CN111172187A - Efficient genetic transformation method for trichoderma viride - Google Patents
Efficient genetic transformation method for trichoderma viride Download PDFInfo
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- HNEGCRMUYSKRRR-UHFFFAOYSA-N Trichodermin Natural products CC(=O)OC1CC2OC3C=C(C)CCC3(C)C1(C)C21CO1 HNEGCRMUYSKRRR-UHFFFAOYSA-N 0.000 description 2
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- YEIGUXGHHKAURB-VAMGGRTRSA-N viridin Chemical compound O=C1C2=C3CCC(=O)C3=CC=C2[C@@]2(C)[C@H](O)[C@H](OC)C(=O)C3=COC1=C23 YEIGUXGHHKAURB-VAMGGRTRSA-N 0.000 description 1
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Abstract
An efficient trichoderma viride genetic transformation method mainly comprises the following steps: A. activating and expanding culture of agrobacterium: the plasmid vector containing the hygromycin resistance gene and the target gene is transferred into T-DNA of competent cells of agrobacterium tumefaciens C58C1 through electric shock transformation; then activating and carrying out expanded culture to obtain agrobacterium liquid; B. preparation of trichoderma viride conidium solution: adding Glucanex muramidase when culturing conidia in liquid; obtaining trichoderma viride conidium solution; C. co-culturing agrobacterium and trichoderma viride; D. preliminary screening and stabilization of transformants: and (3) screening the culture medium by using PDA solid hygromycin, and performing screening culture twice to obtain a stable trichoderma viride transformant. The method can obviously improve the genetic transformation efficiency of trichoderma viride, and is favorable for verifying and analyzing the gene function of trichoderma viride; thereby more quickly and better playing the role of trichoderma viride in preventing and controlling crop diseases and promoting growth.
Description
Technical Field
The invention relates to a genetic transformation method of trichoderma viride.
Background
Trichoderma spp belongs to filamentous fungi, and is also a biological control fungus which is researched most, applied most widely and has the most obvious effect in agriculture at present. Secondary metabolites generated by trichoderma in the metabolic process comprise various antibiotic substances, such as Trichodermin (Trichodermin), Gliotoxin (Gliotoxin), trichoderma viride (Viridin), antibacterial peptide (antibacterial peptide) and the like, and the secondary metabolites have a remarkable antagonistic effect on plant pathogens and have important significance for research and development of disease resistance of plants.
The secondary metabolites of the fungi are respectively regulated and controlled by different gene clusters, the function research is carried out on the genes in the gene cluster of the trichoderma, and the molecular regulation and control mechanism of the secondary metabolites of the trichoderma can be deeply analyzed, so that the development and the application of the secondary metabolites of the trichoderma are promoted.
Genetic transformation is the main technology for realizing gene function research, and the target gene is realized through genetic transformationAnd the function of the gene is verified by knocking out or increasing the gene. Currently, genetic transformation techniques applied to filamentous fungi include electroporation, protoplast-based PEG/CaCl2Transformation, biolistic transformation, agrobacterium-mediated transformation, and the like. Among them, the agrobacterium-mediated genetic transformation technology has been widely used due to its advantages of low cost, simple operation, high transformation efficiency, stable heredity, etc. The agrobacterium-mediated genetic transformation of fungi, the method and principle thereof are as follows: agrobacterium is a gram-negative bacterium in soil, the cells of which have a T-DNA (Transfer DNA, triple helix DNA) in the plasmid vector; inserting a target gene into T-DNA of agrobacterium; after the agrobacterium enters cells of a receptor (filamentous fungi) through infection, the T-DNA of the agrobacterium is shed and inserted into the genome of the receptor; realizing the transfer and integration of exogenous genes (target genes) to the genome of a receptor (filamentous fungi); then, the strain of the transgenic filamentous fungus is obtained by culturing.
In 1998, the agrobacterium-mediated genetic transformation technology was applied to filamentous fungi for the first time and succeeded, and the transformation efficiency was 600 times higher than that of the conventional method. And then, carrying out genetic transformation on the trichoderma by virtue of an agrobacterium-mediated genetic transformation technology in each laboratory, successfully constructing a trichoderma T-DNA random insertion mutant library, and providing an important experimental material for trichoderma gene function research.
However, the transformation efficiency of different Trichoderma species in filamentous fungi varies greatly. Trichoderma viride (Trichoderma virens) has a thick conidium wall, so that agrobacterium is difficult to infect cells entering Trichoderma viride, the genetic transformation efficiency is very low, and the functional verification, development and utilization of important genes of Trichoderma viride (Trichoderma virens) are severely limited. For this reason, development of a high-efficiency genetic transformation method for Trichoderma viride is urgently required.
Disclosure of Invention
The invention aims to provide an efficient trichoderma viride genetic transformation method, which can obviously improve the genetic transformation efficiency of trichoderma viride and is beneficial to verifying and analyzing the gene function of trichoderma viride; thereby more quickly and better playing the role of trichoderma viride in preventing and controlling soil-borne diseases of crops and promoting the growth of the crops and improving the yield of the crops.
The invention aims to realize the technical scheme that the efficient trichoderma viride genetic transformation method comprises the following steps:
A. activating and expanding culture of agrobacterium
The plasmid vector containing hygromycin resistance gene and target gene is transferred into T-DNA of competent cells of agrobacterium tumefaciens C58C1 through electric shock transformation to obtain a positive agrobacterium tumefaciens strain carrying the plasmid vector in the T-DNA; culturing the positive agrobacterium strain for 48-60h at 26-30 ℃ by using an LB solid culture medium; then, selecting a positive agrobacterium strain to an LB liquid culture medium, and carrying out shake culture at 26-30 ℃ for 48-60 h; then, centrifuging at 4000rpm for 5min to collect positive agrobacterium strains; then, performing IM induction culture to resuspend the positive agrobacterium strain, enabling the light absorption value of the suspension to be 0.2-0.3 at the wavelength of 600nm, and performing shaking culture at 28 ℃ for 6h to obtain an agrobacterium liquid after the enlarged culture;
B. preparation of trichoderma viride conidium solution
Activating and culturing Trichoderma viride with PDA plate culture medium at 26-30 deg.C to produce conidia, scraping bacterial surface with sterilized water, and filtering hypha with three layers of lens wiping paper to obtain conidia filtrate; centrifuging the conidium filtrate at 4000-5000rpm for 5-10min, pouring out the supernatant, and collecting conidium; resuspending conidia by using NaCl solution with the concentration of 0.6-0.8M, adding Glucanex muramidase, wherein the addition amount of the Glucanex muramidase is 15-20mg per milliliter of the NaCl solution; then, carrying out shaking culture at 80rpm at 30 ℃ for 2.5-4 h; centrifuging the suspension at 4000rpm, discarding the filtrate to obtain conidium, cleaning the conidium with 0.6-0.8M NaCl solution for 1-2 times, filtering the residue with single layer lens wiping paper to obtain trichoderma viride conidium solution, and adjusting the concentration to 106Per ml;
C. co-culture of Agrobacterium and Trichoderma viride
Mixing the agrobacterium liquid obtained in the step A and the trichoderma viride conidium liquid obtained in the step B in equal volume to obtain a mixed liquid; uniformly coating the mixed solution in a co-culture plate culture medium, and performing dark culture at 20-24 ℃ for 68-76h to obtain a co-culture;
the co-culture plate culture medium is obtained by adding 14-16 g of agar powder into each liter of IM induction culture medium and solidifying;
D. preliminary screening and stabilization of transformants
Adding hygromycin antibiotic with the final concentration of 140-160ug/mL into the PDA solid culture medium to obtain a PDA solid screening culture medium;
taking a PDA solid screening culture medium, covering the PDA solid screening culture medium on the co-culture obtained in the step C, and then culturing at 27-29 ℃ until a trichoderma virens strain grows on the surface of the PDA solid screening culture medium; the grown trichoderma virens strain is a trichoderma virens primary screening transformant with agrobacterium T-DNA inserted into a genome;
selecting trichoderma viride primary screening transformants, inoculating the transformants to another PDA solid screening culture medium, and then culturing at 27-29 ℃ until trichoderma viride strains grow on the surface of the PDA solid screening culture medium; the trichoderma virens strain is a trichoderma virens transformant which grows stably and has agrobacterium T-DNA inserted into a genome.
Compared with the prior art, the invention has the beneficial effects that:
the applicant finds that Glucanex muramidase can carry out enzymolysis on the conidium wall of Trichoderma virens (Trichoderma virens) so that agrobacterium can successfully infect cells entering Trichoderma virens; thereby greatly improving the transformation efficiency of transferring the T-DNA containing the target gene in the agrobacterium plasmid vector into the genome of trichoderma viride. Further being beneficial to knockout of Trichoderma viride genes and construction of random T-DNA insertion mutant libraries, facilitating functional verification, development and utilization of Trichoderma viride (Trichoderma virens) important genes, and promoting development and application of Trichoderma viride secondary metabolites.
Experiments show that the target gene (the candidate gene for synthesizing the collotoxin) is transferred into the genetic transformation operation of trichoderma viride genome:
the method of the invention is used for co-culturing 2x106And (3) screening 22 trichoderma viride transformants positive in resistance detection by hygromycin resistance screening in a PDA solid screening culture medium from trichoderma viride conidia. The identification result of the gene specific primer shows that: these 22 resistance testsAmong the positive transformants, 3 of them were indeed homologous recombinant transformants having the target gene transferred into the target site; 17 ectopic integrative transformants which are not transferred with the target gene at the target position; the genomes of the other 2 transformants were identical to the genome of Trichoderma viride before transformation, and were false positive transformants.
While the 12x10 cultured by the traditional method6Among the trichoderma viride conidia, only 3 trichoderma viride transformants positive in resistance detection were selected by hygromycin resistance screening using PDA solid screening medium. The identification result of the gene specific primer shows that: all 3 ectopic integrative transformants are transferred with target genes at other positions; rather than homologous recombinant transformants having the target gene transferred into the target site.
In a word, the method greatly improves the transformation efficiency of the T-DNA containing the target gene in the agrobacterium plasmid vector to be transferred into the genome of trichoderma viride, and can successfully obtain trichoderma viride transformants with the target gene (homologous recombination) transferred into the target position of the genome only by co-culturing a small amount of trichoderma viride conidia.
Secondly, in the existing trichoderma viride agrobacterium-mediated genetic transformation method, an agrobacterium suspension and a trichoderma viride conidium suspension are coated on a sterilized filter membrane or filter paper, and after co-culture is finished; then the filter membrane or the filter paper is lifted out and cut into strips of 5cm or the whole piece is paved in a hygromycin resistance screening culture medium for screening transformants; the operation is time-consuming and labor-consuming, and the agrobacterium is easy to pollute. The method of the invention directly pours and covers the hygromycin resistance screening culture medium on the co-cultured hyphae, thereby greatly reducing the operation steps and the pollution probability; meanwhile, the co-culture is fully covered by the hygromycin screening culture medium, so that the screening efficiency of positive transformants is obviously improved, and the appearance of false positive transformants is effectively reduced.
Further, the IM induction medium of the invention is prepared by the following method:
preparing MES biological buffer solution with the concentration of 0.9-1.1M, and adjusting the pH value to 5.4-5.6 by NaOH;
600ml of MES biological buffer was added to 400mlWater; then adding 1.5-1.6g of K2HPO4, KH 1.1-1.3g2PO45-7g of MgSO 54·7H2O, 2.5-3.5g NaCl, 9-11mg CaCl2·2H2O, 47-53mg NH4NO34.5-5.5g of glycerol and 4.5-5.5mg of ZnSO4·7H2O, 4.5-5.5mg of CuSO4·5H2O, 4.5-5.5mg of MnSO4·H2O, 4.5-5.5mg of Na2MoO4·7H2O, 4.5-5.5mg of H3BO318-22mg of glucose and 1mg of FeSO4Obtaining mixed liquid;
sterilizing the mixed solution at 121 deg.C under high temperature and high pressure, and adding acetosyringone with final concentration of 200 μ M.
The present invention will be described in further detail with reference to examples.
Detailed Description
Example 1
The invention relates to a high-efficiency trichoderma viride genetic transformation method, which comprises the following steps:
A. activating and expanding culture of agrobacterium
The plasmid vector containing hygromycin resistance gene and target gene is transferred into T-DNA of competent cells of agrobacterium tumefaciens C58C1 through electric shock transformation to obtain a positive agrobacterium tumefaciens strain carrying the plasmid vector in the T-DNA; culturing the positive agrobacterium strain for 48h at 26 ℃ by using an LB solid culture medium; then, picking positive agrobacterium strains to an LB liquid culture medium, and carrying out shake culture for 48h at 26 ℃; then, centrifuging at 4000rpm for 5min to collect positive agrobacterium strains; then, using IM induction culture medium to resuspend the positive agrobacterium strain, leading the light absorption value of the suspension to be 0.2 at the wavelength of 600nm, and then carrying out shaking culture at the temperature of 28 ℃ for 6h to obtain agrobacterium liquid after enlarged culture;
B. preparation of trichoderma viride conidium solution
Activating and culturing Trichoderma viride with PDA plate culture medium at 26 deg.C to produce conidia, scraping bacterial surface with sterilized water, and filtering hypha with three layers of lens wiping paper to obtain conidia filtrate; filtering conidiumCentrifuging at 4000rpm for 10min, pouring out supernatant, and collecting conidium; resuspending conidia by using NaCl solution with the concentration of 0.6M, adding Glucanex muramidase, wherein the addition amount of the Glucanex muramidase is 15mg per milliliter of the NaCl solution; then, shaking and culturing at 30 ℃ and 80rpm for 2.5 h; then, the suspension was centrifuged at 4000rpm, the filtrate was discarded to obtain conidia, the conidia were washed with 0.6M NaCl solution 1 time, and the residue was filtered with single-layer mirror paper to obtain conidia solution of Trichoderma viride, the concentration of which was adjusted to 106Per ml;
C. co-culture of Agrobacterium and Trichoderma viride
Mixing the agrobacterium liquid obtained in the step A and the trichoderma viride conidium liquid obtained in the step B in equal volume to obtain a mixed liquid; uniformly coating the mixed solution in a co-culture plate culture medium, and performing dark culture at 24 ℃ for 68h to obtain a co-culture;
the co-culture plate culture medium is obtained by adding 14 g of agar powder into each liter of IM induction culture medium and solidifying;
D. preliminary screening and stabilization of transformants
Adding hygromycin antibiotic with the final concentration of 140ug/mL into the PDA solid culture medium to obtain a PDA solid screening culture medium;
taking a PDA solid screening culture medium, covering the PDA solid screening culture medium on the co-culture obtained in the step C, and then culturing at 27 ℃ until a trichoderma virens strain grows on the surface of the PDA solid screening culture medium; the grown trichoderma virens strain is a trichoderma virens primary screening transformant with agrobacterium T-DNA inserted into a genome;
selecting and taking out trichoderma viride primary screening transformants, inoculating the trichoderma viride primary screening transformants to another PDA solid screening culture medium, and then, culturing at 27 ℃ until trichoderma viride strains grow on the surface of the PDA solid screening culture medium; the trichoderma virens strain is a trichoderma virens transformant which grows stably and has agrobacterium T-DNA inserted into a genome.
The IM induction culture medium is prepared by the following method:
preparing MES biological buffer solution with the concentration of 0.9M, and adjusting the pH value to 5.4 by NaOH;
adding 6 to 400ml of MES biological buffer00ml of water; 1.5g of K were added2HPO4, 1.2g KH2PO46g of MgSO 24·7H2O, 3g NaCl, 10mg CaCl2·2H2O, 50mg NH4NO35g of glycerol, 5mg of ZnSO4·7H2O, 5mg of CuSO4·5H2O, 5mg of MnSO4·H2O, 5mg of Na2MoO4·7H2O, 5mg of H3BO320mg of glucose, 1mg of FeSO4Obtaining mixed liquid;
sterilizing the mixed solution at 121 deg.C under high temperature and high pressure, and adding acetosyringone with final concentration of 200 μ M.
Example 2
A high-efficiency trichoderma viride genetic transformation method comprises the following steps:
A. activating and expanding culture of agrobacterium
The plasmid vector containing hygromycin resistance gene and target gene is transferred into T-DNA of competent cells of agrobacterium tumefaciens C58C1 through electric shock transformation to obtain a positive agrobacterium tumefaciens strain carrying the plasmid vector in the T-DNA; culturing the positive agrobacterium strain for 60h at 30 ℃ by using an LB solid culture medium; then, selecting a positive agrobacterium strain to an LB liquid culture medium, and carrying out shake culture at 30 ℃ for 60 h; then, centrifuging at 4000rpm for 5min to collect positive agrobacterium strains; then, using IM induction culture medium to resuspend the positive agrobacterium strain, leading the light absorption value of the suspension to be 0.3 at the wavelength of 600nm, and then carrying out shaking culture at the temperature of 28 ℃ for 6h to obtain agrobacterium liquid after enlarged culture;
B. preparation of trichoderma viride conidium solution
Activating and culturing Trichoderma viride with PDA plate culture medium at 30 deg.C to produce conidia, scraping bacterial surface with sterilized water, and filtering hypha with three layers of lens wiping paper to obtain conidia filtrate; centrifuging the conidium filtrate at 5000rpm for 5min, pouring out the supernatant, and collecting conidia; resuspending conidia by using NaCl solution with the concentration of 0.8M, adding Glucanex muramidase, wherein the addition amount of the Glucanex muramidase is 20mg per milliliter of the NaCl solution; then, shaking culture is carried out for 4h at the temperature of 30 ℃ and the rpm of 80;then, the suspension was centrifuged at 4000rpm, the filtrate was discarded to obtain conidia, the conidia were washed with 0.8M NaCl solution 2 times, and the residue was filtered with single-layer mirror paper to obtain conidia solution of Trichoderma viride, the concentration of which was adjusted to 106Per ml;
C. co-culture of Agrobacterium and Trichoderma viride
Mixing the agrobacterium liquid obtained in the step A and the trichoderma viride conidium liquid obtained in the step B in equal volume to obtain a mixed liquid; uniformly coating the mixed solution in a co-culture plate culture medium, and performing dark culture at 20 ℃ for 76h to obtain a co-culture;
the co-culture plate culture medium is obtained by adding 16 g of agar powder into each liter of IM induction culture medium and solidifying;
D. preliminary screening and stabilization of transformants
Adding hygromycin antibiotic with the final concentration of 160ug/mL into the PDA solid culture medium to obtain a PDA solid screening culture medium;
taking a PDA solid screening culture medium, covering the PDA solid screening culture medium on the co-culture obtained in the step C, and then culturing at 29 ℃ until a trichoderma virens strain grows on the surface of the PDA solid screening culture medium; the grown trichoderma virens strain is a trichoderma virens primary screening transformant with agrobacterium T-DNA inserted into a genome;
selecting and taking out trichoderma viride primary screening transformants, inoculating the trichoderma viride primary screening transformants to another PDA solid screening culture medium, and then, culturing at 29 ℃ until trichoderma viride strains grow on the surface of the PDA solid screening culture medium; the trichoderma virens strain is a trichoderma virens transformant which grows stably and has agrobacterium T-DNA inserted into a genome.
The IM induction culture medium is prepared by the following method:
preparing 1.1M MES biological buffer solution, and adjusting the pH value to 5.6 by using NaOH;
adding 600ml of water into 400ml of MES biological buffer; 1.55g of K were added2HPO4, 1.1g KH2PO45g of MgSO 54·7H2O, 2.5g NaCl, 9mg CaCl2·2H2O, 47mg NH4NO34.5g of glycerol, 4.5mg of ZnSO4·7H2O, 4.5mg of CuSO4·5H2O, 4.5mg of MnSO4·H2O, 4.5mg of Na2MoO4·7H2O, 4.5mg of H3BO318mg of glucose, 1mg of FeSO4Obtaining mixed liquid;
sterilizing the mixed solution at 121 deg.C under high temperature and high pressure, and adding acetosyringone with final concentration of 200 μ M.
Example 3
A high-efficiency trichoderma viride genetic transformation method comprises the following steps:
A. activating and expanding culture of agrobacterium
The plasmid vector containing hygromycin resistance gene and target gene is transferred into T-DNA of competent cells of agrobacterium tumefaciens C58C1 through electric shock transformation to obtain a positive agrobacterium tumefaciens strain carrying the plasmid vector in the T-DNA; culturing the positive agrobacterium strain for 54h at 28 ℃ by using an LB solid culture medium; then, selecting a positive agrobacterium strain to an LB liquid culture medium, and carrying out shaking culture at 28 ℃ for 54 h; then, centrifuging at 4000rpm for 5min to collect positive agrobacterium strains; then, using IM induction culture medium to resuspend the positive agrobacterium strain, leading the light absorption value of the suspension to be 0.25 at the wavelength of 600nm, and then carrying out shaking culture at the temperature of 28 ℃ for 6h to obtain agrobacterium liquid after enlarged culture;
B. preparation of trichoderma viride conidium solution
Activating and culturing Trichoderma viride with PDA plate culture medium at 268 deg.C to produce conidia, scraping bacterial surface with sterilized water, and filtering hypha with three layers of lens wiping paper to obtain conidia filtrate; centrifuging the conidium filtrate at 4500rpm for 7min, pouring out the supernatant, and collecting conidium; resuspending conidia by using NaCl solution with the concentration of 0.7M, adding Glucanex muramidase, wherein the addition amount of the Glucanex muramidase is 18mg per milliliter of the NaCl solution; then, shaking culture is carried out for 3h at the temperature of 30 ℃ and the rpm of 80; then, the suspension was centrifuged at 4000rpm, the filtrate was discarded to obtain conidia, the conidia were washed with 0.7M NaCl solution 1 time, and the residue was filtered with single-layer mirror paper to obtain conidia solution of Trichoderma viride, the concentration of which was adjusted to 106Per ml;
C. co-culture of Agrobacterium and Trichoderma viride
Mixing the agrobacterium liquid obtained in the step A and the trichoderma viride conidium liquid obtained in the step B in equal volume to obtain a mixed liquid; uniformly coating the mixed solution in a co-culture plate culture medium, and performing dark culture at 22 ℃ for 72h to obtain a co-culture;
the co-culture plate culture medium is obtained by adding 15 g of agar powder into each liter of IM induction culture medium and solidifying;
D. preliminary screening and stabilization of transformants
Adding hygromycin antibiotic with the final concentration of 150ug/mL into the PDA solid culture medium to obtain a PDA solid screening culture medium;
taking a PDA solid screening culture medium, covering the PDA solid screening culture medium on the co-culture obtained in the step C, and then culturing at 28 ℃ until a trichoderma virens strain grows on the surface of the PDA solid screening culture medium; the grown trichoderma virens strain is a trichoderma virens primary screening transformant with agrobacterium T-DNA inserted into a genome;
selecting and taking out trichoderma viride primary screening transformants, inoculating the trichoderma viride primary screening transformants to another PDA solid screening culture medium, and then, culturing at 28 ℃ until trichoderma viride strains grow on the surface of the PDA solid screening culture medium; the trichoderma virens strain is a trichoderma virens transformant which grows stably and has agrobacterium T-DNA inserted into a genome.
The IM induction medium is prepared by the following method:
preparing MES biological buffer solution with the concentration of 1.0M, and adjusting the pH value to 5.5 by NaOH;
adding 600ml of water into 400ml of MES biological buffer; 1.6g of K were added2HPO4, 1.3g KH2PO47g of MgSO 24·7H2O, 3.5g NaCl, 11mg CaCl2·2H2O, 53mg NH4NO35.5g of glycerol, 5.5mg of ZnSO4·7H2O, 5.5mg of CuSO4·5H2O, 5.5mg of MnSO4·H2O, 5.5mg of Na2MoO4·7H2O, 5.5mg of H3BO322mg of glucose, 1mg ofFeSO4Obtaining mixed liquid;
sterilizing the mixed solution at 121 deg.C under high temperature and high pressure, and adding acetosyringone with final concentration of 200 μ M.
The following is a description of the experimental results of the method of the present invention applied to transfer of the candidate gene for synthesizing gliotoxin into the genome of Trichoderma viride as a target gene.
The experimental results are as follows:
designing primers according to a sequence of a candidate gene (target gene) synthesized by the collotoxin, respectively amplifying by using ultra-fidelity DNA polymerase to obtain fragments of 1016bp at the 5 'end and 892bp at the 3' end of the target gene, using the fragments as a left homologous arm (LF) and a right homologous arm (RF) of gene transfer, assembling the LF and the RF to two sides of a hygromycin resistance gene (hyp) of a modified pGKO2-hyp plasmid vector, and obtaining the plasmid vector containing the hygromycin resistance gene and the target gene.
A plasmid vector containing a hygromycin resistance gene and a target gene and Trichoderma viride T23 were subjected to genetic transformation experiments according to the method of the present invention. The experimental results are as follows:
in co-culture of 2X106And (3) screening 22 trichoderma viride transformants positive in resistance detection by hygromycin resistance screening in a PDA solid screening culture medium from trichoderma viride conidia. The identification result of the gene specific primer shows that: of the 22 transformants positive for the resistance detection, 3 were indeed homologous recombinant transformants having the target gene transferred into the target site; 17 ectopic integrative transformants which are not transferred with the target gene at the target position; the genomes of the other 2 transformants were identical to the genome of Trichoderma viride before transformation, and were false positive transformants.
And 12X10 in the co-culture by using the existing agrobacterium-mediated genetic transformation method6Among the trichoderma viride T23 conidia, only 3 trichoderma viride transformants positive for resistance detection were selected by hygromycin resistance selection using PDA solid selection medium. The identification result of the gene specific primer shows that: all 3 ectopic integrative transformants are transferred with target genes at other positions; rather than homologous recombinant transformants having the target gene transferred into the target site.
In a word, the transformation efficiency of the existing agrobacterium-mediated genetic transformation method in trichoderma viride T23 is extremely low, only a few transformants can be obtained in 6 times of the trichoderma viride T23 conidia co-cultured by the method, the molecular identification results are all ectopic integration insertion, and the target gene transfer mutant of homologous recombination is not identified. The method of the invention only uses one sixth of the prior method to co-culture trichoderma viride conidia to obtain 22 transformants, and the molecular identification result shows that 3 homologous recombination target genes are transferred into mutants. The method greatly improves the transformation efficiency of the trichoderma viride gene. The gene function of trichoderma viride can be verified and analyzed; thereby more quickly and better playing the role of trichoderma viride in preventing and controlling soil-borne diseases of crops and promoting the growth of the crops and improving the yield of the crops.
Claims (2)
1. A high-efficiency trichoderma viride genetic transformation method comprises the following steps:
A. activating and expanding culture of agrobacterium
The plasmid vector containing hygromycin resistance gene and target gene is transferred into T-DNA of competent cells of agrobacterium tumefaciens C58C1 through electric shock transformation to obtain a positive agrobacterium tumefaciens strain carrying the plasmid vector in the T-DNA; culturing the positive agrobacterium strain for 48-60h at 26-30 ℃ by using an LB solid culture medium; then, selecting a positive agrobacterium strain to an LB liquid culture medium, and carrying out shake culture at 26-30 ℃ for 48-60 h; then, centrifuging at 4000rpm for 5min to collect positive agrobacterium strains; then, performing IM induction culture to resuspend the positive agrobacterium strain, enabling the light absorption value of the suspension to be 0.2-0.3 at the wavelength of 600nm, and performing shaking culture at 28 ℃ for 6h to obtain an agrobacterium liquid after the enlarged culture;
B. preparation of trichoderma viride conidium solution
Activating and culturing Trichoderma viride with PDA plate culture medium at 26-30 deg.C to produce conidia, scraping bacterial surface with sterilized water, and filtering hypha with three layers of lens wiping paper to obtain conidia filtrate; centrifuging the conidium filtrate at 4000-5000rpm for 5-10min, pouring out the supernatant, and collecting conidium; then resuspending conidia by using NaCl solution with the concentration of 0.6-0.8M,adding glucarex muramidase, wherein the addition amount of the glucarex muramidase is 15-20mg per ml of NaCl solution; then, carrying out shaking culture at 80rpm at 30 ℃ for 2.5-4 h; centrifuging the suspension at 4000rpm, discarding the filtrate to obtain conidium, cleaning the conidium with 0.6-0.8M NaCl solution for 1-2 times, filtering the residue with single layer lens wiping paper to obtain trichoderma viride conidium solution, and adjusting the concentration to 106Per ml;
C. co-culture of Agrobacterium and Trichoderma viride
Mixing the agrobacterium liquid obtained in the step A and the trichoderma viride conidium liquid obtained in the step B in equal volume to obtain a mixed liquid; uniformly coating the mixed solution in a co-culture plate culture medium, and performing dark culture at 20-24 ℃ for 68-76h to obtain a co-culture;
the co-culture plate culture medium is obtained by adding 14-16 g of agar powder into each liter of IM induction culture medium and solidifying;
D. preliminary screening and stabilization of transformants
Adding hygromycin antibiotic with the final concentration of 140-160ug/mL into the PDA solid culture medium to obtain a PDA solid screening culture medium;
taking a PDA solid screening culture medium, covering the PDA solid screening culture medium on the co-culture obtained in the step C, and then culturing at 27-29 ℃ until a trichoderma virens strain grows on the surface of the PDA solid screening culture medium; the grown trichoderma virens strain is a trichoderma virens primary screening transformant with agrobacterium T-DNA inserted into a genome;
selecting trichoderma viride primary screening transformants, inoculating the transformants to another PDA solid screening culture medium, and then culturing at 27-29 ℃ until trichoderma viride strains grow on the surface of the PDA solid screening culture medium; the trichoderma virens strain is a trichoderma virens transformant which grows stably and has agrobacterium T-DNA inserted into a genome.
2. The method of claim 1, wherein the IM induction medium is formulated by the following method:
preparing MES biological buffer solution with the concentration of 0.9-1.1M, and adjusting the pH value to 5.4-5.6 by NaOH;
adding 600ml of water into 400ml of MES biological buffer; then adding 1.5-1.6g of K2HPO4, KH 1.1-1.3g2PO45-7g of MgSO 54·7H2O, 2.5-3.5g NaCl, 9-11mg CaCl2·2H2O, 47-53mg NH4NO34.5-5.5g of glycerol and 4.5-5.5mg of ZnSO4·7H2O, 4.5-5.5mg of CuSO4·5H2O, 4.5-5.5mg of MnSO4·H2O, 4.5-5.5mg of Na2MoO4·7H2O, 4.5-5.5mg of H3BO318-22mg of glucose and 1mg of FeSO4Obtaining mixed liquid;
sterilizing the mixed solution at 121 deg.C under high temperature and high pressure, and adding acetosyringone with final concentration of 200 μ M.
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