CN1995362B - Gibberella fujikuroi electroporation genetic transformation method - Google Patents

Abstract

The invention provides a method for genetically transforming Gibberalla fujikuroi with exogenous genes using electroporation technology: using fungal cell wall degrading enzymes to treat the cell walls of Gibberella fujikuroi to obtain protoplasts and purify , the purified protoplasts are mixed with an appropriate amount of exogenous gene/deoxyribonucleic acid (DNA) aqueous solution, and then the mixture is electroporated by electroporation, and the electroporated protoplasts can absorb exogenous DNA, thereby realizing exogenous DNA Genetic transformation of Gibberella. The beneficial effects of the present invention are reflected in: it can be used for molecular biology research of Gibberella Fujikura, such as studying the pathogenic molecular mechanism of the fungus to rice and the growth and development mechanism of the fungus; it can be used to carry out genetic engineering to Gibberella fujikura Thereby, genetic improvement and breeding are carried out to improve the synthesis ability of gibberellin.

Description

A kind of electroporation genetic transformation method of Gibberella fujikura

(1) Technical field

The invention relates to the electroporation genetic transformation technology of Gibberella fujikuroi, which is applicable to the fields of genetic engineering of Gibberella fujikuroi, molecular biology research of Gibberella and the like.

(2) Background technology

Gibberella fujikuroi is a gibberellin-producing fungus, which causes bakanae disease due to excessive production of gibberellin in rice. This disease occurs in all rice-producing areas in my country and poses a serious threat to rice production. Gibberellin is a plant growth regulator, which plays a very important role in plant growth and development, and is widely used in fruits, vegetables, rice and other crops. Gibberellin can also be used in beer production industry, clinical medicine and so on.

Gibberella has become one of the filamentous fungi with good development prospects. At present, many studies are trying to improve strains through genetic engineering. At the same time, research on the pathogenic molecular biology and development of Gibberella to rice has also attracted great attention. However, the research and development of genetic transformation methods is the premise of carrying out genetic engineering and molecular biology research on Gibberella.

The genetic transformation of filamentous fungi currently mainly includes lithium acetate-mediated spore transformation, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation and electroporation transformation. In Gibberella (G.fujikuroi), only the first two transformation methods are currently applied—spore transformation method and PEG-mediated protoplast transformation method, and there is no report on transformation by electroporation. The spore transformation method is limited to Gibberella strains that can produce spores. The PEG-mediated protoplast transformation method is widely used, but the transformation steps are many and the efficiency is not high. Electroporation transformation technology has high efficiency in the genetic transformation of microorganisms, etc., and is simple and easy to implement. It has been widely used in genetic engineering and molecular biology research. World-renowned companies such as Bio-rad and Phamacia have developed electroporation instruments.

The present invention utilizes the electroporation technology for the first time to carry out exogenous gene transformation to Fujikura gibberella (G.fujikuroi), finds suitable transformation parameters and optimizes the combination of parameters, the transformation procedure is simpler than other existing methods, and improves Improve the transformation efficiency, this method can be used for genetic engineering and molecular biology research and application of Gibberella.

(3) Contents of the invention

The object of the present invention is to provide a method for genetic transformation of exogenous genes by electroporation (electroporation) technology, and to find a combination of transformation parameters with higher conversion efficiency, which can be used for gibberella genetic engineering And molecular biology research and other fields to solve the shortcomings of other genetic transformation methods.

The technical solution adopted by the present invention to achieve the purpose of the invention is: utilize fungal cell wall degrading enzymes to process the cell wall of Gibberella, obtain protoplasts and purify, purify protoplasts and appropriate amount of exogenous gene/deoxyribonucleic acid (DNA) aqueous solution mixing, and then using an electroporation apparatus to perform electric shock treatment on the mixture, and the electric shock treated protoplasts can absorb exogenous DNA, thereby realizing the genetic transformation of exogenous DNA to Gibberella.

The electroporation genetic transformation method of described Fujikura Gibberella fujikuroi is carried out as follows:

1) Suspend the protoplasts of Gibberella Fujikura in 1M sorbitol solution A, count them under a microscope, and adjust the protoplast concentration in the protoplast body fluid to 10 ⁷ -10 ⁸ /mL;

2) The exogenous DNA used for transformation is dissolved in sterile deionized water to a concentration of 5-10 μg/ul, and 10-15 μg DNA is added to each 100 ul of protoplast body fluid, mixed evenly, and ice-bathed for 30-40 minutes to form a mixed solution;

3) Add the mixture obtained in step 2) into the pre-cooled electric shock cup, and after 5-10 minutes in ice bath, perform electric shock with an electroporator;

4) After electric shock, add sorbitol solution B with a concentration of 1M to the electric shock cup, ice-bath for 30min-1h, suspend and spread on the MYG solid regeneration medium plate, culture at 28°C for 12h, cover the regeneration plate with the above-mentioned The soft agar MYG regeneration medium with an antibiotic final concentration of 50 μg/mL corresponding to exogenous DNA was cultivated at 28°C until transformants appeared; the sorbitol solution B solution contained 10mM Tris-Cl, 50mM CaCl ₂ , and pH7. 5; The final concentration of the MYG solid regeneration medium is: maltose 5g/L; yeast extract 5g/L; glucose 10g/L; agar powder 15g/L; sucrose 0.5mol/L; pH6.5; the soft agar The final concentration of MYG regeneration medium is: maltose 5g/L; yeast extract 5g/L; glucose 10g/L; agar powder 8g/L; pH6.5.

6) Subculture the subculture on the soft agar MYG regeneration medium containing the antibiotics corresponding to the exogenous DNA at a final concentration of 100 μg/ml, and store after stable growth. The final concentration of the soft agar MYG regeneration medium is: maltose 5g/L; yeast extract 5; glucose 10g/L; agar powder 8g/L; pH6.5.

The preparation method of the protoplast of described Gibberella fujikura is as follows:

(1) The mixed enzyme of Dryslase and Lying enzyme (Sigma) with a mass ratio of 3:7 was dissolved in 1M _MgSO solution to make a cell wall degrading enzyme solution with a final concentration of 15mg/ml, and the enzyme solution was sterilized by filtration Afterwards, add about 0.9-1.1 g of wet bacteria per 10 mL of enzyme solution, place it on a shaker at 30 °C with a rotation speed of 50-60 rpm, and enzymolyze free protoplasts for 3-4 hours to obtain protoplast enzymatic hydrolysis solution;

(2) Centrifuge the protoplast enzymolysis solution, pour out the upper layer protoplast body liquid, filter to remove mycelia fragments, drip sterile deionized water into the filtrate containing protoplasts so that the concentration of MgSO is _0.5M , and centrifuge to obtain protoplasts precipitation.

The preparation method of the wet thalline of described Gibberella fujikura is as follows:

a. Carry out Gibberella Fujikura strain activation on the PDA medium plate, culture temperature 26-28 ℃, described PDA medium final concentration is: potato 200g/L, sucrose 20g/L, agar powder 15g/L; pH Naturally, sterilized at 121°C for 18 minutes;

b. Get the activated Gibberella fujikura strain and inoculate it into the MYG liquid medium containing glass beads, and place it on a shaker with a temperature of 26-28° C. and a rotating speed of 200 rpm for 20 h. The final concentration of the MYG liquid medium is: Maltose 5g/L; yeast extract 5g/L; glucose 10g/L; pH6.5;

c. Carry out expanded culture in MYG liquid culture medium by the inoculum amount of 10% of the culture solution obtained in step b for 20h;

d. filter the culture solution obtained in step c with a sterilized single-layer filter cloth miracloth (Calbiochem), rinse the mycelium on the filter cloth with sterile deionized water, repeat 2 to 4 times, remove the residual culture solution, and then Rinse with 1M MgSO ₄ for 2 to 4 times, and collect the wet cells.

Since most fungi are screened with hygromycin at present, the exogenous DNA described in the present invention is preferably plasmid pAN7-1 with hygromycin resistance gene, and the corresponding antibiotic is hygromycin. Other plasmids with hygromycin resistance genes, such as plasmids pCB1003, pCB1004 (Anne M. Carroll, et al, Fungal Genetics Newsletter 1994, 41:22), etc., are also applicable to the present invention. Any other DNA or plasmid that can transform fungi can also be transformed by the method described in the present invention.

Specifically, the method is carried out as follows:

1) Suspend the protoplasts of Gibberella Fujikura in 1M sorbitol solution A, count them under a microscope, and adjust the protoplast concentration in the protoplast body fluid to 10 ⁷ /mL;

2) The exogenous plasmid pAN7-1 (Punt et al.1987 Gene 56: 117-124) used for transformation was cut into a linear shape by restriction enzymes, purified and dissolved in sterile deionized water at 5-10 μg/uL, every 100uL Add 10 μg DNA to the protoplast body fluid, mix well, and ice-bath for 30 minutes to form a mixture;

3) Add 40uL of the mixed solution obtained in step 2) to the pre-cooled 2cm electric shock cup and bathe in ice for 5 minutes, then prepare the electroporation instrument, set the electric shock parameters as voltage 850V, capacitance 25μF, resistance 250Ω, and perform electric shock;

4) After the electric shock, add 1 mL of 1M sorbitol solution B to the electric shock cup, bathe in ice for 30 min to 1 h, suspend and spread on the MYG solid regeneration plate, 200uL/dish, incubate at 28°C for 12 hours, and cover the regeneration plate with moisture Soft agar MYG regeneration medium with a final concentration of 50 μg/mL of mycin, 10 mL/plate, cultivated at 28 ° C until transformants appear; the sorbitol solution B solution contains 10 mM Tris-Cl, 50 mM CaCl ₂ , and pH 7.5 ;

5) Transformants were subcultured on soft agar MYG regeneration medium containing hygromycin with a final concentration of 100 μg/mL, and stored after stable growth.

The beneficial effect of the present invention is reflected in that it can be used in the molecular biology research of the gibberella fujikura, such as the research on the pathogenic molecular mechanism of the fungus to rice and the growth and development mechanism of the fungus. It can be used to carry out genetic engineering on gibberellin so as to carry out genetic improvement and breeding, and improve the synthesis ability of gibberellin.

(4) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1: Genetic transformation of exogenous hygromycin resistance gene to Gibberella fujikuroi by electroporation

Using the method described in the Summary of the Invention, the exogenous plasmid pAN7-1 containing the hygromycin resistance gene was introduced into Gibberella, and the stably transformed engineered bacteria were obtained through hygromycin screening, and the transformation efficiency was higher than that mediated by lithium acetate. The spore transformation method is simpler than the transformation method mediated by polyethylene glycol PEG. The specific method is as follows:

1. Preparation of Gibberella protoplasts:

1) Activating Gibberella fujikura species on a PDA medium plate at a culture temperature of 26-28°C. The final concentration of PDA medium is: potato, 200g/L; sucrose, 20g/L; agar powder, 15g/L; pH is natural. Sterilize at 121°C for 18 minutes.

2) Take a small piece and inoculate it into MYG liquid medium (containing about 40 glass beads), and place it in a shaker at a temperature of 26-28° C. and a rotation speed of 200 rpm for 20 hours. The final concentration of the MYG liquid medium: maltose 5g/L; yeast extract 5g/L; glucose 10g/L; pH6.5;

3) The culture solution in step 2) is expanded and cultivated in MYG liquid medium for 20 hours according to the inoculum amount of 10%. Final concentration of MYG liquid medium: maltose 5g/L; yeast extract 5g/L; glucose 10g/L; pH6.5;

4) Filter the culture solution in step 3) with a sterilized single-layer filter cloth miracloth (Calbiochem), rinse the mycelium on the filter cloth with sterile deionized water several times, remove the residual culture solution as much as possible, and then use 1M MgSO ₄ Rinse 3 times to collect wet cells.

5) Prepare a cell wall degrading enzyme solution, dissolve the mixed enzyme in 1M MgSO ₄ with a mass ratio of Dryslase: Lying enzyme = 3:7, the final concentration is about 15 mg/ml, and add Gibberella wet bacteria to the enzyme solution after filtering and sterilizing For each 10ml of enzyme solution, add 1 gram of wet bacteria. Place a shaker at 30° C. at a speed of 60 rpm to enzymatically hydrolyze the free protoplasts for 4 hours.

6) The protoplast enzymatic solution was centrifuged at 500rpm for 10min, and the upper protoplast liquid was filtered through a sterilized double-layer filter cloth miracloth to remove mycelium fragments, and sterile deionized water was slowly dripped into the filtrate containing protoplasts to adjust the concentration of MgSO ₄ The concentration was 0.5M, centrifuged at 3500rpm for 10min, and the protoplasts were precipitated.

7) Protoplast precipitation was suspended with 1M sorbitol solution, centrifuged at 3500rpm for 10min, and repeated once.

8) The precipitated protoplasts were suspended in 100 μL of an appropriate amount of 1M sorbitol solution, examined and counted under a microscope, and the concentration of protoplasts was adjusted to about 10 ⁷ /ml for the following transformations.

2. Electroporation to transform protoplasts with exogenous DNA:

1) DNA preparation. The exogenous plasmid pAN7-1 (Punt et al. 1987 Gene56: 117-124) used for transformation was cut into a line by restriction enzymes, purified and dissolved in sterile deionized water at a concentration of 10ug/ul for transformation.

2) Add 10ug DNA to 100ul protoplast body fluid, mix well, and after 30min in ice bath, take 40ul of the mixture and add it to a pre-cooled electric shock cup (2cm). After 5min in ice bath, prepare the electroporator and set the electric shock parameters for electric shock. Voltage, 850V; Capacitance, 25μF; Resistance, 250Ω.

3) Add 1ml of 1M sorbitol solution (containing 10mM Tris-Cl, pH 7.5, 50mMCaCl ₂ ) after electric shock, bathe in ice for 1h, suspend and spread on MYG solid regeneration plate, 200ul/dish. After culturing at 28°C for 12 hours, the regeneration plate was covered with soft agar MYG regeneration medium containing hygromycin (50ug/ml), 10ml/plate. Continue culturing at 28°C to observe the appearance of transformants. The final concentration of the MYG solid regeneration medium is: maltose 5g/L; yeast extract 5g/L; glucose 10g/L; agar powder 15g/L; sucrose 0.5mol/L; Medium: maltose 5g/L; yeast extract 5g/L; glucose 10g/L; agar powder 8g/L; pH6.5.

4) Transformants were subcultured on the above-mentioned MYG solid regeneration medium with increased hygromycin concentration (100ug/ml), and stored after stable growth.

result:

The transformation rate is more than 8 transformants/ug DNA, compared to the lithium acetate-mediated spore transformation method (1-2 transformants/ ug DNA), there is a greater improvement.

Claims (5)

1. an electroporation genetic transformation method of gibberella fujikura, is characterized in that described method is carried out as follows: 1) Suspend the protoplasts of Gibberella Fujikura in 1M sorbitol solution A, count them under a microscope, and adjust the protoplast concentration in the protoplast body fluid to 10 ⁷ -10 ⁸ /mL; 2) The exogenous DNA used for transformation is dissolved in sterile deionized water to a concentration of 5-10 μg/ul, and 10-15 μg DNA is added to each 100 ul of protoplast body fluid, mixed evenly, and ice-bathed for 30-40 minutes to form a mixed solution; 3) Add the mixture obtained in step 2) into the pre-cooled electric shock cup, and after 5-10 minutes in an ice bath, perform electric shock with an electroporator. Electric shock parameters: voltage is 850V, capacitance is 25μF, and resistance is 250Ω; 4) After electric shock, add sorbitol solution B with a concentration of 1M to the electric shock cup, ice-bath for 30min-1h, suspend and spread on the MYG solid regeneration medium plate, culture at 28°C for 12h, cover the regeneration plate with the above-mentioned The soft agar MYG regeneration medium with an antibiotic final concentration of 50 μg/mL corresponding to exogenous DNA was cultivated at 28°C until transformants appeared; the sorbitol solution B solution contained 10mM Tris-Cl, 50mM CaCl ₂ , and pH7. 5; The final concentration of the MYG solid regeneration medium is: maltose 5g/L; yeast extract 5g/L; glucose 10g/L; agar powder 15g/L; sucrose 0.5mol/L; pH6.5; the soft agar The final concentration of MYG regeneration medium is: maltose 5g/L; yeast extract 5g/L; glucose 10g/L; agar powder 8g/L; pH6.5; 5) Transformants are subcultured on soft agar MYG regeneration medium containing the antibiotics corresponding to the exogenous DNA at a final concentration of 100 μg/mL, and stored after stable growth. The final concentration of the soft agar MYG regeneration medium is: maltose 5g/L; yeast extract 5g/L; glucose 10g/L; agar powder 8g/L; pH6.5. 2. the electroporation genetic transformation method of Gibberella Fujikura as claimed in claim 1, is characterized in that the preparation method of the protoplast of described Gibberella Fujikura is as follows: (1) Dissolve the mixed enzyme of Driselase and Lysing enzyme with a mass ratio of 3:7 in 1M MgSO ₄ solution to make a cell wall degrading enzyme solution with a final concentration of 15mg/ml. Add 0.9 to 1.1 g of wet bacteria to the enzyme solution, place it on a shaker at 30°C with a rotation speed of 50 to 60 rpm, and enzymolyze free protoplasts for 3 to 4 hours to obtain a protoplast enzymatic solution; (2) Centrifuge the protoplast enzymolysis solution, pour out the upper layer protoplast body liquid, filter to remove mycelia fragments, drip sterile deionized water into the filtrate containing protoplasts so that the concentration of MgSO is _0.5M , and centrifuge to obtain protoplasts precipitation. 3. the electroporation genetic transformation method of Gibberella Fujikura as claimed in claim 2, is characterized in that the preparation method of the wet thalline of described Gibberella Fujikura is as follows: a. Carry out Gibberella Fujikura strain activation on the PDA medium plate, culture temperature 26～28 ℃, described PDA medium final concentration is: potato 200g/L, sucrose 20g/L, agar powder 15g/L; pH Naturally, sterilized at 121°C for 18 minutes; b. Get the activated Gibberella fujikura strain and inoculate it into the MYG liquid medium containing glass beads, and place it on a shaker with a temperature of 26-28° C. and a rotating speed of 200 rpm for 20 h. The final concentration of the MYG liquid medium is: Maltose 5g/L; yeast extract 5g/L; glucose 10g/L; pH6.5; c. Carry out expanded culture in MYG liquid culture medium by the inoculum amount of 10% of the culture solution obtained in step b for 20h; d. Filter the culture solution obtained in step c with a sterilized single-layer filter cloth miracloth, rinse the mycelium on the filter cloth with sterile deionized water, repeat 2 to 4 times, remove the residual culture solution, and then use 1M MgSO ₄ Rinse 2 to 4 times to collect wet bacteria. 4. the electroporation genetic transformation method of Fujikura gibberella as claimed in claim 1, is characterized in that described exogenous DNA is the plasmid pAN7-1 with hygromycin resistance gene, and described antibiotic is hygromycin Mycin. 5. the electroporation genetic transformation method of gibberella fujikura as claimed in claim 1, is characterized in that described method is carried out as follows: 1) Suspend the protoplasts of Gibberella Fujikura in 1M sorbitol solution A, count them under a microscope, and adjust the protoplast concentration in the protoplast body fluid to 10 ⁷ /mL; 2) The exogenous plasmid pAN7-1 used for transformation was cut into a linear shape by restriction enzymes, and after purification, it was dissolved in sterile deionized water at 5-10 μg/uL, and 10 μg DNA was added to each 100 uL of protoplast body fluid, mixed evenly, and kept in an ice bath for 30 minutes into a mixture; 3) Add 40uL of the mixed solution obtained in step 2) into the 2cm electric shock cup pre-cooled on ice and bathe in ice for 5 minutes, then prepare the electroporation instrument, set the electric shock parameter voltage to 850V, capacitance to 25μF, and resistance to 250Ω, and perform electric shock; 4) After the electric shock, add 1 mL of 1M sorbitol solution B to the electric shock cup, bathe in ice for 30 min to 1 h, suspend and spread on the MYG solid regeneration plate, 200uL/dish, incubate at 28°C for 12 hours, and cover the regeneration plate with moisture Soft agar MYG regeneration medium with a final concentration of 50 μg/mL of mycin, 10 mL/plate, cultivated at 28 ° C until transformants appear; the sorbitol solution B solution contains 10 mM Tris-Cl, 50 mM CaCl ₂ , and pH7. 5; 5) Transformants were subcultured on soft agar MYG regeneration medium containing hygromycin with a final concentration of 100 μg/mL, and stored after stable growth.