Described soft agar MYG regeneration culture medium final concentration is: maltose 5g/L; Yeast extract paste 5g/L; Glucose 10g/L; Agar powder 8g/L; PH6.5.
6) beggar's succeeding transfer culture on the soft agar MYG regeneration culture medium that contains with the corresponding microbiotic final concentration 100 μ g/ml of described foreign DNA is preserved behind the stable growth, and described soft agar MYG regeneration culture medium final concentration is: maltose 5g/L; Yeast extract paste 5; Glucose 10g/L; Agar powder 8g/L; PH6.5.
The preparation method of the protoplastis of described gibberella fujikuroi bacterium is as follows:
(1) Dryslase and Lying enzyme (Sigma) mass ratio are that 3: 7 mixed enzyme is dissolved in 1M MgSO
4In the solution, making final concentration is the cell wall degrading enzyme liquid of 15mg/ml, and every 10mL enzyme liquid added about 0.9～1.1g wet thallus after described enzyme liquid was sterilized after filtration, put 30 ℃ shaking table, rotating speed is 50～60rpm, and the free protoplastis 3～4h of enzymolysis gets the protoplastis enzymolysis solution;
(2) the protoplastis enzymolysis solution is centrifugal, inclining upper strata protoplasma body fluid, removes by filter the mycelia relic, splashes into aseptic deionized water and make MgSO in containing the filtrate of protoplastis
4Concentration be 0.5M, centrifugal protoplastis precipitation.
The preparation method of the wet thallus of described gibberella fujikuroi bacterium is as follows:
A. carry out the gibberella fujikuroi actication of culture on the PDA culture medium flat plate, culture temperature 26-28 ℃, described PDA substratum final concentration is: potato 200g/L, sucrose 20g/L, agar powder 15g/L; The pH nature was sterilized 18 minutes down at 121 ℃;
B. learn from else's experience activatory gibberella fujikuroi bacterial classification inoculation to the MYG liquid nutrient medium that contains granulated glass sphere, placing temperature is that 26～28 ℃, rotating speed are that the shaking table of 200rpm is cultivated 20h, and described MYG liquid nutrient medium final concentration is: maltose 5g/L; Yeast extract paste 5g/L; Glucose 10g/L; PH6.5;
C. step b gained nutrient solution is carried out enlarged culturing 20h by 10% inoculum size at the MYG liquid nutrient medium;
D. the individual layer filter cloth miracloth (Calbiochem) of step c gained nutrient solution with sterilization filtered, the mycelium with on the aseptic deionized water flushing filter cloth repeats 2～4 times, removes residual culture, uses 1M MgSO again
4Wash 2～4 times, collect wet thallus.
Use hygromycin selection because fungi is most at present, so the foreign DNA described in the present invention preferably has the plasmid pAN7-1 of hygromycin gene, its corresponding microbiotic is a Totomycin.Other has the plasmid of hygromycin gene, and as plasmid pCB1003, (Fungal Genetics Newsletter 1994 41:22) etc., is equally applicable to the present invention to pCB1004 for Anne M.Carroll, et al.Other any DNA or plasmid that can transform fungi also can adopt method of the present invention to transform.
Concrete, described method is carried out as follows:
1) the gibberella fujikuroi protoplastis is suspended with 1M Sorbitol Solution USP A, with the microscopy counting, the protoplastis concentration of adjusting protoplasma body fluid is 10
7/ mL;
2) the exogenous plasmid pAN7-1 (Punt et al.1987 Gene 56:117-124) that is used to transform is cut into wire through restriction enzyme, be dissolved in aseptic deionized water 5～10 μ g/uL behind the purifying, add 10 μ g DNA in every 100uL protoplasma body fluid, mixing, ice bath 30min becomes mixed solution;
3) 2cm of precooling electric shock cup, middle adding step 2) behind the gained mixed solution 40uL ice bath 5min, prepare electroporation apparatus, setting shock parameters voltage is 850V, electric capacity 25 μ F, and resistance is 250 Ω, shocks by electricity;
4) adding 1mL concentration in the electric shock back electric shock cup is 1M Sorbitol Solution USP B, ice bath 30min～1h, coat the solid regenerated flat board of MYG after the suspension, the 200uL/ ware, behind 28 ℃ of cultivation 12h, cover the soft agar MYG regeneration culture medium that contains Totomycin final concentration 50 μ g/mL on regenerated plate, the 10mL/ flat board is cultured to transformant at 28 ℃ and occurs; Contain 10mMTris-Cl, 50mM CaCl in the described Sorbitol Solution USP B solution
2, and pH7.5;
5) transformant is preserved behind the stable growth containing succeeding transfer culture on the soft agar MYG regeneration culture medium that the Totomycin final concentration is 100 μ g/mL.
Beneficial effect of the present invention is embodied in: can be used for the molecular biology research of gibberella fujikuroi, as study this bacterium to pathogenic molecular mechanism of paddy rice and the mechanism etc. of growing of this bacterium.Carry out the genetic improvement breeding thereby can be used for that gibberella is implemented genetically engineered, improve the synthesis capability of Plant hormones regulators,gibberellins.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: utilize electroporation gibberella fujikuroi (Gibberella fujikuroi) to be carried out the genetic transformation of the mould plain gene of external source moisture resistance
Utilize the method described in the summary of the invention, the exogenous plasmid pAN7-1 that will contain hygromycin gene has imported gibberella, obtained the engineering bacteria of stable conversion by hygromycin selection, transformation efficiency is higher than the spore conversion method of Lithium Acetate mediation, and is simpler than the conversion method step of polyoxyethylene glycol PEG mediation.Concrete grammar is as follows:
1. the preparation of gibberella protoplastis:
1) on the PDA culture medium flat plate, carries out the gibberella fujikuroi actication of culture, 26～28 ℃ of culture temperature.PDA substratum final concentration is: potato, 200g/L; Sucrose, 20g/L; Agar powder, 15g/L; The pH nature.Sterilized 18 minutes down at 121 ℃.
2) get 1 fritter and be seeded to MYG liquid nutrient medium (containing about 40 granulated glass spherees), placing temperature is that 26～28 ℃, rotating speed are that the shaking table of 200rpm is cultivated 20h.Described MYG liquid nutrient medium final concentration: maltose 5g/L; Yeast extract paste 5g/L; Glucose 10g/L; PH6.5;
3) with step 2) nutrient solution carries out enlarged culturing 20h by 10% inoculum size at the MYG liquid nutrient medium.MYG liquid nutrient medium final concentration: maltose 5g/L; Yeast extract paste 5g/L; Glucose 10g/L; PH6.5;
4) the individual layer filter cloth miracloth (Calbiochem) of step 3) nutrient solution with sterilization filtered, with the mycelium on the aseptic deionized water flushing filter cloth repeatedly, remove residual culture as far as possible, use 1M MgSO then
4Wash 3 times, collect wet thallus.
5) preparation cell wall degrading enzyme liquid, be Dryslase with the quality proportioning: Lying enzyme=3: 7 mixed enzyme is dissolved in 1M MgSO
4, final concentration is about 15mg/ml, and enzyme liquid adds the gibberella wet thallus after the degerming after filtration, adds 1 gram wet thallus in every 10ml enzyme liquid.Put 30 ℃ shaking table, rotating speed is the free protoplastis 4h of enzymolysis under the 60rpm.
6) the protoplastis enzymolysis solution is with the centrifugal 10min of 500rpm, and upper strata protoplasma body fluid removes by filter the mycelia relic through the double-layer filter cloth miracloth of sterilization, slowly splashes into aseptic deionized water and regulate MgSO in containing the filtrate of protoplastis
4Concentration to 0.5M, the centrifugal 10min of 3500rpm, the precipitation protoplastis.
7) the protoplastis precipitation suspends with the 1M Sorbitol Solution USP, and the centrifugal 10min of 3500rpm repeats 1 time.
8) the protoplastis precipitation is suspended in an amount of 1M Sorbitol Solution USP 100 μ L, microscope inspection inspection and counting, and the concentration of adjusting protoplastis is 10
7About/ml, be used for following conversion.
2. electroporation carries out the foreign DNA conversion to protoplastis:
1) DNA preparation.The exogenous plasmid pAN7-1 that is used to transform (Punt et al.1987 Gene56:117-124) is cut into wire through restriction enzyme, is dissolved in aseptic deionized water behind the purifying, and concentration 10ug/ul is used for transforming.
2) add 10ug DNA in the 100ul protoplasma body fluid, mixing behind the ice bath 30min, is got the electric shock cup (2cm) that the 40ul mixed solution is added to precooling, behind the ice bath 5min, prepares electroporation apparatus, sets shock parameters and shocks by electricity.Voltage, 850V; Electric capacity 25 μ F; Resistance, 250 Ω.
3) to add 1ml concentration be that the Sorbitol Solution USP of 1M (contains 10mM Tris-Cl, pH7.5,50mMCaCl electric shock back
2), ice bath 1h coats the solid regenerated flat board of MYG, the 200ul/ ware after the suspension.Behind 28 ℃ of cultivation 12h, on regenerated plate, cover the soft agar MYG regeneration culture medium that contains Totomycin (50ug/ml), 10ml/ flat board.Continuation is cultivated the appearance of observing transformant at 28 ℃.The solid regenerated substratum final concentration of described MYG is: maltose 5g/L; Yeast extract paste 5g/L; Glucose 10g/L; Agar powder 15g/L; Sucrose 0.5mol/L; PH6.5; Described soft agar MYG regeneration culture medium: maltose 5g/L; Yeast extract paste 5g/L; Glucose 10g/L; Agar powder 8g/L; PH6.5.
4) transformant succeeding transfer culture on the solid regenerated substratum of above-mentioned MYG that improves Totomycin concentration (100ug/ml) is preserved behind the stable growth.
The result:
Transformation efficiency is greater than 8 transformants/more than the ug DNA, with respect to LESLIE and DICKMAN (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1991, and the spore conversion method (1-2 transformant/ug DNA) of Lithium Acetate mediation p.1423-1429) improves a lot.
Claims (5)
1. the electroporation genetic transforming method of a gibberella fujikuroi is characterized in that described method carries out as follows:
1) the gibberella fujikuroi protoplastis is suspended with 1M Sorbitol Solution USP A, with the microscopy counting, the protoplastis concentration of adjusting protoplasma body fluid is 10
7-10
8/ mL;
2) to be dissolved in aseptic deionized water to concentration be 5～10 μ g/ul to the foreign DNA that is used to transform, and adds 10～15 μ gDNA in every 100ul protoplasma body fluid, mixing, and ice bath 30～40min becomes mixed solution;
3) in the electric shock cup of precooling, add step 2) the gained mixed solution, behind ice bath 5～10min, shock by electricity shock parameters with electroporation apparatus: voltage is 850V, and electric capacity is 25 μ F, and resistance is 250 Ω;
4) adding concentration in the electric shock back electric shock cup is the Sorbitol Solution USP B of 1M, ice bath 30min～1h, coat the solid regenerated culture medium flat plate of MYG after the suspension, behind 28 ℃ of cultivation 12h, on regenerated plate, cover the soft agar MYG regeneration culture medium that contains with the corresponding microbiotic final concentration 50 μ g/mL of described foreign DNA, be cultured to transformant at 28 ℃ and occur; Contain 10mM Tris-Cl, 50mM CaCl in the described Sorbitol Solution USP B solution
2, and pH7.5; The solid regenerated substratum final concentration of described MYG is: maltose 5g/L; Yeast extract paste 5g/L; Glucose 10g/L; Agar powder 15g/L; Sucrose 0.5mol/L; PH6.5; Described soft agar MYG regeneration culture medium final concentration is: maltose 5g/L; Yeast extract paste 5g/L; Glucose 10g/L; Agar powder 8g/L; PH6.5;
5) transformant succeeding transfer culture on the soft agar MYG regeneration culture medium that contains with the corresponding microbiotic final concentration 100 μ g/mL of described foreign DNA is preserved behind the stable growth, and described soft agar MYG regeneration culture medium final concentration is: maltose 5g/L; Yeast extract paste 5g/L; Glucose 10g/L; Agar powder 8g/L; PH6.5.
2. the electroporation genetic transforming method of gibberella fujikuroi as claimed in claim 1 is characterized in that the preparation method of protoplastis of described gibberella fujikuroi bacterium is as follows:
(1) Driselase and Lysing enzyme mass ratio are that 3: 7 mixed enzyme is dissolved in 1M MgSO
4In the solution, making final concentration is the cell wall degrading enzyme liquid of 15mg/ml, and every 10mL enzyme liquid added 0.9～1.1g wet thallus after described enzyme liquid was sterilized after filtration, put 30 ℃ shaking table, rotating speed is 50～60rpm, and the free protoplastis 3～4h of enzymolysis gets the protoplastis enzymolysis solution;
(2) the protoplastis enzymolysis solution is centrifugal, inclining upper strata protoplasma body fluid, removes by filter the mycelia relic, splashes into aseptic deionized water and make MgSO in containing the filtrate of protoplastis
4Concentration be 0.5M, centrifugal protoplastis precipitation.
3. the electroporation genetic transforming method of gibberella fujikuroi as claimed in claim 2 is characterized in that the preparation method of wet thallus of described gibberella fujikuroi bacterium is as follows:
A. on the PDA culture medium flat plate, carry out the gibberella fujikuroi actication of culture, 26～28 ℃ of culture temperature, described PDA substratum final concentration is: potato 200g/L, sucrose 20g/L, agar powder 15g/L; The pH nature was sterilized 18 minutes down at 121 ℃;
B. learn from else's experience activatory gibberella fujikuroi bacterial classification inoculation to the MYG liquid nutrient medium that contains granulated glass sphere, placing temperature is that 26～28 ℃, rotating speed are that the shaking table of 200rpm is cultivated 20h, and described MYG liquid nutrient medium final concentration is: maltose 5g/L; Yeast extract paste 5g/L; Glucose 10g/L; PH6.5;
C. step b gained nutrient solution is carried out enlarged culturing 20h by 10% inoculum size at the MYG liquid nutrient medium;
D. the individual layer filter cloth miracloth of step c gained nutrient solution with sterilization filtered, the mycelium with on the aseptic deionized water flushing filter cloth repeats 2～4 times, removes residual culture, uses 1M MgSO again
4Wash 2～4 times, collect wet thallus.
4. the electroporation genetic transforming method of gibberella fujikuroi as claimed in claim 1 is characterized in that described foreign DNA is the plasmid pAN7-1 that has hygromycin gene, and described microbiotic is a Totomycin.
5. the electroporation genetic transforming method of gibberella fujikuroi as claimed in claim 1 is characterized in that described method carries out as follows:
1) the gibberella fujikuroi protoplastis is suspended with 1M Sorbitol Solution USP A, with the microscopy counting, the protoplastis concentration of adjusting protoplasma body fluid is 10
7/ mL;
2) the exogenous plasmid pAN7-1 that is used to transform is cut into wire through restriction enzyme, is dissolved in aseptic deionized water 5～10 μ g/uL behind the purifying, adds 10 μ g DNA in every 100uL protoplasma body fluid, mixing, and ice bath 30min becomes mixed solution;
3) adding step 2 in the 2cm of the precooling on ice electric shock cup) behind the gained mixed solution 40uL ice bath 5min, prepare electroporation apparatus, setting shock parameters voltage is 850V, electric capacity 25 μ F, and resistance is 250 Ω, shocks by electricity;
4) adding 1mL concentration in the electric shock back electric shock cup is 1M Sorbitol Solution USP B, ice bath 30min～1h, coat the solid regenerated flat board of MYG after the suspension, the 200uL/ ware, behind 28 ℃ of cultivation 12h, cover the soft agar MYG regeneration culture medium that contains Totomycin final concentration 50 μ g/mL on regenerated plate, the 10mL/ flat board is cultured to transformant at 28 ℃ and occurs; Contain 10mM Tris-Cl, 50mM CaCl in the described Sorbitol Solution USP B solution
2, and pH7.5;
5) transformant is preserved behind the stable growth containing succeeding transfer culture on the soft agar MYG regeneration culture medium that the Totomycin final concentration is 100 μ g/mL.
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| CN102352321A (en) * | 2011-10-09 | 2012-02-15 | 江西新瑞丰生化有限公司 | Preparation and regenerating method for protoplast produced by gibberellin |
| CN104388418A (en) * | 2014-11-21 | 2015-03-04 | 郑州大学 | Method for breeding gibberellic acid high-producing strains by performing low-energy ion induced mutation on gibberella |
| CN106244620A (en) | 2016-09-09 | 2016-12-21 | 福州大学 | It is independent of medium and directly converts the method that foreign DNA enters aspergillus niger resting spore |
Citations (3)
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|---|---|---|---|---|
| WO1992017598A1 (en) * | 1991-03-29 | 1992-10-15 | The Board Of Trustees Of The University Of Illinois | Production fo transgenic soybean plants |
| CN1584035A (en) * | 2004-05-22 | 2005-02-23 | 浙江大学 | Expressions of recombined swine lactoferrin and its peptide by pichia |
| CN1718732A (en) * | 2004-07-07 | 2006-01-11 | 新疆农业科学院微生物应用研究所 | A kind of electric method for transformation that shuttle plasmid is imported brevibacterium flavum |
-
2006
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992017598A1 (en) * | 1991-03-29 | 1992-10-15 | The Board Of Trustees Of The University Of Illinois | Production fo transgenic soybean plants |
| CN1584035A (en) * | 2004-05-22 | 2005-02-23 | 浙江大学 | Expressions of recombined swine lactoferrin and its peptide by pichia |
| CN1718732A (en) * | 2004-07-07 | 2006-01-11 | 新疆农业科学院微生物应用研究所 | A kind of electric method for transformation that shuttle plasmid is imported brevibacterium flavum |
Non-Patent Citations (3)
| Title |
|---|
| JP特开平11-56360A 1999.03.02 |
| 朱平 等.赤霉菌原生质体DNA转化.菌物系统19 2.2000,19(2),283-287. |
| 朱平等.赤霉菌原生质体DNA转化.菌物系统19 2.2000,19(2),283-287. * |
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