CN119242450A - A method for mining new functional genes of asexually reproducing filamentous fungi and its application - Google Patents

Abstract

The present invention discloses a method for mining new functional genes of asexually propagated filamentous fungi and its application, which relates to the field of applied microbial technology, with a deposit number of CGMCC No.12166; deposit unit: China General Microbiological Culture Collection Management Center; deposit date: April 11, 2016. The present invention discloses a method for mining new functional genes of asexually propagated filamentous fungi: S1, constructing a Trichoderma strain A that is defective in urea synthesis but has hygromycin resistance; S2, obtaining a random mutant strain B; S3, constructing a temporary pseudo-"diploid" by a protoplast fusion method; S4, performing random complementation of random mutant chromosome genes through homologous recombination of two sets of chromosomes in the nucleus, and obtaining phenotype complemented mutants through phenotypic change screening; S5, using resequencing technology to determine the key mutation sites of complementation and reversely verifying the gene function through a gene editing method. The method of the present invention provides a new method for studying the functional genes of asexually propagated filamentous fungi.

Description

Method for excavating novel functional genes of asexual propagation filamentous fungi and application thereof

Technical Field

The invention relates to the technical field of applied microorganisms, in particular to a method for excavating a novel functional gene of asexual propagation filamentous fungi and application thereof.

Background

Fungi include the three major classes of yeasts, molds and mushrooms (macrofungi), which belong to different subgenera of the fungi kingdom, molds being the generic name for filamentous fungi. In recent years, with the continuous improvement of sequencing technology, whole genome sequencing of filamentous fungi has been very convenient, research on gene functions has become a hotspot in research in the field of fungi, but genome annotation is obtained based on sequence comparison, and the premise of the comparison is that known functional genes need to be in a database, and in recent decades, although gene knockout, RNA interference, overexpression and yeast two-hybrid technology have been successfully used for research on gene functions of filamentous fungi, most of the research is research on reverse genetics depending on genes with known functions, originality is not strong, and most of the gene functions in the filamentous fungi and the function coupling effect of the gene functions with other genes (namely by multi-gene knockout) are not clear. Therefore, from the beginning of phenotype, the development of new functional genes is one of the most original works in the field of research on filamentous fungi, however, most of the propagation of filamentous fungi is performed by means of asexual spores (node spores, chlamydospores, sporangiospores and conidia) formed by the differentiation of mycelium, and it is difficult to find new functional genes by hybridization, backcrossing, gene function complementation, etc. based on biological phenotype as in the case of sexual propagation organisms, and to resolve the functional coupling relationship with other genes, which becomes the bottleneck of molecular biology research on filamentous fungi worldwide.

Disclosure of Invention

The invention aims to solve the problems and provide a method for excavating novel functional genes of asexual propagation filamentous fungi and application thereof.

In order to achieve the purpose, the technical scheme of the invention is that the asexual propagation type filamentous fungus is a trichoderma wild strain, the preservation number of the trichoderma wild strain is CGMCC No.12166, the preservation unit is China general microbiological culture Collection center, and the preservation date is 2016, 4 and 11.

The invention further discloses a method for excavating a novel functional gene of the asexual propagation type filamentous fungi, which comprises the following steps:

S1, constructing a trichoderma strain A which is defective in uridine synthesis but has hygromycin resistance;

s2, obtaining a random mutant B, and screening a random mutant B with acid resistance by irradiating a wild strain with X rays;

S3, preparing and fusing protoplasts, and constructing temporary pseudo-diploids by a protoplast fusion method, wherein two sets of chromosomes of the temporary pseudo-diploids are respectively from a trichoderma strain A and a random mutant strain B;

S4, screening and phenotype verification, carrying out random complementation on chromosome genes of random mutants through homologous recombination by two sets of chromosomes in the nucleus, and obtaining phenotype complementation mutants through phenotype change screening;

s5, re-sequencing and gene editing verification, determining the complementary key mutation sites by using a re-sequencing technology, and reversely verifying the gene function by using a gene editing method.

Preferably, in step S1, the trichoderma strain a is constructed by performing gene knockout with hygromycin as a biomarker for the key gene ura3 (A1 a 105144) for trichoderma uridine synthesis.

Preferably, in the step S2, the random mutant B is obtained by taking 200 μl of spore suspension with a concentration of 1×10 ⁷ in a 96-well plate, irradiating with X-ray, diluting the irradiated spore suspension, and spreading on a screening medium under required conditions, and screening to obtain mutant B with a remarkably changed phenotype.

Preferably, the total dose of the X-ray irradiation is 250Gy, and the total irradiation is performed three times, and the three doses are 84Gy, 84Gy and 82Gy respectively.

Preferably, the protoplast fusion process and verification method comprises preparing protoplasts from Trichoderma strain A and random mutant strain B, performing fusion by PEG-mediated method, screening regenerated transformant with hygromycin-containing oligotrophic medium, performing monospore separation, removing heterokaryon transformant, retaining temporary quasi-diploid, and verifying with specific primers.

Preferably, the protoplast fusion method is PEG-mediated protoplast fusion with gradually reduced concentration, and the specific process comprises the steps of 5 minutes at room temperature in 9.5 percent PEG+Ca ²⁺ hypertonic solution, 5 minutes on ice in 7 percent PEG+Ca ²⁺ hypertonic solution, and 20 minutes on ice in 5 percent PEG+Ca ^{2 +} hypertonic solution.

Preferably, the specific primers comprise E-U3-F, E-hygb-R, E-U3-F, E-U3-R.

Preferably, a basal salt glucose medium containing hygromycin B is used as a temporary pseudo "diploid" selective medium.

Preferably, in step S4, a basal salt glucose medium is used to screen strains in which the functional gene of interest is complemented.

Preferably, in the step S5, the specific process of determining the complementary key mutation site by using the resequencing technology is that the mutation sites of the complementary strain C are compared with the mutation sites of the mutant strain B after the mutation C and the initial mutant strain B, the phenotype of which is changed after the last complementary is cultured on a PDA, are extracted to perform mapping analysis, and the mutation sites of the mutant strain C and the initial mutant strain B are different from each other, namely the mutation sites of the mutant strain C and the initial mutant strain B are the range of the key gene.

Compared with the prior art, the beneficial effect of this scheme:

1) According to the invention, the proper dosage of random mutation of the trichoderma reesei of the asexual propagation type filamentous fungus is found for the first time through research, and the optimal culture medium is utilized to screen the mutant and the resequencing technology is combined to realize the detection of the mutation site while the mortality rate of spores of the asexual propagation type filamentous fungus reaches 99.9% by X-rays, so that an experimental model from phenotype to functional genes is established.

2) The invention realizes the screening of fusion strains by constructing a strain A with defective uridine synthesis but hygromycin resistance and a random mutant B with uridine synthesis capacity and screening protoplast fusion strains by utilizing the characteristic of functional complementation.

3) The invention utilizes the characteristic of temporary pseudo-diploid strain instability to lead to automatic separation of chromosomes for the first time, thereby realizing recovery after random complementation of the chromosomes.

4) The invention uses the characteristic of chromosome random anaplerotic to intensively study the initial mutant strain and the last phenotype anaplerotic strain, and finds out the target gene by comparing the mutation sites of the initial mutant strain and the last phenotype anaplerotic strain and further verifying by using a gene editing technology.

Drawings

FIG. 1 is a schematic diagram of a gene ura3 knockout and validation strategy in an embodiment of the invention;

FIG. 2 is a schematic diagram of a single-core multi-chromosome validation strategy and PCR validation results in an embodiment of the present invention;

FIG. 3 is a schematic diagram of a protoplast fusion process and a separation strategy according to an embodiment of the invention;

FIG. 4 is a schematic representation of phenotype and re-sequencing results of mutant and complementing strains in an example of the invention;

FIG. 5 is a graph showing the comparison of phenotypes of the complementation strain and the dominant mutant strain after editing and verifying the differential mutation sites in the examples of the present invention.

Detailed Description

In order that those skilled in the art will better understand the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, wherein it is to be understood that the illustrated embodiments are merely exemplary of some, but not all, of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.

Examples:

The asexual propagation type filamentous fungi is a trichoderma wild strain, the preservation number of the trichoderma wild strain is CGMCC No.12166, the preservation unit is China general microbiological culture Collection center, and the preservation date is 2016, 4, 11.

The acid response novel functional gene mining method of the asexual propagation filamentous fungi comprises the following steps:

S1, constructing a trichoderma strain A which is defective in uridine synthesis but has hygromycin resistance;

In this example, the specific construction procedure for trichoderma strain a (tool strain a) was as follows:

s101, preparing transformation fragments

Four pairs of primers were designed for the gene ura3 (A1A 105144), wherein the first pair of primers U3-up-F (CTGTCAGGCAATTAGCACAGG) and U3-up-R (CGAGCCTACCTAGTAC ACACAATCGGACTAACAAGCGCCATCAATGCC) amplified a 1500bp length fragment upstream of the ura3 gene from the Trichoderma genome, the second pair of primers U3-do-F (GATAACCATCGCATG CAAGGTCGATTCCCATTCTTTCGGTGCAGCTCTTCAG) and U3-do-R (GTCTCTGAT CGTCCTACCAATCTGG) amplified a 1500bp length fragment downstream of the ura3 gene from the Trichoderma genome, and the third pair of primers HygB-F (CGATTGTGTGTACTAGGTAGGCTCG) and Hyg B-R (GGAATCGACCTTGCATGCGATG) amplified an open reading frame for the hygromycin B phosphotransferase gene from the pcDNA1 plasmid, which was hygromycin resistance expression cassette. The fourth pair of primers E-U3-F (CGGGACTGTTGGAGTAGGTTC) and E-HygB-R (GGTCGTTTCTGACACAACGG TG) are homologous recombination verification primers. The three fragments of the upstream of the ura3 gene, the hygromycin B resistance expression frame and the downstream of the ura3 gene are designed by primers so that two adjacent fragments have a base complementary overlapping region of about 25bp, wherein the upstream end is in complementary overlapping with the hygromycin initial end, the hygromycin end is in complementary overlapping with the downstream initial end, and the ura3 gene knockout fragment of the upstream fragment of the ura3 plus the hygromycin resistance expression frame plus the downstream fragment of the ura3 can be obtained by overlap extension PCR.

S102, transforming and knocking out uridine synthesis gene U3

After the wild trichoderma is sporulated on a PDA culture medium, the trichoderma is diluted to 10 ⁷ and uniformly coated on the PDA culture medium covered with the ibuprofen for culturing for 16 hours, after the spores are just germinated, the thalli are placed in a hypertonic solution containing trichoderma cell wall lyase to prepare protoplasts, the ura3 gene knockout fragment is introduced into the trichoderma wild type protoplasts, the protoplasts are subjected to rich culture on a 1M sucrose-containing PDA culture medium, after 20 hours, the lower sucrose PDA culture medium is covered by a hygromycin B-containing PDA upper layer culture medium, after 48 hours, strains capable of growing on the upper layer are picked up and inoculated on a hygromycin B-containing PDA plate for culturing. After 48 hours of growth of the transformed strain, mycelia of the transformed strain were picked and subjected to PCR verification of homologous recombination after high temperature lysis with a lysate. And (3) continuously culturing the homologous recombination transformant strain, and carrying out single spore purification on a PDA plate containing hygromycin B and uridine after the transformant strain is subjected to light spore production to obtain the homozygous ura3 homologous recombination gene knockout transformant.

S2, obtaining a random mutant B, and screening a random mutant B with acid resistance by irradiating a wild strain with X-rays.

In this example, the specific screening procedure for acid-tolerant random mutant B was as follows:

S201X-ray irradiation of spores

NJAU4742 on PDA, eluting with sterile water to obtain spore solution, diluting spore solution to 1×10 ⁷ pieces/mL, mixing 100 μl spore suspension with concentration of 1×10 ⁷ and acetic acid-sodium acetate buffer solution with pH of 5.0 at ratio of 1 to 1, and irradiating with X-ray (total dose 250Gy, three times of irradiation, 84Gy and 82 Gy) in 96-well plate at intervals of 10 min.

S202, screening acid-resistant mutants

Diluting the irradiated mutant sporozoites to different concentrations, coating the same amount on a corresponding screening culture medium to find the optimal concentration for screening, and screening to obtain mutant B with obviously changed phenotype. The mutant strain has various growth indexes obviously superior to that of wild type under the condition of screening, the growth speed of the acid-resistant mutant strain screened by the invention on a culture medium with pH of 2.2 is obviously superior to that of the wild type, the growth diameter is 2-3cm larger than that of the wild type, and the thickness of hypha is not reduced.

S3, preparing and fusing protoplasts, constructing temporary quasi-diploid by a protoplast fusion method, wherein two sets of chromosomes of the temporary quasi-diploid are respectively from a trichoderma strain A and a random mutant strain B.

In this example, the specific procedure for protoplast preparation and fusion is as follows:

S301, protoplast preparation

The spore liquid of the template strain A and the spore liquid of the mutant strain B with the concentration of 1X 10 ⁷/mL are taken, the spore liquid of the template strain A is evenly coated on a PDA culture medium covered with the ibuprofen and containing uridine for culturing for 19 hours, and the spore liquid of the mutant strain B is evenly coated on the PDA culture medium covered with the ibuprofen and cultured for 16 hours. After two strains of spores are germinated, the spores are placed in a hypertonic solution containing trichoderma cell wall lysate, and the protoplasts are prepared by pyrolysis at 28 ℃ and 100 rpm. Filtering the lysate with sterile four-layer gauze, preserving the filtrate, centrifuging at 2000rpm in a centrifuge for 10min, keeping 2-3mL supernatant, centrifuging again under the same condition, and discarding the supernatant to obtain protoplast solid.

S301 fusion of protoplasts

The protoplast fusion mutants were screened using a decreasing concentration of PEG mediated method and double pressure. Protoplast of a single strain is prepared (see patent number 201810031220.9 for a specific method), then protoplast fusion is induced by PEG with gradually reduced concentration, the specific process is that 9.5% PEG+CaCl2 hypertonic solution is firstly used as a screening medium at room temperature (25 ℃) for 5 minutes, then 7% PEG+CaCl2 hypertonic solution is used as 5 minutes on ice, then 5% PEG+CaCl2 hypertonic solution is used as 5 minutes on ice, finally 25% PEG and CaCl 2 hypertonic solution are added to a certain volume according to the proportion of 1 to 4, and then the mixture is subjected to rich culture on a PDA plate with 1M sucrose, and after 16 hours, an upper layer is covered with a basic salt glucose culture medium containing hygromycin B as a screening medium, and an upper layer transformant is selected and inoculated on the PDA plate containing hygromycin B. After 48 hours of growth, the mycelium of the transformed strain was picked and subjected to PCR verification of homologous recombination after high temperature lysis with lysis solution. The two pairs of verification primers, the first pair of primers E-U3-F (CGGGACTGTTGGAGTAGGTTC) and E-HygB-R (GGTCGTTTCTGACACAACGGTG), were used to verify the chromosomal genotype of the tool strain A, and the second pair of primers E-U3-F (CGGGACTGTTGGAGTAGGTTC) and E-U3-R (CATCCAATGCAATGCATGCGAG) were used to verify the chromosomal genotype of the mutant strain B. When only one of the corresponding size bands can be amplified by PCR using two pairs of primers, it is presumed that the hygromycin B resistance gene expression cassette is randomly inserted into the chromosome of the strain B or the ura3 open reading frame is randomly inserted into the chromosome of the strain A, and when both pairs of primers can amplify the corresponding size bands by PCR, we consider that the mutant has the following conditions that 1. The protoplast of the mutant contains only a single nucleus and the cell nucleus contains chromosomes of various genotypes and comes from the strain A and the strain B, respectively, 2. The protoplast of the mutant contains a multinuclear and the cell nucleus comes from the strain A and the strain B, respectively, 3. The mutant has the coexistence of the single nucleus and the multinuclear. the protoplast fusion efficiency of the selected mutants on the screening medium was about 60% by PCR verification. After the hyphae of the fusion strain are lysed, the corresponding size bands can be amplified by PCR with both pairs of primers, which are sporulated and monosporated on a hygromycin B-containing PDA. Since Trichoderma spores belong to conidia, the spores of which contain only one nucleus, the offspring of fusion strains of heterokaryons are theoretically single resistant strains, they cannot grow on double selective media, whereas diploid fusion strains theoretically exist with diploid offspring. The data on single spores on plates also support the conclusion that when mutants were post-sporulation diluted to 107 spores/mL and 30 microliters were blotted onto PDA plates containing hygromycin B, the number of single spores was only 4-5, indicating that most spores of the fusion strain were only one resistant. The mutant obtained by monosporation can be considered as a fusion strain of single-core multichromosomes, which is then allowed to continue to grow spores on hygromycin B-containing PDA, in order to allow the chromosomes of the acid-tolerant mutant strain B to recombine as homogeneously as possible with the chromosomes of strain a during polyploid phase and thus to restore the disrupted genes. The second single spore still requires 10 ⁷ spores per mL to reach 4-5 strains on PDA plates containing hygromycin B, which indicates that polyploid trichoderma fusion strains are unstable and can be differentiated into haploids in the hypha growth process. In order to obtain haploid strain of homozygous strain B after multiple passages of polyploid state and homologous recombination, sucking 4 x 102 spores/mL 30 microliter spore liquid after spore production of fusion strain, coating on basic salt glucose culture medium, wherein the number of monospore is 6-7, and the result accords with the probability that only one half of polyploid is strain B after differentiation, and the experimental data partially prove that the polyploid growth condition is deduced before. By homologous recombination of polyploids we obtained a complementing haploid strain C with a lower growth capacity under acidic conditions than strain B and wild type, which suggests that some favorable mutations during the complementing process are successfully complemented and some mutations that are unfavorable for growth under acidic conditions are still retained. This result demonstrates the feasibility of protoplast fusion of Trichoderma strain NJAU-4742, and at the same time, since random mutation sites are often more than 100 sites after irradiation, the screening cannot be performed by single gene complementation, but large-scale gene complementation after protoplast fusion provides a feasible method for screening Trichoderma specific genes, namely, firstly randomly mutating and screening mutant strains with obvious phenotype, then carrying out protoplast fusion complementation genes with strain A, and finally carrying out re-sequencing screening related genes after the obviously phenotypically complemented strains are complemented.

S4, screening and phenotype verification, wherein random mutant chromosome genes are randomly complemented through homologous recombination by two sets of chromosomes in the nucleus, and phenotype-complemented mutants are obtained through phenotype change screening.

In this example, the screening method of the functional anaplerotic strain is as follows:

The transient pseudo-diploid strain is subjected to a plurality of single spore separations (increasing the probability of homologous recombination, i.e. increasing the probability of the functional gene being complemented) on the hygromycin-containing oligotrophic medium, each single spore separation requiring PCR primer verification (the resistance gene may be integrated into the mutant or template strain genome by random insertion and homologous recombination, thereby generating a heterozygote that needs to be removed), and finally the phenotype-restored transformant is selected on the oligotrophic medium of the corresponding selection condition (compared with WT), at which point the transient pseudo-diploid strain is subjected to chromosome separation, and the selected transformant is the strain C with the target functional gene being complemented, since the strain providing the complementing template is U3 defective and cannot grow on the oligotrophic medium.

S5, re-sequencing and gene editing verification, determining the complementary key mutation sites by using a re-sequencing technology, and reversely verifying the gene function by using a gene editing method.

In this embodiment, the resequencing and gene editing verification specifically includes the following steps:

s501 resequencing analysis of functional anaplerotic Strain

And (3) culturing the mutant C with the phenotype changed after the last compensation and the initial mutant B on a PDA, extracting hypha for mapping analysis, comparing the mutation sites of the mutant C with the mutation sites of the initial mutant B after data analysis, wherein the mutation sites of the compensation strain C are smaller than the mutation sites of the mutant B, and the mutation sites which are different from each other are the range of the key genes.

S502, functional gene knockout verification

And C and B mutation sites are compared, the range of key functional genes is found, then B is taken as a starting strain, and genes in the range are singly complemented by a PEG (polyethylene glycol) -mediated method. By comparing phenotypes under the corresponding screening conditions, a key gene was found that brings the B phenotype closer to C. After finding the gene, knocking out the gene by taking the wild type as an original strain, and verifying that if the phenotype is close to B after the wild type knocks out the gene, the gene is the key gene.

15 Different mutation sites exist in the anaplerotic strain and the dominant mutant strain, and editing verification is carried out on the 15 mutation sites. The phenotype was verified by knockout of the genes involved in these 15 mutation sites by homologous recombination using hygromycin as a biomarker. Four pairs of primers were designed for gene asr (A1A 106651), wherein the first pair of primers asr1-up-F (CTACCGGCCTAGAGGC AATTTC) and asr1-up-R (GGTAGCTCTCATCGATTGGTCGCGGGCTAGTTATGCGTA TAGCAG) amplified a fragment of 1500bp length upstream of the asr gene from the Trichoderma genome, the second pair of primers asr-do-F (CCATAGTACCACCCCGCCATAAAGCAAATTCTTCTACGGGG AGGGAC) and U3-do-R (GATAAGCCCGGGGATGGTATTG) amplified a fragment of 1500bp length downstream of the asr gene from the Trichoderma genome, and the third pair of primers HygB-F (CGATTGT GTGTACTAGGTAGGCTCG) and HygB-R (GGAATCGACCTTGCATGCGATG) amplified an open reading frame for the hygromycin B phosphotransferase gene from the pcDNA1 plasmid, as hygromycin resistance expression cassettes. The fourth pair of primers E-asr-F (GTTGTTTGGACTCGAAGCGC) and E-HygB-R (GGTCGTTTCTGACACAACGGTG) are homologous recombination verification primers. The three fragments of asr gene upstream, hygromycin B resistance expression frame and asr gene downstream make adjacent two fragments have base complementary overlapping region of about 25bp by means of primer design, in which the upstream end and hygromycin initial end are complementary overlapped, and the hygromycin end and downstream initial end are complementary overlapped, and the gene knockout fragment of asr1 upstream fragment+hygromycin resistance expression frame + asr1 downstream fragment can be obtained by means of overlap extension PCR. The result shows that the deletion of one of the genes can obviously improve the growth capacity of the trichoderma acid stress, which is consistent with the trend of mutant strains. (as shown in FIG. 5)

The above specific embodiments are provided for illustrative purposes only and are not intended to limit the invention, and modifications, no inventive contribution, will be made to the embodiments by those skilled in the art after having read the present specification, as long as they are within the scope of the patent statutes.

Claims (12)

1. An asexually reproducing filamentous fungus, characterized in that: the asexually reproducing filamentous fungus is a wild strain of Trichoderma, the wild strain of Trichoderma has a deposit number of CGMCC No. 12166; the deposit unit is China General Microbiological Culture Collection Center; the deposit date is April 11, 2016. 2. A method for mining new functional genes of asexually propagated filamentous fungi according to claim 1, characterized in that it comprises the following steps: S1. Construction of Trichoderma strain A that is deficient in urea synthesis but resistant to hygromycin; S2, obtaining a random mutant strain B, irradiating the wild-type strain with X-rays and screening out a random mutant strain B with acid resistance; S3, protoplast preparation and fusion, constructing a temporary pseudo-"diploid" by protoplast fusion method; the two sets of chromosomes of the temporary pseudo-"diploid" are respectively from Trichoderma strain A and random mutant strain B; S4, screening and phenotypic verification, random complementation of random mutant chromosome genes through homologous recombination of two sets of chromosomes in the nucleus, and screening of phenotypic changes to obtain phenotypic complement mutants; S5. Resequencing and gene editing verification: Use resequencing technology to determine the key mutation sites to be complemented and reversely verify the gene function through gene editing methods. 3. The method according to claim 2, characterized in that: in step S1, the construction of the Trichoderma strain A is to knock out the key gene ura3 (A1A105144) of Trichoderma uridine synthesis using hygromycin as a biomarker. 4. The method according to claim 2, characterized in that: in step S2, the method for obtaining the random mutant strain B is: taking 200 μl of a spore suspension with a concentration of 1×10 ⁷ on a 96-well plate and irradiating it with X-rays, diluting the irradiated spore suspension and applying it to a screening medium with required conditions, and screening to obtain a mutant strain B with a significant phenotypic change. 5. The method according to claim 4 is characterized in that the total dose of X-ray irradiation is 250 Gy, and the total irradiation is three times, and the three doses are 84 Gy, 84 Gy and 82 Gy respectively. 6. The method according to claim 2 is characterized in that: in step S3, the process and verification method of the protoplast fusion are as follows: after preparing protoplasts from Trichoderma strain A and random mutant strain B respectively, the protoplasts are fused using a PEG-mediated method, the regenerated transformants are screened using a hygromycin-containing oligotrophic medium and then single spore separation is performed, the transformants of heterokaryons are removed, a temporary "diploid" is retained, and verification is performed using specific primers. 7. according to the described method of claim 6, it is characterized in that: described protoplast fusion method is the PEG mediation protoplast fusion that reduces gradually through concentration, and concrete process is: 9.5% PEG+Ca ²⁺ hypertonic solution room temperature 5 minutes; 7% PEG+Ca ²⁺ hypertonic solution on ice 5 minutes; 5% PEG+Ca ²⁺ hypertonic solution on ice 20 minutes. 8. The method according to claim 6, characterized in that the specific primers include: E-U3-F, E-hygb-R; E-U3-F, E-U3-R. 9. The method according to claim 5, characterized in that a basic salt glucose medium containing hygromycin B is used as a temporary pseudo-"diploid" screening medium. 10. The method according to claim 2, characterized in that: in step S4, a basic salt glucose medium is used to screen the strain in which the target functional gene is complemented. 11. The method according to claim 2 is characterized in that: in step S5, the specific process of determining the key mutation sites of complementation by using resequencing technology is: the mutant C whose phenotype changed after the last complementation and the initial mutant strain B are cultured on PDA and then the mycelium is extracted for mapping analysis, and the mutation sites of the two are compared after data analysis. The mutation sites of complementation strain C are less than those of mutant strain B, and the difference in mutation sites between the two is the range of key genes. 12. Application of the method according to any one of claims 2 to 11 in mining new functional genes in response to acid.