Application of SRR1 gene in regulation and control of basidiospore morphogenesis in post-meiosis stage

Abstract

The invention discloses an application of an SRR1 gene in regulation and control of basidiospore morphogenesis in a post-meiosis stage, and belongs to the technical field of genetic engineering. Compared with a wild type, a constructed SRR1 gene deletion mutant Srr1 has the advantages that the pileus of a mature sporocarp of a Srr1 strain is white, and the Srr1 strain loses sporulation ability. Micromorphological analysis shows that a basidium of the Srr1 strain contains four cell nucleuses, basidium peduncle development is abnormal, the top end cannot expand to the final shape and size of basidium, and the result shows that the SRR1 gene causes a spore-free phenotype by influencing basidium peduncle development in the post-meiosis stage. Research results of the invention provide theoretical basis and gene resources for creation of spore-free strains, and are helpful for perfecting fungal development biology theoretical research related to sexual reproduction.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to the application of the SRR1 gene in regulating the morphogenesis of basidiospores in the post-meiotic stage. Background Art

[0002] Basidiospores are sexual spores produced by basidiomycetes. Among the basidiomycete fungi widely cultivated in edible mushroom production, especially the edible fungi of the genus Pleurotus (such as Pleurotus ostreatus), the released basidiospores can not only damage the precision technical instruments in the production facilities, but also trigger an occupational respiratory disease called "mushroom worker's lung", seriously threatening the health of practitioners. Basidiospores also have the risk of enabling the escape of strains from the artificial culture environment and establishing invasive populations in the natural environment, which may have unpredictable effects on the genetic diversity of natural populations in the local ecosystem. In addition, the large amount of released basidiospores will also affect the commercial properties (due to the attachment of basidiospores to the fruiting bodies) and cause the spread of edible mushroom diseases, etc. Therefore, the commercial demand for sporeless strains of edible fungi is becoming increasingly urgent.

[0003] The process of basidiospore formation involves complex biological mechanisms, which is a continuous and complex process occurring at the microscopic level. Its precise time points, locations, and regulatory mechanisms are all difficult to directly define and observe. This characteristic greatly increases the research difficulty, resulting in relatively slow progress in the research on the formation mechanism of basidiospores. In particular, the research on the molecular mechanism of basidiospore morphogenesis in the post-meiotic stage is almost blank. In-depth analysis of the molecular mechanism of basidiospore formation is not only the key to promoting the molecular breeding process of sporeless strains of edible fungi, but also helps to better understand the sexual reproduction process of basidiomycetes and provide important theoretical support for fungal developmental biology.

[0004] Aposporous mutants play an important role in cultivating aposporous strains. So far, naturally occurring aposporous mutants have been found in various basidiomycetes. Aposporous mutants have also been successfully obtained through artificial mutagenesis processes, such as ultraviolet (UV) irradiation or the application of chemical mutagens (such as nitrosoguanidine). These aposporous mutants are mainly used as breeding materials for developing commercially cultivated aposporous strains. However, there are still relatively few aposporous strains that have been successfully commercialized, only three strains: Pleurotus ostreatus SPOPPO, Pleurotus eryngii, and Agrocybe cylindracea. The reason is that aposporous mutants are prone to introducing other undesirable traits during the mutation process, resulting in defects in fruiting body traits, yield, etc. It is very difficult to restore the yield and quality of these mutants to a level suitable for commercial cultivation through traditional breeding methods such as artificial selection and cross-breeding. Therefore, targeting the functional genes that control basidiospore formation and using various commercially cultivated strains for directed molecular breeding of aposporous strains has become a research hotspot. Although some genes related to basidiospore formation have been discovered currently, there are still a large number of unknown genes waiting to be explored. Exploring the key genes that control basidiospore formation and analyzing their molecular regulatory mechanisms are of crucial significance for promoting the molecular breeding process of aposporous strains and their commercial applications. Summary of the Invention

[0005] In view of the above-mentioned prior art, the object of the present invention is to provide the application of the SRR1 gene in regulating the morphogenesis of basidiospores in the post-meiotic stage, laying a foundation for deeply analyzing the molecular mechanism of basidiospore formation and promoting the molecular breeding process of aposporous edible fungi strains.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, there is provided the application of the SRR1 gene in any one of the following (1)-(4):

[0008] (1) Regulating the spore-producing ability of basidiomycetes;

[0009] (2) Regulating the development of basidiospores in basidiomycetes;

[0010] (3) Regulating the development of basidiophores in the post-meiotic stage of basidiomycetes;

[0011] (4) Cultivating new varieties of aposporous basidiomycetes;

[0012] The SRR1 gene is a DNA molecule shown in any one of the following i)-iii):

[0013] i) A DNA molecule with a nucleotide sequence shown in SEQ ID NO.1;

[0014] ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i);

[0015] iii) A DNA molecule having 80% or more homology with the DNA fragment defined in i) or ii) and encoding a protein functionally equivalent to the protein shown in SEQ ID NO.2.

[0016] The regulation of the sporulation ability of basidiomycetes specifically refers to: regulating the number of basidiospores produced by basidiomycetes.

[0017] Basidiospore morphogenesis refers to: the structural change process of the formation, development and maturation of basidiospores during the reproduction process of fungi in the phylum Basidiomycota. The present invention verifies that the SRR1 gene can regulate basidiospore morphogenesis in the post-meiotic stage. Specifically, by knocking out the SRR1 gene, the top of the basidiophore cannot expand, resulting in a sporeless phenotype.

[0018] In the second aspect of the present invention, there is provided the use of the protein encoded by the SRR1 gene in any one of the following (1)-(4):

[0019] (1) Regulating the sporulation ability of basidiomycetes;

[0020] (2) Regulating the development of basidiospores of basidiomycetes;

[0021] (3) Regulating the development of basidiophores in the post-meiotic stage of basidiomycetes;

[0022] (4) Cultivating new varieties of sporeless basidiomycetes;

[0023] The amino acid sequence of the protein encoded by the SRR1 gene is as shown in SEQ ID NO.2.

[0024] In the third aspect of the present invention, there is provided the use of the knockout recombinant expression vector of the SRR1 gene or the genetically engineered bacterium containing the knockout recombinant expression vector of the SRR1 gene in any one of the following (1)-(4):

[0025] (1) Regulating the sporulation ability of basidiomycetes;

[0026] (2) Regulating the development of basidiospores of basidiomycetes;

[0027] (3) Regulating the development of basidiophores in the post-meiotic stage of basidiomycetes;

[0028] (4) Cultivating new varieties of sporeless basidiomycetes.

[0029] In the fourth aspect of the present invention, there is provided a method for regulating the sporulation ability of basidiomycetes in the post-meiotic stage. The method affects the normal development of basidiophores in the post-meiotic stage by knocking out the SRR1 gene or inhibiting the expression of the SRR1 gene, thereby reducing the sporulation ability of basidiomycetes;

[0030] Alternatively, the sporulation ability of basidiomycetes can be improved by overexpressing the SRR1 gene or enhancing the expression of the SRR1 gene.

[0031] The effect on the normal development of basidia after meiosis specifically refers to that the tip of the basidium cannot expand to the final shape and size of the basidiospore.

[0032] In the fifth aspect of the present invention, a method for cultivating a new variety of asexual-spore basidiomycetes is provided, including the following steps: by knocking out the SRR1 gene in a wild-type basidiomycete variety, affecting the development of basidia after meiosis, a new variety of asexual-spore basidiomycetes with lost sporulation ability is obtained.

[0033] Advantages of the present invention:

[0034] The present invention for the first time discovers that the SRR1 gene has the biological function of regulating the morphogenesis of basidiospores after meiosis. In basidiomycetes, compared with the wild type, the deletion mutant △Srr1 obtained by constructing a CRISPR / Cas9 knockout vector of the SRR1 gene has a white pileus color in the mature fruiting body and loses the sporulation ability. Microscopic morphological analysis shows that the basidia of the △Srr1 strain contain 4 nuclei, the development of the basidia is abnormal, and the tip cannot expand to the final shape and size of the basidiospore. This result indicates that the SRR1 gene leads to the asexual-spore phenotype by affecting the development of basidia after meiosis. The research results of the present invention provide a theoretical basis and gene resources for the creation of asexual-spore strains, and contribute to the improvement of the theoretical research on fungal developmental biology related to sexual reproduction. Description of the Drawings

[0035] Figure 1 For the mining of functional genes for the formation of Coprinopsis cinerea basidiospores; wherein, Figure 1 A in it shows 7 genes that are specifically highly expressed in the gill part of the fruiting body of Coprinopsis cinerea in the young mushroom stage (VM: mycelium stage, HN: hyphal knotting stage, P1: primordium stage 1, P2: primordium stage 2, YFB_S: young fruiting body stipe, YFB_C: young mushroom cap, YFB_L: young mushroom gill, FB_S: mature stipe, FB_CL: mature cap and gill); Figure 1 B in it shows the statistical results of the expression levels of the 7 genes in the gills of young mushrooms compared with the average expression levels in other periods; Figure 1 C in it shows the expression levels of the SRR1 homologous gene in Laccaria bicolor (Lacbi2_453082) at different developmental stages (M: mycelium stage, P_cap: top of primordium, P_stipe: lower part of primordium, YFB_cap: young mushroom cap, YFB_stipe: young mushroom stipe. FB_.cap: fruiting body cap, FB_stipe: fruiting body stipe); Figure 1In D, the expression levels of the SRR1 homologous gene at different developmental stages of Agrocybe aegerita (Agrae_CAA7260214) are shown (YVM: mycelium stage, P1: primordium stage 1, P2: primordium stage 2, YFB: young fruiting body, MFB: mature fruiting body, PFB: fruiting body after sporulation); Figure 1 In E, the expression levels of the SRR1 homologous gene at different developmental stages of Pleurotus ostreatus are shown (VM: mycelium stage, P1: primordium stage 1, P2: primordium stage 2, YFB_C: young fruiting body cap, YFB_S: young fruiting body stalk, FB_C: mature fruiting body cap, FB_L: mature fruiting body gill, FB_S: mature fruiting body stalk, FB_V: mature fruiting body veil); Figure 1 In G, Cc_451915 is the conserved domain.

[0036] Figure 2 is the phylogenetic analysis of the SRR1 homologous gene; among them, Figure 2 In A, it is the phylogenetic analysis of the SRR1 homologous gene, and the asterisk marks indicate the branches with more than 70% bootstrap support; Figure 2 In B, it is the conservation of the DNA-binding domain with unknown function and the flanking regions of SRR1; Figure 2 In C, it is the conservation analysis of the DNA-binding domain of the SRR1 homologous gene.

[0037] Figure 3 is the construction of the SRR1 gene knockout vector; among them, Figure 3 In A, it is the map of the plasmid for gene editing; Figure 3 In B, it is the schematic diagram of the screening primers and the positions of gRNA; Figure 3 In C, compared with the wild type (WT), the △Srr1 strain has a deletion of 1,666 base pairs (bp);

[0038] Figure 4 is the functional analysis of the SRR1 gene; Figure 4 In A, it is the fruiting body morphology of the ΔSrr1 and wild type strains (WT) at different developmental stages; Figure 4 In B, it is the morphology of the fruiting body and gills; Figure 4 In C, it is the microscopic images of cystidia and pseudocystidia; Figure 4 In D, the fluorescence microscope images using Hoechst staining show the number of nuclei in the basidia. Detailed implementation manners

[0039] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0040] As mentioned above, a large number of spores released by fruiting bodies during the production of edible fungi can cause allergic reactions in the lungs of practitioners and also pose risks such as disease transmission. Cultivating sporeless strains of edible fungi is the key to solving this problem. Mining the key genes that control basidiospore formation and analyzing their molecular regulatory mechanisms are important prerequisites for promoting the molecular breeding process of sporeless strains.

[0041] Coprinopsis cinerea has a short life cycle and is easy to culture, with a clear genetic background and simple gene manipulation. The process of basidiospore formation has been studied most comprehensively and in detail in it, so it is widely used as a model species for studying the growth and development of basidiomycetes.

[0042] Basidia are usually formed by the swelling of the apical cells of secondary hyphae. Basidiospores grow at the front end of the basidium and are connected to the basidium by sterigmata. The development of Coprinopsis cinerea basidiospores begins with meiosis, during which four haploid nuclei are formed and then migrate to the developing initial basidiospores. This process can be divided into four stages: Stage 1 (initiation stage), characterized by the spherical swelling at the top of the basidiospore stalk; Stage 2 (asymmetric growth stage), the basidiospore is located at a specific position on the basidiospore stalk due to the initial asymmetric growth towards the abaxial side; Stage 3 (equal swelling stage), the spore swells spherically, the endoplasmic reticulum develops, the number of Golgi vesicles, other types of vesicles and mitochondria increases, and the number of wall layers increases; Stage 4 (elongation stage), the spore grows longitudinally, the number of Golgi vesicles decreases, but new microtubule structures appear inside the spore. A pore cap is formed at the top of the basidiospore, covering the germ pore, the wall thickens significantly, and it begins to color within 2 hours, and the color of the cap turns black. The completion of morphogenesis is marked by the strong ejection of basidiospores. During this process, the basidiospores are ejected from the basidium by the mechanical force generated by the combination with Bleuler’s drop.

[0043] Some genes related to chromosome behavior during meiosis have been identified in Coprinopsis cinerea, including rad9, rad11, rad12, spo11 and MER3. The deletion of these genes affects DNA replication before meiosis, chromosome condensation, synapsis, recombination, homologous chromosome pairing and meiosis spindle formation, resulting in the inability to form basidiospores.

[0044] In Pleurotus ostreatus, two meiosis-related genes, rad51 and dmc1, have also been successfully cloned. Disrupting the two genes by homologous recombination both results in a sporeless phenotype. MSH4 is an important gene controlling the meiosis process in yeast. Okuda et al. obtained a sporeless mutant strain of Pleurotus pulmonarius, and through identification, it was found that the defect of the homologous gene STPP1 of MSH4 was the reason for the mutant to lose the ability to produce spores.

[0045] In Schizophyllum commune, after artificially disrupting the MSH4 homologous gene (defined as scMSH4), the number of basidiospores decreased significantly, only 0.1% of that of the wild type (Lavrijssen et al., 2020). Yoneyama identified a C6 zinc finger transcription factor in Pleurotus cornucopiae by next-generation sequencing (NGS) technology and TILLING technology. The deletion of this gene led to a sporeless phenotype by inhibiting the meiosis process (Yoneyama et al., 2020).

[0046] Although preliminary progress has been made in the study of the basidiospore formation mechanism, the vast majority of functional genes reported so far that are involved in basidiospore morphogenesis are mainly related to the meiosis process. The formation of basidiospores still involves complex developmental processes in the post-meiotic stage, such as the swelling of basidiophores and the migration of nuclei. However, genes necessary for basidiospore formation in the post-meiotic stage have not been reported yet.

[0047] Based on this, the present invention provides the application of the SRR1 gene in regulating basidiospore morphogenesis in the post-meiotic stage. First, through comparative transcriptome analysis of different tissue parts and different developmental stages of Coprinopsis cinerea, the present invention identified a gene SRR1 that was specifically highly expressed in the gill samples of fruiting bodies at the young mushroom stage. SRR1 is a C2H2-type zinc finger transcription factor and plays an important role in basidiospore formation.

[0048] The nucleotide sequence of the SRR1 gene is shown in SEQ ID NO.1, as follows:

[0049] SEQ ID NO.1:

[0050]

[0051] The amino acid sequence of the protein encoded by the SRR1 gene is shown in SEQ ID NO.2 as follows:

[0052] SEQ ID NO.2:

[0053] MAGTAAAHPQPLHQYDFQSNSVFNPGNSPTHNSFPIHHQAGPSNLTSTGLAGLGIQLQCPGHHQDPHHHKSSFSPKTYLPDPGDQYISFSPRIESTYSSFGGGAVPPLGVDDLAHGGEDMSTIWPLLDPSTSGSVDLDLGPLGLDTMSTTVPIVPPPASSMASGSGSTGGGSSDEDLSIESLSAAAGLSVEEFTAKITEGAQYALKHLAEMESGLSDGTGSTSLDPLAMSMSMSAQDLYFDNTIPSLDALAPFPDFGGLPEPEMWSLQQTCLGINPADVMPGPVLSSTVSSLDDMFSGSLDYTANRLDEQQATAPISEFSMHANAFNPDGTISMPLSALAAPTRSSRYEPNNAHPHRHQHYRAQKQVGLGFGQASTKPDDGDQLSDYEPPAFLSPSSSEYSPSFGTTRQFQTRTTRTNKRKVAQNEVVEPKPPVATSSKDTSDHLPPIDLGSPVFDAHRGIELEDLKARAERYRLRNQGREYDKRWLLSFAGKLSNKGELIEEFRCYVVGCQQVNKRRDHILIHVGAHLDQRPFKCIHCSARFLRKNECKRHELSHTGVRPFTCNICPYPGTTFVRQDLLRRHMKRTHQVDEEKENNKPSTSTMSFRPRKKVKSF*

[0054] Phylogenetic analysis showed that the SRR1 gene is highly conserved in Agaricomycetes. Using gene editing technology to knockout SRR1, the results showed that: compared with the wild type, the cap color of the mature fruiting body of the knockout mutant △Srr1 strain was white and it lost the ability to produce spores. Microscopic morphological analysis showed that: the basidia of the △Srr1 strain contained 4 nuclei, and the basidiophores developed abnormally and the top could not expand to the final shape and size of the basidiospores, indicating that SRR1 led to the sporeless phenotype by affecting the development of basidiophores in the post-meiotic stage.

[0055] The following will further describe in detail the specific implementation manners of the present invention in conjunction with embodiments. The following detailed descriptions are all illustrative and are intended to provide further explanations for the present application, rather than limiting the scope of the present invention.

[0056] Example 1: SRR1 is an important candidate gene controlling basidiospore formation

[0057] Transcriptome data of different developmental stages and different tissue parts of Coprinopsis cinerea (mycelium stage, primordium stage 1, primordium stage 2, young mushroom stipe, young mushroom cap, young mushroom gill, mature mushroom stipe, mature mushroom cap, mature mushroom gill) were analyzed to screen genes highly specifically expressed in the gill part of the fruiting body at the young mushroom stage. A total of 7 genes highly expressed in the gills were identified. Among them, the gene Cc_451915 was specifically expressed in the gills and was not detected in all other tissues and developmental stages ( Figure 1 A). At the same time, compared with the other 6 genes, the fold change of the expression level of the Cc_451915 gene in the gills compared to the average expression level in other periods was also the highest, which was 8.12-fold ( Figure 1 B). According to the reported basidiomycete genome and transcriptome data, through homologous gene alignment and transcriptome data analysis, it was found that the homologous genes of Cc_451915 also had similar expression patterns in other basidiomycetes. For Laccaria bicolor, it was highly specifically expressed in the cap part of its fruiting body ( Figure 1 C); for Cyclocybe aegerita, it was highly specifically expressed at the fruiting body stage ( Figure 1 D); for Pleurotus ostreatus, it was highly specifically expressed in the gill part of its fruiting body ( Figure 1 E). Based on the above results, it was speculated that Cc_451915 might be involved in basidiospore formation. Through conservative domain query, the Cc_451915 gene encodes a polypeptide of 684 amino acids, containing three C2H2-type zinc finger domains ( Figure 1 G), indicating that it is a transcription factor of the C2H2-type zinc finger family, and it was named Sporulation-Related Regulator 1, SRR1.

[0058] Example 2: The function of SRR1 is highly conserved in Agaricomycetes

[0059] To evaluate the evolutionary pattern and conservation of SRR1 homologous genes in Agaricomycetes, phylogenetic analysis was performed on SRR1 homologous genes searched from 189 genomes of Agaricomycetes. The results showed that SRR1 was highly conserved in Agaricomycetes and evolved after Russulales separated from Agaricomycetes. There was clear homology in Agaricales, Boletales, Russulales, Polyporales, etc. SRR1 mostly existed as a single-copy gene ( Figure 2 A). Sequence analysis showed that the SRR1 protein sequence was highly conserved in three C2H2-type DNA binding domains and the region about 50 amino acids (aa) upstream ( Figure 2 B and Figure 2 C).

[0060] Example 3: Construction of SRR1 knockout strain (△Srr1) and detection of its traits

[0061] (1) Construction of Srr1 knockout strain

[0062] The △Srr1 strain was constructed using the CRISPR-Cas9 gene editing RNP system.

[0063] The specific construction process was as follows: First, using the pUC19 plasmid as a template, the homologous recombination template vector Srr1Up-PABA-Srr1Down was constructed by the Gibson assembly method, where PABA (natural para-aminobenzoic acid synthase gene, pab1) was used as a positive selection marker. The Srr1 gene was knocked out by a two-target method, and sgRNAs were designed and synthesized at positions near the start codon and stop codon upstream and downstream of the Srr1 gene, respectively. The in vitro assembled RNP complex, sgRNA, and the homologous recombination template vector Srr1UpPABA-Srr1Down were genetically transformed by the PEG-mediated method, and the transformants with the PABA marker successfully replacing the Srr1 gene fragment were screened for subsequent experiments. During the construction process, the two RNPs targeted the front end and the end of the gene respectively, resulting in a deletion of 1,666 base pairs (bp) ( Figure 3 C). The primer sequences used in the construction process are shown in Table 1.

[0064] Table 1: Primer sequences:

[0065]

[0066] (2) Trait detection of △Srr1

[0067] A fruiting experiment was carried out on the △Srr1 strain and the wild type WT (Coprinopsis cinerea), and the results showed that there were no phenotypic differences between the two during the mycelial stage, the number of sporidia, and the early stage of fruiting body development.Figure 4 A). The gills, cystidia and pseudoparaphyses showed no difference from those of the WT, and their typical autolysis process was also similar to that of the WT. Compared with the wild type, the color of the fruiting body cap of the △Srr1 strain at the mature stage was white, indicating the loss of sporulation ability. Microscopic observation showed that the development of basidiospores in the △Srr1 strain was arrested at the second stage, that is, the basidiophores could not further expand to reach the final shape and size of the basidiospores. The nuclei were stained and observed using a fluorescence microscope. The results showed that the basidia of the mutant contained four nuclei, indicating that meiosis had been completed. Figure 4 D). In summary, the development of basidiospores in the Srr1 mutant was arrested after meiosis, indicating that SRR1 is an important gene regulating the morphogenesis of basidiospores after meiosis, and the deletion of SRR1 led to the arrest of basidiophore development.

[0068] In summary, the SRR1 gene can regulate the morphogenesis of basidiospores in the post-meiotic stage of Coprinopsis cinerea and has application value in creating sporeless strains.

[0069] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. Use of the SRR1 gene in any one of the following (1)-(4): (1) Regulating the sporulation ability of basidiomycetes; (2) Regulating the development of basidiospores in basidiomycetes; (3) Regulating the development of basidia after meiosis in basidiomycetes; (4) Cultivating new varieties of asexual-sporing basidiomycetes; The SRR1 gene is a DNA molecule shown in any one of the following i)-iii): i) A DNA molecule with a nucleotide sequence shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i); iii) A DNA molecule having 80% or more homology with the DNA fragment defined in i) or ii) and encoding a protein functionally equivalent to the protein shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The regulation of the sporulation ability of basidiomycetes specifically refers to: regulating the number of basidiospores produced by basidiomycetes.

3. Use of the protein encoded by the SRR1 gene in any one of the following (1)-(4): (1) Regulating the sporulation ability of basidiomycetes; (2) Regulating the development of basidiospores in basidiomycetes; (3) Regulating the development of basidia after meiosis in basidiomycetes; (4) Cultivating new varieties of asexual-sporing basidiomycetes; The amino acid sequence of the protein encoded by the SRR1 gene is shown in SEQ ID NO.

2.

4. Use of the knockout recombinant expression vector of the SRR1 gene according to claim 1 or a genetically engineered bacterium containing the knockout recombinant expression vector of the SRR1 gene in any one of the following (1)-(4): (1) Regulating the sporulation ability of basidiomycetes; (2) Regulating the development of basidiospores in basidiomycetes; (3) Regulating the development of basidia after meiosis in basidiomycetes; (4) Cultivating new varieties of asexual-sporing basidiomycetes.

5. A method for regulating the sporulation ability of basidiomycetes in the post-meiotic stage, characterized in that, The method affects the normal development of basidia after meiosis by knocking out the SRR1 gene or inhibiting the expression of the SRR1 gene, thereby reducing the sporulation ability of basidiomycetes; Alternatively, the sporulation ability of basidiomycetes is improved by overexpressing the SRR1 gene or enhancing the expression of the SRR1 gene.

6. The method according to claim 5, characterized in that, The specific meaning of affecting the normal development of basidia after meiosis is: the top of the basidium cannot expand to the final shape and size of the basidiospore.

7. A method for cultivating a new variety of a basidiomycete without spores, characterized in that, It includes the following steps: by knocking out the SRR1 gene in a wild-type basidiomycete variety, affecting the development of basidia after meiosis, and obtaining a new variety of asexual-sporing basidiomycetes that has lost the sporulation ability.

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