LeRho1 gene expression vector and method for improving heat resistance and trichoderma resistance of lentinus edodes

Abstract

The invention discloses a LeRho1 gene expression vector and a method for improving heat resistance and trichoderma resistance of lentinus edodes. The LeRho1 gene expression vector is a recombinant expression vector, a target gene for coding the LeRho1 protein is inserted into the recombinant expression vector, and the amino acid sequence of the LeRho1 protein is shown as SEQ ID NO. 1. The invention finds that the LeRho1 gene has a positive regulation effect on the heat resistance of the shiitake mushroom strain, and the LeUSPA and LeUSPH genes mediate the thermal response of the shiitake mushroom strain through negative regulation. Therefore, according to the method, the LeRho1 gene overexpression vector or LeUSPA and LeUSPH gene silencing vectors are constructed and transferred into the shiitake mushroom strain for genetic transformation, the heat resistance and trichoderma resistance of the shiitake mushroom strain can be remarkably improved, and the transformation efficiency is high.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, relates to a LeRho1 gene expression vector and a method for improving the heat resistance and Trichoderma resistance of shiitake mushrooms. Background Technology

[0002] The shiitake mushroom (Lentinula edodes) industry has become an important pillar industry of my country's agriculture, playing a crucial role in increasing farmers' income. However, shiitake mushrooms are low-temperature fungi, with an optimal temperature range of 22-28℃ for mycelial growth. The differentiation and development of the fruiting bodies (i.e., edible shiitake mushrooms), which determine the final yield and quality, require even lower temperatures (usually 12-22℃) and a larger temperature difference (10℃). This low-temperature fruiting biological characteristic creates a sharp contradiction with the frequent occurrence of extreme high temperatures in summer against the backdrop of global warming. Reports of high temperatures adversely affecting shiitake mycelial growth, even causing substrate rot, reduced yield, and serious economic losses have received widespread attention. Therefore, mitigating the impact of high-temperature stress on shiitake mushrooms, a crop highly sensitive to temperature, is an urgent problem to be solved.

[0003] Heat stress is a potent activator of the cell membrane-bound (CWI) pathway. In the model fungus *Saccharomyces cerevisiae*, the typical CWI pathway begins with a group of transmembrane sensor proteins on the cell membrane, such as Wsc1, Wsc2, Wsc3, and Mid2. However, no homologs of cell membrane surface receptors involved in heat sensing (such as Wsc orthologs) were found in the genome of *Lentinula edodes*, and the Wsc / Mid sensor was also absent. This suggests that Agaricales fungi may have evolved novel heat stress sensing elements, alternative signal activation mechanisms, or unique receptor-effector coupling patterns, but this remains unclear.

[0004] The core of genetic transformation technology in edible fungi lies in combining molecular biology and genetic engineering techniques to stably integrate the target gene into the genome of the host strain, obtaining strains with directed changes in genetic traits. However, compared to the model species of fungi (yeast), Agaricales fungi (such as shiitake mushrooms) have complex genetic backgrounds and complex cell walls, making it difficult to introduce recombinant DNA fragments into cells. Although there have been some successful studies on genetic transformation in edible fungi, the efficiency and stability of genetic transformation are still significantly lower than those of model species. In practical applications, there are still issues such as high false positive rates, high proportion of chimeras in transformants, and recombinant DNA integration into the genome without protein expression. As a globally important wood-rotting edible fungus, shiitake mushrooms still face significant technical bottlenecks in the construction of genetic transformation systems compared to closely related species such as enoki mushrooms, oyster mushrooms, and Ganoderma lucidum. Currently, the genetic transformation vector systems widely used in research are still limited to single models. Therefore, current research urgently needs to overcome technical bottlenecks such as low transformation efficiency and insufficient diversity of genetic transformation vectors to provide reliable technical support for molecular breeding and functional genomics research in shiitake mushrooms. Summary of the Invention

[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a LeRho1 gene expression vector and a method for enhancing the heat resistance and Trichoderma resistance of *Lentinula edodes*. The aim is to reveal, for the first time, the dynamic response network of *Lentinula edodes* at the mRNA and protein phosphorylation levels to heat stress using transient transcriptome sequencing and phosphoproteomics techniques. Three key heat-resistance candidate genes (LeRho1, LeUSPA, and LeUSPH) were screened and identified. Experiments showed that the LeRho1 gene has a positive regulatory effect on the heat resistance of *Lentinula edodes* strains, while the LeUSPA and LeUSPH genes mediate the heat response of *Lentinula edodes* strains through negative regulation. By constructing a LeRho1 gene overexpression vector or a LeUSPA / LeUSPH gene silencing vector and transferring it into *Lentinula edodes* strains for genetic transformation, the heat resistance and Trichoderma resistance of *Lentinula edodes* strains can be significantly improved with high transformation efficiency. This solves the technical problems of low transformation efficiency and insufficient diversity of genetic transformation vectors in existing *Lentinula edodes* genetic transformation methods.

[0006] To achieve the above objectives, according to a first aspect of the present invention, a LeRho1 gene expression vector is provided, which is a recombinant expression vector, into which a target gene encoding the LeRho1 protein is inserted, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0007] Preferably, the LeRho1 gene expression vector has the nucleotide sequence of the target gene LeRho1 as shown in SEQ ID NO. 2.

[0008] Preferably, the LeRho1 gene expression vector, wherein the recombinant expression vector further integrates the Kozak sequence unique to shiitake mushrooms, the Kozak sequence as shown in SEQ ID NO. 5 "-GCCATC-", which is directly linked to the translation start site "ATG" of the target gene LeRho1.

[0009] Preferably, the LeRho1 gene expression vector also carries an resistance gene, a gene encoding a tag protein, or a gene encoding a fluorescent protein for screening positive transformants; the gene encoding the tag protein or the gene encoding the fluorescent protein is linked downstream of the target gene LeRho1.

[0010] Preferably, the LeRho1 gene expression vector is a recombinant plasmid expressing a fusion protein, wherein the fusion protein is a LeRho1-tag protein or a LeRho1-fluorescent protein; the tag protein includes a Flag tag, an HA tag, and a Myc tag; and the fluorescent protein includes green fluorescent protein (GFP) and red fluorescent protein (mCherry).

[0011] Preferably, the LeRho1 gene expression vector is a recombinant plasmid carrying the target gene LeRho1, the resistance gene, and the fluorescent protein gene, and the promoter used to drive the resistance gene is the LeEF1α promoter, the sequence of which is shown in SEQ ID NO. 6.

[0012] Preferably, the LeRho1 gene expression vector, wherein the recombinant plasmid carrying the target gene LeRho1, the resistance gene, and the fluorescent protein gene as shown in SEQ ID NO. 2, is prepared according to the following method: The CaMV35S promoter of the pCAMBIA1300 vector was replaced with the lentinan leEF1α promoter to construct the pCAMBIA1300-E backbone vector; the pCAMBIA1300-E backbone vector itself carries a resistance gene, which is linked downstream of the leEF1α promoter; the resistance gene includes the resistance gene Hyg. The Kozak sequence shown in SEQ ID NO.5 was linked to the translation start site "ATG" of the target gene LeRho1. The target gene LeRho1 and the fluorescent protein gene were then inserted into the pCAMBIA1300-E backbone vector by EcoRI / BamHI double digestion to obtain the recombinant plasmid.

[0013] According to a second aspect of the present invention, a method for improving the heat resistance of shiitake mushroom strains is provided, comprising the steps of transferring a LeRho1 gene expression vector as described in the present invention, or a gene silencing vector for silencing LeUSPA and / or LeUSPH, into shiitake mushroom strains to prepare transformants, and screening for positive transformants.

[0014] Preferably, in the method, the gene silencing vector is a hairpin-structured RNAi silencing vector, which includes an interfering fragment and a loop connecting the interfering fragment; The gene silencing vector used to silence LeUSPA contains the interfering fragment as shown in SEQ ID NO. 10; A gene silencing vector for silencing LeUSPH, wherein the interfering fragment is the sequence shown in SEQ ID NO. 11.

[0015] Preferably, in the method, the loop connecting the interfering fragment is an intron sequence of a gene highly expressed in shiitake mushrooms, as shown in SEQ ID NO. 12.

[0016] According to a third aspect of the present invention, a method for enhancing the resistance of *Trichoderma* to *Lentinula edodes* strains is provided, comprising the steps of transferring the LeRho1 gene expression vector as described in the present invention into *Lentinula edodes* strains to prepare transformants, and screening for positive transformants.

[0017] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: This invention has discovered that the LeRho1 gene has a positive regulatory effect on the heat resistance of shiitake mushroom strains, while the LeUSPA and LeUSPH genes mediate the heat response of shiitake mushroom strains through negative regulation. Experiments have confirmed that by constructing LeRho1 gene overexpression vectors or LeUSPA and LeUSPH gene silencing vectors and transferring them into shiitake mushroom strains for genetic transformation, the heat resistance and Trichoderma resistance of shiitake mushroom strains can be significantly improved, and the transformation efficiency is high. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the technical route.

[0019] Figure 2 This document presents the conserved motif distribution, protein domains, promoter region cis-regulatory elements (800 bp), and gene structure analysis of the lentinan small GTPase gene. Figure A shows a schematic diagram of the conserved motifs of the lentinan small GTPase protein; the numbers in the colored boxes represent different motif numbers, and the black lines indicate non-conserved sequences. The bottom bar indicates the gene and protein lengths. Figure B shows the domain analysis of the lentinan small GTPase protein based on the NCBI conserved domain database. Figure C shows the analysis of the cis-regulatory elements of the promoter sequences (800 bp) of 34 lentinan small GTPase genes using the PlantCARE online tool. Figure D is a diagram illustrating the gene structure analysis.

[0020] Figure 3This is a conserved sequence analysis of predicted small GTPase protein from shiitake mushrooms. Note: G1 motif: GXXXXGK[T / S]; G2 motif: T; G3 motif: DxxG; G4 motif: [N / T]KXD; G5 motif: [C / S]A[K / L / T]).

[0021] Figure 4 This is a phylogenetic tree and subfamily classification of small GTPase proteins from shiitake mushrooms, straw mushrooms, bicolor wax mushrooms, Aspergillus fumigatus, Saccharomyces cerevisiae, Aspergillus nidus, rice blast fungus, and Cryptococcus neoformans. Note: The phylogenetic tree was constructed based on the small GTPase proteins of the following species: shiitake mushroom (red circle), straw mushroom (cyan circle), bicolor wax mushroom (orange circle), Aspergillus fumigatus (yellow circle), Saccharomyces cerevisiae (purple circle), Aspergillus nidulans (blue circle), rice blast fungus (dark green circle), and Cryptococcus neoformans (green circle). The phylogenetic tree was constructed using the maximum likelihood method with IQ-TREE v2.2.2 software. Members of each subfamily were marked with different colors, and the branch expansion value of all branches was greater than 50 (marked with red triangles of different sizes).

[0022] Figure 5 It represents the relative expression levels of small G protein family genes in shiitake mushrooms under heat stress.

[0023] Figure 6 This section describes the construction of a vector for overexpressing LeRho1 in shiitake mushrooms and the screening of transformants. In the figure, A is a schematic diagram of the gene expression vector; B is the relative expression level of the LeRho1 gene in shiitake mushroom transformants; C is the observation of LeRho1-GFP by laser confocal microscopy (the red arrow indicates the region with strong eGFP signal aggregation); and D is the detection of anti-Flag protein immunoblotting in shiitake mushroom mycelium.

[0024] Figure 7This is a PCR gel imaging verification of pCAMBIA-E-LeRho1-eGFP and pCAMBIA-E-LeRho1-Flag plasmids. Figure A shows the PCR identification results of *E. coli* colonies, with 1-24 representing the pCAMBIA-E-LeRho1-eGFP plasmid and 25-48 representing the pCAMBIA-E-LeRho1-Flag plasmid. Figure B shows the PCR identification results of *Agrobacterium* colonies, with 1-4 representing the pCAMBIA-E-LeRho1-Flag plasmid and 5-8 representing the pCAMBIA-E-LeRho1-eGFP plasmid. Figure C shows the PCR identification results of *Lentinula edodes* transformants, with 1-10 representing the pCAMBIA-E empty vector plasmid, 11 representing the pCAMBIA-E positive control, 12 representing the negative control, and 13-48 representing the pCAMBIA-E-Flag transformant. IA1300-E-eGFP transformant; D represents PCR identification results of shiitake mushroom transformants, 1-33 represent pCAMBIA1300-E-eGFP transformants, 34 represents pCAMBIA1300-E-eGFP positive control, 35 represents YS3334 negative control, and 36 represents blank control; 37-48 represent pCAMBIA-E-LeRho1-Flag transformants; E represents PCR identification results of shiitake mushroom transformants, 1-48 represent pCAMBIA-E-LeRho1-Flag transformants; F represents identification results of shiitake mushroom transformants, 1-6 represent pCAMBIA-E-LeRho1-Flag transformants, 7 represents pCAMBIA-E-LeRho1-Flag plasmid positive control, 8 represents YS3334 negative control, and 9 represents blank control.

[0025] Figure 8 It is the pCAMBIA1300-E-LeRho1-eGFP expression vector.

[0026] Figure 9 It is the pCAMBIA1300-E-LeRho1-Flag expression vector.

[0027] Figure 10 This is an assessment of the stress resistance of LeRho1 overexpression transformants in shiitake mushroom mycelium. Figure A shows the mycelial growth status of the overexpressing strains under environmental stress; B shows the comparison of the relative mycelial recovery rate between the LeRho1 overexpressing strains and the empty vector control strain after heat shock treatment (P<0.05); C shows the comparison of the relative mycelial recovery rate between the LeRho1 overexpressing strains and the empty vector control strain after Trichoderma infection (P<0.05); D shows the comparison of the relative mycelial recovery rate between the LeRho1 overexpressing strains and the empty vector control strain after light stress (P<0.05); E shows the comparison of the relative mycelial recovery rate between the LeRho1 overexpressing strains and the empty vector control strain after cold stress (P<0.05).

[0028] Figure 11These are the WB quality control results and the GFPCO-IP results.

[0029] Figure 12 This is a GO enrichment analysis of the protein identified by LeRho1-GFP immunoprecipitation.

[0030] Figure 13 The top image shows an electrophoresis image of Escherichia coli colony PCR verification; the bottom image shows an electrophoresis image of Agrobacterium colony PCR verification.

[0031] Figure 14 It is a LeUSPA hairpin structure gene silencing vector.

[0032] Figure 15 It is a LeUSPH hairpin structure gene silencing vector.

[0033] Figure 16 This is a confocal fluorescence microscopy observation of LeRho1 with LeUSPA-mCherry and LeUSPH-mCherry. In the figure, A to C represent the co-expression of LeRho1-GFP and LeUSPA-mCherry; D to F represent the co-expression of LeRho1-GFP and LeUSPH-mCherry.

[0034] Figure 17 This diagram shows the validation of gene silencing transformants of the LeUSPA / LeUSPH gene in the YS3334 strain of *Lentinula edodes*. Figure A shows the PCR validation of the LeUSPA gene RNAi transformant: -: negative control (empty vector transformant); +: positive control; NTC: blank control. Figure B shows the validation of the LeUSPH gene RNAi transformant: -: negative control (empty vector transformant); +: positive control; NTC: blank control. Figure C shows the qRT-PCR results of the LeUSPA gene silencing transformant: CK is the empty vector transformant, *: P<0.05, **: P<0.01, ****P<0.0001. Figure D shows the qRT-PCR results of the LeUSPH gene silencing transformant: CK is the empty vector transformant, *: P<0.05, **: P<0.01, ****P<0.0001.

[0035] Figure 18 This is an assessment of the heat resistance of LeUSPA / LeUSPH gene-silenced transformants of shiitake mushroom mycelium. In the figure, A represents the mycelial growth phenotype of the transformants after heat stress; B represents the relative ratio of mycelial growth rate after stress to before stress. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0037] The technical route of the present invention is as follows Figure 1 As shown, the system systematically analyzed the regulatory mechanisms of high temperature and moderate-high temperature stress on the mycelial growth of *Lentinula edodes* strains (YS3334, S606, YS55, YS3355, YS3357, and YS48), and screened out the wild-type *Lentinula edodes* strains YS3334 (heat-resistant strain) and YS48 (heat-sensitive strain) suitable for further in-depth research on heat tolerance mechanisms. Using transient transcriptome sequencing and phosphoproteomics, the dynamic response network of *Lentinula edodes* to heat stress at the mRNA and protein phosphorylation levels was revealed for the first time, and three key candidate heat tolerance genes (LeRho1, LeUSPA, and LeUSPH) were screened.

[0038] Based on transcriptome analysis of *Lentinula edodes*, the 200 genes with the highest expression levels in the *Lentinula edodes* transcriptome were screened. By statistically analyzing the high-frequency bases six bases upstream of their translation initiation sites, a *Lentinula edodes*-specific Kozak sequence was formed, as shown in SEQ ID NO. 5, "-GCCATC-", which was used to promote and optimize the translation initiation process of the LeRho1 protein. Furthermore, based on transcriptome analysis, we also identified that the expression level of the LeEF1α gene was extremely high and stable before and after heat stress (average TPM_TMM: 5047.19). Therefore, the LeEF1α promoter-driven hygromycin resistance gene (Hyg) was used as a selection marker, significantly improving the efficiency of *Lentinula edodes* transgenic screening and the stability of exogenous gene expression.

[0039] We constructed the LeRho1-eGFP fusion protein expression vector and the LeRho1-Flag fusion expression vector by integrating the conserved sequence "-GCCATC-" (a shiitake-specific Kozak sequence) 6 bp upstream of the translation start site of the highly expressed gene in shiitake mushrooms. Experiments revealed and confirmed that overexpression of LeRho1 in shiitake mushroom strains transformed into positive transformants not only enhanced cell wall thickness and significantly improved heat resistance, but also increased resistance to Trichoderma atroviride, photosensitivity, and cold sensitivity. This demonstrates that the LeRho1 protein has the potential to serve as a key target for multiple resistances in shiitake mushroom molecular breeding.

[0040] Using Co-IP / MS to identify LeRho1 interacting proteins, a novel dual-fluorescent labeling co-expression vector system was developed to achieve co-localization of target proteins in live cells, confirming that LeRho1 protein co-localizes with LeUSPA and LeUSPH proteins, respectively. The effects of LeUSPA and LeUSPH gene expression vectors and LeUSPA and LeUSPH gene silencing vectors on the heat resistance of *Lentinula edodes* strains were investigated. The results showed that silencing LeUSPA and LeUSPH genes significantly improved the heat resistance of *Lentinula edodes* strains, confirming that LeUSPA and LeUSPH genes mediate the heat response through negative regulation.

[0041] Based on this discovery, the present invention provides a LeRho1 gene expression vector, which is a recombinant expression vector, into which the target gene encoding the LeRho1 protein is inserted, and the amino acid sequence of the LeRho1 protein is shown in SEQ ID NO. 1.

[0042] In some embodiments, the LeRho1 gene expression vector carries the target gene LeRho1 as shown in SEQ ID NO. 2. Preferably, it also integrates a Kozak sequence unique to shiitake mushrooms, the Kozak sequence as shown in SEQ ID NO. 5 "-GCCATC-", which is directly linked to the translation initiation site "ATG" of the target gene LeRho1 to promote and optimize the translation initiation process of the LeRho1 protein.

[0043] The "-GCCATC-" Kozak sequence shown in SEQ ID NO. 5 of this invention is based on the 200 genes with the highest expression levels in the shiitake mushroom transcriptome of this invention. It is formed by statistically analyzing the high-frequency base combinations of the 6 bases upstream of their translation initiation sites. The Kozak sequence is only 6 bp and is directly added to the translation initiation site (ATG) of the LeRho1 gene during primer synthesis. It is directly linked to the LeRho1 gene to promote and optimize the protein translation initiation process. Its functions are reflected in the following aspects: (1) Identifying the start codon: The Kozak sequence surrounds the start codon AUG (ATG in DNA). The small subunit of the ribosome scans the mRNA, looking for a suitable AUG as the translation initiation site. The presence of the Kozak sequence acts as a "signal" to help the ribosome more accurately identify the true start codon, rather than other possible AUGs in the sequence (which may be located in the wrong reading frame or position).

[0044] (2) Enhanced translation initiation efficiency: Beyond recognition, Kozak sequences can significantly improve translation initiation efficiency. A consensus-compliant strong Kozak sequence can attract translation initiation factors, promoting more efficient assembly of the ribosome complex at the initiation site, thereby initiating protein synthesis more quickly. This directly affects the final protein yield.

[0045] (3) Define the translation start site: In the presence of multiple potential AUG sites, the strongest Kozak sequence usually determines which AUG is selected as the primary translation start site, which helps ensure that the protein is synthesized from the correct amino acid (methionine).

[0046] Furthermore, the LeRho1 gene expression vector also carries an resistance gene, a gene encoding a tag protein, or a gene encoding a fluorescent protein (i.e., a fluorescent protein gene); the gene encoding the tag protein or the gene encoding the fluorescent protein is linked downstream of the target gene LeRho1 for screening positive transformants.

[0047] In some embodiments, the LeRho1 gene expression vector is a recombinant plasmid expressing a fusion protein, which is a LeRho1-tag protein. The tag protein includes Flag-tag, HA-tag, Myc-tag, etc. For example, the DNA sequence encoding the Flag-tag is fused to a specific position (N-terminus or C-terminus) of the target gene LeRho1 using genetic engineering techniques, thereby expressing the fusion protein. When this recombinant gene is expressed in cells, the resulting target protein will carry this Flag, facilitating the screening of positive transformants. In some embodiments, the Flag sequence is fused to the C-terminus of the protein encoded by the LeRho1 gene.

[0048] In some embodiments, the LeRho1 gene expression vector is a recombinant plasmid expressing a fusion protein, wherein the fusion protein is LeRho1-fluorescent protein; the fluorescent protein includes green fluorescent GFP protein, green fluorescent eGFP protein, and red fluorescent mCherry protein. For example, the DNA sequence encoding green fluorescent eGFP protein is fused to a specific position (N-terminus or C-terminus) of the "target gene LeRho1" using genetic engineering techniques, thereby expressing the "fusion protein." In this way, when and where the target gene is expressed, and where the expressed protein is located in the cell, can be directly and in real time observed through the appearance and location of fluorescence. In some embodiments, the DNA sequence encoding green fluorescent eGFP protein is fused to the C-terminus of the protein encoded by the LeRho1 gene.

[0049] In some embodiments, the LeRho1 gene expression vector is a recombinant plasmid expressing the fusion protein, prepared according to the following method: The Kozak sequence was directly added to the translation start site (ATG) of the LeRho1 gene during primer synthesis and linked directly thereto. The 1137 bp gene sequence of the LeRho1 gene (with the 3' and 5' untranslated regions removed) was ligated, along with either the eGFP sequence (as shown in SEQ ID NO. 3) or the Flag sequence (as shown in SEQ ID NO. 4), into the linearized pCAMBIA1300-E vector to form recombinant plasmids. In some embodiments, the recombinant plasmids constructed are pCAMBIA1300-E-LeRho1-eGFP or pCAMBIA1300-E-LeRho1-Flag.

[0050] In some embodiments, the LeRho1 gene expression vector is a recombinant plasmid carrying the target gene LeRho1, a resistance gene, and a fluorescent protein gene. The promoter used to drive the resistance gene is the LeEF1α promoter, the sequence of which is shown in SEQ ID NO. 6. LeEF1α directly links the resistance gene through homologous recombination. In this invention, its transcriptome shows extremely high expression levels (average TPM_TMM: 5047.19), and the gene expression level remains relatively constant after heat stress. Using this to drive the expression of the resistance gene can significantly improve the efficiency of transgenic screening of shiitake mushrooms and the stability of exogenous gene expression.

[0051] In some embodiments, the recombinant plasmid carrying the target gene LeRho1, the resistance gene, and the fluorescent protein gene as shown in SEQ ID NO.2 is prepared according to the following method: The CaMV35S promoter of the pCAMBIA1300 vector was replaced with the lentinan leEF1α promoter to construct the pCAMBIA1300-E backbone vector; the pCAMBIA1300-E backbone vector itself carries a resistance gene, which is linked downstream of the leEF1α promoter; the resistance gene includes the resistance gene Hyg. The Kozak sequence shown in SEQ ID NO.5 was linked to the translation start site "ATG" of the target gene LeRho1. The target gene LeRho1 and the fluorescent protein gene were inserted into the pCAMBIA1300-E backbone vector by EcoRI / BamHI double digestion to obtain the recombinant plasmid.

[0052] In addition, the present invention provides a method for improving the heat resistance and Trichoderma resistance of shiitake mushroom strains, which includes the steps of transferring the LeRho1 gene expression vector as described in the present invention into shiitake mushroom strains to prepare transformants, and screening for positive transformants. The positive transformant refers to a shiitake mushroom strain that has successfully incorporated the LeRho1 gene expression vector as described in the present invention and is able to normally express the target gene LeRho1.

[0053] In addition, the present invention also provides a method for improving the heat resistance of shiitake mushroom strains, which includes... The steps involve transferring gene silencing vectors used to silence LeUSPA and / or LeUSPH into shiitake mushroom strains to prepare transformants and screening for positive transformants.

[0054] The positive transformant refers to a shiitake mushroom strain that has been successfully introduced with a gene silencing vector for silencing LeUSPA and / or LeUSPH, thereby silencing the LeUSPA and / or LeUSPH genes in the shiitake mushroom strain and improving the heat resistance of the shiitake mushroom strain.

[0055] In some embodiments, the gene silencing vector is a hairpin-structured RNAi silencing vector, comprising an interfering fragment and a loop connecting the interfering fragment, wherein the loop connecting the interfering fragment is an intron sequence of a lentinan gene highly expressed in shiitake mushrooms, as shown in SEQ ID NO. 12; wherein the gene silencing vector for silencing LeUSPA has the interfering fragment as shown in SEQ ID NO. 10; and the gene silencing vector for silencing LeUSPH has the interfering fragment as shown in SEQ ID NO. 11.

[0056] The following are examples. 1. Test materials Tested strains: *Lentinula edodes* binukaryotic strains YS3334, S606, YS55, YS3355, YS3357, and YS48; *Trichoderma viride* strain 92-1; plasmid pCAMBIA1300-g (the original 35S promoter was replaced by the Legpd promoter to activate the hygromycin B phosphotransferase gene), preserved by the Institute of Applied Mycology, Huazhong Agricultural University. *Lentinula edodes* strains YS3334, S606, YS55, YS3355, YS3357, and YS48 were screened and preserved by the Institute of Applied Mycology, Huazhong Agricultural University, and are all documented in existing scientific literature, belonging to known and obtainable strains.

[0057] Escherichia coli (E. coli) competent cells Trans1-T1 were purchased from TransGen Biotechnology, and Agrobacterium tumefaciens (EHA105) competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0058] O1L. Liquid MYG medium formulation does not contain agar.

[0059] LB (Leydig Meat Extract) Medium: To prepare one liter of medium, add the following to 950 ml of deionized water: 10 g tryptone, 5 g yeast extract, and 10 g NaCl. Shake the container until the solutes dissolve. Adjust the pH to 7.0 with 5 mol / L NaOH. Make up the volume to 1 L with deionized water and autoclave at 15 psi for 21 min.

[0060] Alternatively, adjust the pH to 6.7-7.0 using NaOH.

[0061] Alternatively, adjust the pH to 5.6 with NaOH.

[0062] Alternatively, adjust the pH to 5.6 with NaOH.

[0063] Reagents: Carboxin (Solepro), hygromycin (Roche), kanamycin (Guosai Biotechnology), cephalosporin (Carboxin), EcoRI, BamHI, KpnI, SbfI, RsrII, and AseI endonucleases (New England). Phanta Super-Fidelity DNA Polymerase, HiScript II One Step RT-PCR Kit, and AceQ™ QpcrSYBR Green Master Mix were purchased from Novizan Biotechnology (Nanjing) Co., Ltd.; pClone007 Blunt Simple Vector Kit was purchased from TsingKe; Hlingene agarose gel extraction kit was purchased from Shanghai Huiling Biotechnology Co., Ltd.; RNAisoPlus was purchased from Takara Bio Engineering (Dalian) Co., Ltd.; pEASY®-Basic Seamless Cloning and Assembly Kit was purchased from TransGen Biotechnology.

[0064] 2. Test methods: (1) Measurement of mycelial growth rate: Mycelial growth capacity determination of wild-type strains: Wild-type strains and empty vector strains were inoculated on MYG culture dishes and incubated at 25℃. The diameter of hyphae was determined by the cross-cross method, and one-way ANOVA (Alpha=0.05) was performed using GraphPadPrism software, with 5 replicates for each treatment.

[0065] Determination of mycelial growth recovery ability after heat shock: Evaluation method of heat / cold resistance and photosensitivity of shiitake mushrooms: Shiitake mushroom mycelial blocks were inoculated on MYG solid medium plates and pre-cultured at 25℃ in the dark for 5 days to complete the pre-culture; after pre-culture, the two vertical diameters of the colony were measured by the cross-cross method (denoted as D1A and D1B); the plates were treated as follows: a) Heat resistance test: treated at 38℃ for 1 day and then transferred to 25℃ for 5 days to recover growth; b) Cold resistance test: treated at 15℃ for 5 days and then transferred to 25℃ for 5 days to recover growth; c) Photosensitivity test: treated at 3500 lx light intensity for 1 day and then transferred to 25℃ in the dark for 5 days to recover. After the recovery period, the two vertical diameters of the colonies were measured again (denoted as D2AD2B), and the recovery growth after stress was calculated: DA = D2A - D1A; DB = D2B - D1B. The relative growth rate (%) = [(DA / recovery days) / (D1A / pre-culture days)] × 100 (recovery days = 5 days, pre-culture days = 5 days). Statistical analysis: One-way ANOVA was performed using GraphPadPrism 10.3.0 software, with a significance level set at p < 0.05. This experiment assessed the stress tolerance of shiitake mushrooms by measuring the mycelial recovery growth, and strict temperature and light control ensured the accuracy of the results.

[0066] (2) Evaluation method of resistance to Trichoderma in shiitake mushrooms: After 10 days of dark culture of MYG plate at 25℃, the central part of the aged mycelium block with a diameter of 8mm was removed and an equal amount of activated Trichoderma mycelium block was inoculated; after 5 days, the photos were taken and the infection area (S) of Trichoderma was calculated by ImageJ software. The smaller the S value, the stronger the resistance of shiitake mushrooms to Trichoderma.

[0067] (3) Transcriptome analysis of heat-resistant and heat-sensitive strains of shiitake mushrooms under heat stress ① Transcriptome sample preparation: Fresh shiitake mushroom mycelial blocks were inoculated onto MYG culture dishes lined with cellophane and incubated in the dark at 25°C for 7 days. After incubation, the culture dishes were opened and placed in a 38°C incubator for heat shock. The heat shock times were 0 min, 1 min, 15 min, 30 min, 45 min, 60 min, and 120 min. Each treatment was performed in triplicate, resulting in a total of 42 samples.

[0068] ② Strand-Specific RNA Library Construction and Sequencing: After successful RNA extraction and quality control, the RNA samples were delivered to Fraser Genetics (GBI) Wuhan Company for strand-specific RNA library construction and sequencing. After library construction, preliminary quantification was performed using Qubit 3.0 to dilute the library. Subsequently, the insert size was detected using an Agilent 2100. Once the insert size met expectations, the effective concentration of the library was accurately quantified using Q-PCR to ensure library quality. After passing the library detection, DNB (DNA Nanoball) was prepared, loaded onto a sequencing chip, and subjected to next-generation sequencing using an MGI high-throughput sequencer. The generated data consisted of 150bp paired-end data.

[0069] ③ Gene quantification, differential gene screening, and functional enrichment analysis: The raw sequencing data were of high quality. Data preprocessing was performed using Trimmomatic v0.39. Adapter sequences were cut to filter low-quality reads (SLIDINGWINDOW:4:15, MINLEN:36) and obtain high-quality clean reads. After quality control, the data were re-examined using FastQCv0.11.9, confirming that the base quality distribution, sequencing error rate, and sequence GC content all met the requirements for subsequent analysis.

[0070] Genome alignment was performed using HISAT2 Clean reads were aligned to the *Lentinula edodes* reference genome (NCBI accession: GCA_015476405.1). Gene expression was quantified using HTSeq-count v0.13.5. Count the number of aligned exon regions. Differential expression analysis was performed using the DESeq2v1.36.0 package. After standardization using TMM (trimmed mean of M-values), the fold change and significance level were calculated using the Wald test. The differential gene screening criteria were set as follows: |log2(FoldChange)|≥1 and P-value<0.05.

[0071] Functional enrichment analysis was performed using the clusterProfiler R package (v4.4.0). GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment was performed, and the annotation database was constructed based on the annotation information of the *Lentinula edodes* reference genome (using a self-built org.Le.eg.db package). The enrichment results were visualized using the enrichplotR package (v1.16.1). Bubble charts and pathway topology networks were plotted, with a significant enrichment threshold set at P-value < 0.05. Time series analysis was performed using ClusterGVis software (refer to Zhang J. ClusterGVis: One-step to Cluster and Visualize Gene Expression Matrix. [DS]. 2022). The optimal soft threshold selected for WGCNA analysis was 12.

[0072] (4) Phosphorylated proteomics analysis of heat-resistant strain YS3334 under heat stress ①Proteome Sample Preparation and Processing Beads were incubated in EP tubes at 25°C with shaking for 20 min for phosphorylation enrichment, centrifuged at 2000×g for 1 min, and the supernatant was removed. Beads were washed with TFA / CAN, and the solution was transferred to a C8 desalting column for peptide elution. TFA was added to the eluent to adjust the pH, and desalting was performed using an SDB-RPS desalting column. After vacuum drying, the residue was stored at -20°C.

[0073] An analytical gradient was established using mobile phase B (0.1% formic acid in ACN). The flow rate of the liquid phase was set to 300 nL / min. Peptides were introduced into the mass spectrometer via a Captive Spray nano-ion source for DDA scanning. TIMS was enabled, and PASEF scanning mode was used. Each scan cycle time was 1.1 s, consisting of one MS1 scan and 10 PASEF MS / MS scans, with each PASEF MS / MS scan containing 12 MS / MS spectra.

[0074] ③ Data Analysis: Mass spectrometry data were retrieved using Fragpipe (21.1) software with the MSFragger database retrieval algorithm. The database used for retrieval was the Lentinula_edodes proteome reference database in Uniprot. The main retrieval parameters were as follows: non-standard quantitative analysis was performed; variable modifications were selected as Oxidation (M), Phospho (STY), and Acetyl (Protein N-term); fixed modifications were selected as Carbamidomethyl (C); and enzyme digestion was selected as Trypsin / P. Search results were screened using a 1% FDR (Functional Data Reduction) standard for protein and peptide analysis. For functional annotation and enrichment analysis, based on the principle that proteins with the same or similar sequences have similar functions, the Diamond program in Egg-NOG software was used to obtain the GO, KEGG, and COG database annotation information for the submitted proteins. After obtaining the annotation information for all proteins identified by mass spectrometry, relevant information of differentially expressed proteins was extracted, and their categories and numbers were statistically analyzed. Functional enrichment analysis was then performed using a hypergeometric test to screen out significant functional categories relevant to the experiment. Protein-protein interaction networks of differentially expressed proteins in the comparative group were analyzed based on the STRING database. Motifs of differentially phosphorylated sites in the comparative group were analyzed using MoMo software (http: / / meme-suite.org / tools / momo).

[0075] Dissolve in O and store at -20℃ for later use.

[0076] (6) RNA extraction was performed using the RNAisoPlus kit (TaKaRa): 0.1g of shiitake mushroom mycelium was rapidly ground into powder in liquid nitrogen and transferred to an RNAse-free 1.5mL centrifuge tube. 600μL of RNAisoPlus was added, and the mixture was vortexed and allowed to stand at room temperature for 5min. 1 / 5 volume of chloroform was added, and the mixture was inverted and mixed for 20s. The mixture was allowed to separate into layers and allowed to stand at room temperature for 5min. The mixture was then centrifuged at 13,000×g for 10min at 4℃. The supernatant was transferred to a new RNAse-free 1.5mL centrifuge tube, and 2 / 3 volume of isopropyl alcohol was added. After mixing the ethanol by inversion, allow it to stand at room temperature for 10 min to precipitate, then centrifuge at 13,000 x g for 10 min at 4 °C. Discard the supernatant, add 1 mL of 70% ethanol to wash the precipitate (wash twice), discard the supernatant, centrifuge at 13,000 × g for 2 min at 4 °C, and remove the residual ethanol with a pipette tip. Dry the precipitate at room temperature on a clean bench for no more than 5 min. Add 30 µL of LRNase-free ddH2O, incubate in a 65 °C water bath, and store at -80 °C for transcriptome sequencing. Measure the concentration using a micro-ultraviolet spectrophotometer. Detect RNA integrity using 1% agarose gel electrophoresis.

[0077] The reverse transcription reaction system and procedures for RNA using the HiScript II QRT SuperMix for qPCR (+gDNAwiper) kit are as follows: Add 0 to a total volume of 8 μL, mix gently, incubate at 42 °C for 2 min; dilute the reverse transcription product 10-fold for subsequent qRT-PCR analysis.

[0078] (7) Laser confocal microscopy Fresh transformant mycelial samples were treated using a slide-climbing culture method: a coverslip with attached mycelia was placed upside down on a slide, and a 20% glycerol aqueous solution was injected between the two as an anti-fluorescence quencher. Fluorescence imaging was performed using an Olympus FV3000 confocal laser scanning microscopy system (equipped with an ultra-high sensitivity GaAsP detector), and the target protein localization information was obtained through a multi-channel fluorescence acquisition mode. 510 nm and 610 nm were used as excitation light for eGFP and mCherry, respectively. The PMTVoltage for the 510 nm excitation light was set to 505 V, and the PMTVoltage for the 610 nm excitation light was set to 484 V, with a Laser Transmissivity of 21.8%.

[0079] Approximately 0.6-0.8. After the mycelia of *Lentinula edodes* strain YS3334 were activated on MYG solid medium for 7 days, 5mm × 5mm mycelial blocks were cut using a sterile, sharp blade and immersed in the activated *Agrobacterium* solution for 20 minutes, shaking once every 5 minutes. Then, the mycelial blocks were inoculated onto Co-IM solid medium containing 200 μmol / L LAS and co-cultured at 25°C for 2 days. Afterward, the mycelial blocks were removed from the co-culture medium and transferred to MYG solid medium containing 9 μg / mL hygromycin and 300 μg / mL cefotaximemycin, and incubated at 25°C for approximately 10 days. Mycelial germination was observed, and positive single colonies were screened.

[0080] 3. Primer sequence information used in the experiment Table 1 Primer Information Primer Name Sequence (5'-3') BC_eF1a-kpnI_NployA_R atcgggaattGGTACCATGACCGCAGAAGAGGCCC B_ef1a_F caggctttttcatTGCCTGAATGAGAGGAAGTATGTT POE-Rho1-Flag_F GGGAAATTCGAGCTCGAATTcTTAGACGACAACACACTTGCTACCC POE-Rho1-Flag_R gcccccggggatggcggatccATGGATTACAAGGACGACGATGACAAGTCTGAAATTAGGAGGAAACTCGTTATC HR-Rho1-GFP_F gcccttgctcaccatGGATCCGACGACAACACACTTGCTACCCT HR-Rho1-GFP_R gcccccggggatggcggatccATGTCTGAAATTAGGAGGAAACTCG HRPS_GPDp_F TTCGAATTCGAGCTCGGTACCCGAAGTTTGAGGTGGTTGCG HR_PSGPDp_mcherry_R CCCTTGCTCACCATGGATCCGCCATCcCCGGGGG HRPS_gpdp_MCHERRY_F GGATGGCGGATCCATGGTGAGCAAGGGCGAGG HRPS_MCHERRY_nployA_R ggggaaattcgagctcgaattcCTACTTGTACAGCTCGTCCATGCC HRPS_mcherry_Nploy_F gctgtacaagtagGAATTCGAGCTCGAATTTCCC HRPS_mcherry_Npoly_R ccagtgccaagcttgcatgcctgcaggAATTCCCGATCTAGTAACATAGATGA suHr_PSM_LeUSPA_mCherry_F ctcattccatgtcttttcagGCCCCCGGgGATGGCATGAATGTACGTGATCAGTAATTATATCCG suHr_PSM_LeUSPA_mCherry_R cctcctcGCCCTTGCTCACCATGGATCCGGAGCCACCGCCACCAGAGCCACCACCGCCTTGTTGCAAAGAAGCCGTGC suHr_PSM_LeUSPH_mCherry_F ctcattccatgtcttttcagGCCCCCGGgGATGGCATGCACTCTTCGGGACACTCG suHr_PSM_LeUSPH_mCherry_R ctcctcGCCCTTGCTCACCATGGATCCGGAGCCACCGCCACCAGAGCCACCACCGCCGTCAAAATGTGTACCCCTTTTGG PeRnai_intron7_F gggaaattcgagctcgaattcCTATTATAATAATCGATAAATGCTTCAAGTC PeRnai_Intron7_R gcccccggggatggcggatccGTGCTGTTAGATTATCGGCTTGTATT Example 1: Study on the biological functions of LeRho1 shiitake mushroom under heat stress 1.1 Identification of LeRho1 protein and phylogenetic analysis of the Lentinus edodes small G protein family To achieve accurate identification of LeRho1 protein from *Lentinula edodes*, this study first conducted a systematic screening of the entire family, and then specifically isolated LeRho1. Based on all HMM model files (PF00071, PF0025, PF04675, and PF08477) for all small GTPases, this study first used a local HMM search method to scan the *Lentinula edodes* genome (LentinulaedodesW1-26v1.0) in the United Genome Research Institute's MycoCosm database with default parameters. Preliminary prediction yielded 198 candidate small GTPase proteins from *Lentinula edodes*. The conserved domain distribution of the predicted proteins was visualized using TBtools software based on BatchCD-search, which was used for precise screening of small GTPase proteins from *Lentinula edodes*. Figure 2 (B). After conserved domain analysis and redundant sequence removal, 34 members of the lentinan small GTPase superfamily were finally identified. All members contain a conserved G-box motif (B). Figure 3 The motif was visualized using DNAMAN v9.0 software. Protein motif type and structure determine its function. This study identified conserved motifs of small GTPases from shiitake mushrooms using the online MEME program and visualized their structures using TBtools. Figure 2 (A). A total of 10 conserved motifs were identified, and their spatial arrangement was basically consistent: almost all motifs were enriched at the C-terminus of the amino acid sequence, indicating that this region is a conserved GTP-binding domain; while no motifs were distributed at the N-terminus, suggesting that it is the main region for functional differentiation.

[0081] Analysis of the protein characteristics of small GTPases from shiitake mushrooms showed that: the protein length ranged from 128 (LeRan) to 609 (LeMitRho2) amino acid residues; the molecular weight ranged from 14.0 kDa (LeRan) to 66.0 kDa (LeMitRho2) (Table 2); the isoelectric point ranged from 4.75 (LeYpt7) to 9.32 (LeArf1), with 29 members having an isoelectric point below 7; 19 members had an instability index greater than 40; and 27 members had an aliphatic index greater than 80, indicating that this family of proteins has good stability and is beneficial for maintaining normal function under stress conditions; the average hydrophilicity index of 34 members was less than 0, suggesting that they have strong hydrophilic characteristics.

[0082] Table 2. Identification characteristics of small GTPases in shiitake mushrooms protein amino acid count molecular weight Theoretical pI Instability Index aliphatic index hydrophobic LeSec4 208 23098.01 5.29 42.33 84.42 -0.327 LeYpt7 203 22969.85 4.75 35.08 76.8 -0.38 LeYpt6 211 23469.59 5.29 44.57 79.48 -0.298 LeCdc42 192 21281.56 5.81 30.71 89.69 -0.075 LeYpt52 207 22807.75 6.42 40.4 74.54 -0.454 LeRac 196 21629.94 8.53 30.55 88.01 -0.196 LeArf6 181 20658.75 5.95 35.7 101.27 -0.138 LeRho1 194 21568.98 6.89 53.98 93.35 -0.125 LeArf1 182 20629.87 9.32 43.47 87.8 -0.369 LeYpt31 211 22981.81 5.95 27.87 88.25 -0.208 LeRab3 222 24224.2 5.35 45.29 80.72 -0.287 LeRho4 182 20553.56 7.67 42.98 86.76 -0.351 LeArl1 187 20679.7 5.12 48.51 100.53 -0.053 LeYpt1 232 26103.64 7.65 39.84 82.72 -0.237 LeArf2 182 20552.48 7.01 35.87 90.44 -0.007 LeYpt32 246 27137.47 6.6 43.49 81.3 -0.397 LeRas5 217 24007 5 37.45 72.67 -0.389 LeVPS21 256 27237.36 4.93 47.26 77.85 -0.397 LeRho3 229 25942.51 6.39 39.36 90.13 -0.172 LeRhb1 189 20663.64 5.48 33.46 92.28 -0.024 LeRho2 223 24720.08 5.87 42.06 76.95 -0.406 LeCin4 169 19019.52 5.56 33.67 91.12 -0.292 LeRsR1 187 21192.55 8.97 40.7 102.62 -0.129 LeMiRho2 601 66153.2 6.42 64.63 54.36 -0.859 LeSar1 189 21507.84 5.66 46.38 106.3 -0.01 LeTEM1 225 24917.6 6.43 36.09 88.36 -0.007 LeRas9 192 21901.08 8.62 41.45 79.69 -0.393 LeRhb2 202 22855.35 6.83 47.83 98.37 -0.077 LeArl2 414 46218.74 8.19 56.3 84.78 -0.25 LeMiRho1 627 70054.88 5.49 36.68 89.55 -0.183 LeGtr1 334 37205.46 6.16 53.31 84.67 -0.113 LeRan 128 14696.8 5.71 33.45 87.58 -0.393 LeArl3 155 17853.88 5.58 54.33 80.45 -0.686 LeGtr2 562 61438.07 6.32 59.08 84.96 -0.209 Note: All gene numbers have been replaced with the names of the identified proteins.

[0083] To elucidate the evolutionary relationships of small GTPase proteins in shiitake mushrooms, this study constructed a phylogenetic tree using the amino acid sequences of small GTPases from three basidiomycetes (strawberry, *Agaricus bisporus*, and *Cryptococcus neoformans*) and four ascomycetes (*Aspergillus fumigatus*, *Blastomyces oryzae*, *Aspergillus nidus*, and *Saccharomyces cerevisiae*), with *Saccharomyces cerevisiae* Sec13 and *Lentinula shiitake* LeSec13 as outgroups. Phylogenetic analysis showed that the shiitake mushroom small GTPase superfamily clustered into five branches, named Rho, Arf, Ras, Rab, and Ran based on the evolutionary sequence of the branch nodes. Figure 4 The Ran branch and the Rab branch are closely related in evolution. The number of members in each branch is as follows: 7 in the Rho subfamily, 10 in the Arf subfamily, 5 in the Ras subfamily, 10 in the Rab subfamily, and 1 in the Ran subfamily. Given that the functions of members of the small GTPase superfamily of Saccharomyces cerevisiae have been systematically analyzed, all homologous genes of Lentinus shiitake are named after their orthologous genes in Saccharomyces cerevisiae (which are at the same branch node in the phylogenetic tree) (Takai Y, Sasaki T and Matozaki T), and the orthologous protein LeRho1 of the unique yeast ScRho1 has been identified (amino acid sequence as shown in SEQ ID NO. 1 “MSEIRRKLVIVGDGACGKTCLLIVFSKGTFPEVYVPTVFENYVADVEVDGKHVELALWDTAGQEDYDRLRPLSYPDSHVILICFAVDSPDSLDNVQEKWISEVMHFCAGLPIILVGCKKDLRRDPRVIEELRKTSQRPVTPEEGMAVAQKIGAKHYLECSAKSGEGVREVFQYATRAALLSRPGKKKGSKCVVV”).

[0084] 1.2 Characteristics of LeRho1 and Lentinus edodes small G protein family genes The exon-intron structure of the small GTPase gene in shiitake mushroom was visualized and analyzed using TBtools software. Figure 2 The results showed that the LeRho1 gene has 6 exons and 4 introns, with its 3' untranslated region (UTR) being longer than its 5' UTR. Members of the same subfamily within the small G protein family exhibited similar gene structures, with highly conserved exon numbers, arrangement patterns, and lengths. The 3' UTR of most genes was longer than their 5' UTR, with LeRho4, LeGtr1, LeRhb1, and LeVps21 showing significantly longer 3' UTRs than other members. The Arf subfamily gene structure (except for LeMitRho1 or LeMitRho2) differed significantly from the other four subfamilies: the exon lengths of LeArf2, LeSar1, and LeArl1 were significantly longer than other members, and LeArf2 contained 11 introns (far exceeding the family average).

[0085] 1.3 Analysis of LeRho1 and its promoter transcriptional regulatory elements To investigate the expression regulation mechanism of LeRho1 and lentinan small GTPase family genes in response to environmental stress, this study systematically identified cis-regulatory elements in the promoter region of this gene family. Three types of cis-regulatory elements were detected in the upstream region of the 800 bp gene. Figure 4 The stress response elements include light-responsive elements, drought-induced elements, low-temperature-responsive elements, hypoxia-specific inducible elements, damage-responsive elements, and MYB-binding elements; hormone-responsive elements include auxin-responsive elements, methyl jasmonicate-responsive elements, gibberellin-responsive elements, and salicylic acid-responsive elements; enhancer regions are widely present in the promoter regions of all lentinan small GTPase genes. The LeRho1 promoter region specifically identified environmental stress response, low-temperature response, light response, and diurnal rhythm regulation elements. Notably, this promoter region contains six enhancer regions, consistent with the high expression level of the LeRho1 gene revealed by transcriptome data in this study (transcript abundance TMM value: 896-1319, ranked 148 / 10365th in the genome). Heat stress causes significant damage to fungi; RT-qPCR analysis showed that most members of the lentinan small GTPase family were upregulated, indicating that lentinan cells actively respond to heat stress. Of these, 25 genes (LeRho1, LeRac, LeCdc42, LeMiRho1, LeMiRho2, LeArf1, LeArf2, LeArf3, LeArl1, LeArl2, LeArl3, LeGtr1, LeGtr2, LeCin4, LeRan, and all genes of the shiitake Rab family) were upregulated. Figure 5 It is worth noting that although no heat shock response element was identified in the LeRho1 gene, its expression is still regulated by heat stress.

[0086] Example 2: Construction of LeRho1-eGFP / LeRho1-FLAG fusion protein expression vector and screening of transformants Target gene cloning: Primers for the target gene's open reading frame (ORF) were designed using Primer5 software, and the LeRho1 gene fragment was amplified using YS3334 DNA and cDNA as templates. The PCR amplification system is shown in Table 3.

[0087] Table 3 High-fidelity enzyme PCR amplification system Reagent Name Volume (μL) 2×PhantaMax buffer 20 dNTP mixture 1 PhantaMax high-fidelity DNA polymerase 1 upstream primer F 1 Downstream primer R 1 Template DNA (50 ng / µL) 4 <![CDATA[ddH2O]]> up to 40μL Reaction parameters: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 30 s / min, cycle number 33, 72℃ extension for 5 min. The target band was detected and recovered by 1% agarose gel electrophoresis, and the target gene fragment was recovered using the HLingene agarose gel recovery kit. The recovered target band was ligated into the enzyme-digested vector using Basicmix and transformed into *E. coli* Trans1-T1 competent cells. Single colonies were picked for PCR verification and sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for sequencing.

[0088] To screen for shiitake mushroom transformants with high LeRho1 expression, we constructed two fusion protein expression vectors: LeRho1-GFP and LeRho1-Flag. Figure 6 (A). This vector system employs a novel overexpression design and integrates the Kozak sequence "-GCCATC-", where the Kozak sequence is directly added to the translation start site (ATG) of the LeRho1 gene as shown in SEQ ID NO. 2 during primer synthesis, thus directly linking it to the gene.

[0089] The 1137 bp gene sequence of LeRho1 (with the 3' and 5' untranslated regions removed) was ligated, along with the eGFP sequence (as shown in SEQ ID NO. 3) and the Flag sequence (as shown in SEQ ID NO. 4), into the linearized pCAMBIA1300-E vector to form two recombinant plasmids: pCAMBIA1300-E-LeRho1-eGFP. Figure 8 ) and pCAMBIA1300-E-LeRho1-Flag ( Figure 9 Twenty-four positive single colonies from each vector were selected for PCR amplification and verification. The results showed that 8 single colonies with a 2.17kb band were found in the pCAMBIA1300-E-LeRho1-eGFP vector, and 16 single colonies with a 1.47kb band were found in the pCAMBIA1300-E-LeRho1-Flag vector. Figure 7 (A). The positive plasmid was transformed into Agrobacterium EHA105, and the Agrobacterium positive colonies were consistent with the E. coli colony bands ( Figure 7 (B)

[0090] YS3334 strain was genetically transformed using pCAMBIA1300-E-LeRho1-eGFP and pCAMBIA1300-E-LeRho1-Flag vectors. A single infection yielded 69 pCAMBIA1300-E-LeRho1-eGFP candidate transformants and 65 pCAMBIA1300-E-LeRho1-Flag candidate transformants. DNA extraction and target fragment verification showed that 61 of the pCAMBIA1300-E-LeRho1-eGFP transformants were positive (positive rate 88.4%, 61 / 69). Figure 7 Of the C, D); 58 transformants of pCAMBIA1300-E-LeRho1-Flag were positive (positive rate 89.2%, 58 / 65); Figure 7 (D, E, F). Three positive transformants from each vector were randomly selected for RT-qPCR detection. The results showed that the mRNA expression levels of G16, G22 (eGFP line), F60 (eGFP line), and F25 (Flag line) of YS3334-LeRho1 were significantly higher than those of the empty vector control (YS3334). Figure 6 (B). Laser confocal microscopy analysis showed that the LeRho1-eGFP fusion protein in the YS3334-LeRho1-eGFP transformant exhibited a high-intensity fluorescence signal (B). Figure 6 (C). Western blot analysis further confirmed that the YS3334-LeRho1-Flag transformants F25, F42, and F60 all expressed the LeRho1-Flag fusion protein, with F25 and F42 showing higher expression levels, and F60 showing a highly significant expression level. Figure 6 (D).

[0091] Example 3: Effect of LeRho1 on the stress resistance of shiitake mycelium (1) LeRho1 enhances the heat stress resistance of shiitake mushroom mycelium To investigate the effect of LeRho1 overexpression on the heat-resistant shiitake mushroom strain YS3334, 5-day-old YS3334-CK (empty vector control), YS3334-G13, YS3334-G16, YS3334-G22 (eGFP-labeled transformant), YS3334-F25, YS3334-F45, and YS3334-F60 (Flag-labeled transformant) were subjected to heat shock at 38℃ for 24 h, followed by 5 days of recovery culture. After 5 days of recovery culture, the relative mycelial growth rate was calculated using the formula [(recovery days) / pre-culture days] × 100%. The results showed that ( Figure 10The heat resistance of YS3334-G16, YS3334-G22, YS3334-F60 and YS3334-F25 was significantly higher than that of YS3334-CK (P<0.01), with an average relative recovery growth rate of 76.7% (F25) to 115.2% (G22).

[0092] Cold tolerance test: The strains were cultured at 15°C for 5 days, followed by a recovery culture at 25°C for 5 days. Under low temperature stress, the mycelial growth rate of the LeRho1 transgenic strains (YS3334-G16, YS3334-F60, YS3334-F25, and YS3334-F42 of Lentinus edodes) was significantly reduced compared with the control group YS3334-CK.

[0093] (2) LeRho1 enhances the resistance of shiitake mushroom mycelium to Trichoderma infection. After 10 days of dark incubation of *Lentinula edodes* mycelium on MYG plates at 25°C, the central portion of an 8mm diameter aged mycelial block was removed, and an equal amount of activated *Trichoderma* mycelial blocks were inoculated. Photos were taken 5 days later, and the *Trichoderma* infection area (S) was calculated using ImageJ software. The infection areas of LeRho1-G13, G16, F60, and F25 were significantly smaller than the control. Figure 10 In the control group (G22), the overexpression area ranged from 0.05 cm² to 0.18 cm² (F25), with the control group showing an increase in Trichoderma resistance of 57.2%-88.1%. Figure 10 (A, C)

[0094] (3) LeRho1 enhances the light stress resistance of shiitake mushroom mycelium Light sensitivity test: The cells were exposed to light at 25°C and 3500 lx for one day, followed by recovery culture at 25°C in darkness for five days. Light stress resistance: Under light stress, except for LeRho1-G13, the recovery rate of hyphal diameter in all transformants was significantly lower than that in the control group. Figure 10 The relative mycelial growth rates of the LeRho1 overexpressing transformants ranged from 57.8% (LeRho-G16) to 69.7% (LeRho1-G22), with the control group at 93.0%.

[0095] It is worth noting that although YS3334-G13 exhibits a strong GFP fluorescence signal ( Figure 6 However, LeRho1 overexpression did not show enhanced resistance in any stress tests, suggesting an abnormal T-DNA insertion site or the expression of only a truncated eGFP protein, leading to loss of LeRho1 function. In conclusion, LeRho1 overexpression significantly enhances the heat resistance, resistance to Trichoderma, and photosensitivity of shiitake mushrooms, confirming its potential as a key target for molecular breeding of edible fungi.

[0096] Example 4: Screening of LeRho1 interacting proteins under heat stress (1) Western blot of proteins Protein extraction: Protein extraction conditions were optimized. After grinding with liquid nitrogen, RIPALysis Buffer (TargetMol, C0045) containing 1 mM PMSF was added. After lysis on ice for 30 min, the protein was sonicated at low frequency at 4 °C for 3 min and the supernatant was collected by centrifugation.

[0097] Protein denaturation: Take 40 μL of protein supernatant and add 10 μL of 5-fold protein loading buffer (ABclonal, RM00001), and denature at 100℃ for 5 min. Prepare a 10% SDS-PAGE gel, load 10 μL of protein, and separate by electrophoresis at 150V. Marker (ABclonal, RM19001).

[0098] Transfer: Semi-dry transfer method, 0.45μm PVDF membrane (Millipore, IPVH00010), constant voltage 15V, 15min.

[0099] Sealing: 4% BSA (BioFroxx, 4240GR500), sealing for 1.5h.

[0100] Primary antibody binding: Anti-GFP (ABclonal, AE011), incubated overnight at 4°C, washed 6 times with TBST for 5 min each time.

[0101] Secondary antibody binding: Goat Anti-Rabbit IgG H&L (Jingjie Biotechnology, PTM-6261), incubated with secondary antibody for 1.5 h, washed 6 times with TBST for 5 min each time.

[0102] Color development: ECL chemiluminescent substrate (ABclonal, RM02867).

[0103] Western blot analysis showed that the LeRho1-GFP fusion protein exhibited a clear band at 48.57 kDa. Figure 11 (Middle left).

[0104] To elucidate the protein-protein interaction network of LeRho1 under heat stress, the YS3334-G22 transformant with optimal protein expression was selected for pre- and post-heat shock co-precipitation (Co-IP) analysis. The specific Co-precipitation results of the LeRho1-eGFP fusion protein are as follows: Magnetic bead preparation: Add an appropriate amount of magnetic beads (ABclonal) to 1 mL of pre-chilled PBS, gently mix for 5 min on a mixer, remove the supernatant on a magnetic rack, and repeat 3 times. Immunoprecipitation: Add 800 μL of pre-chilled PBS and 200 μL of protein supernatant, and incubate overnight at 4°C by inversion.

[0105] Denaturation elution: Discard the supernatant, add 50 μL of protein loading buffer, and denature the protein for Western blot detection. Electrophoresis: Prepare a 12% SDS-PAGE gel, separate by electrophoresis at 150V, using an ABclonal RM19001 marker. Transfer, blocking, primary antibody binding, secondary antibody binding, and color development are the same as for Western blot.

[0106] The target protein was significantly enriched in the Co-IP product. Figure 11 (Middle right, lanes 3, 4) The IgG negative control showed no extraneous bands in the target molecular weight region. Figure 11 (Right-middle lane, lane 5). The bands obtained by immunoprecipitation (IP) were analyzed by mass spectrometry, and the mass spectrometry identification results were subjected to GO (Gene Ontology) enrichment analysis.

[0107] Comprehensive analysis suggests that the functional proteins identified by Rho1-GFP immunoprecipitation may be involved in: redox-driven transmembrane transporter activity, GTP binding, GTPase activity, phosphatidylinositol binding, fungal cell wall formation, transmembrane transporter complex assembly, and carboxylic acid metabolism and oxidative phosphorylation. This indicates that LeRho1 may be involved in cell membrane signal transduction, phosphatidylinositol coupling, GTPase activation, and fungal cell wall synthesis under heat stress.

[0108] Quantitative analysis of protein components identified by LeRho1-GFP immunoprecipitation screened potential stress-response proteins, cell wall integrity (CWI) signaling pathway proteins, and cell wall polysaccharide synthesis-related proteins, and an expression abundance heatmap was constructed. The identified stress-response proteins included members of the stress channel protein family LeUSPA and LeUSPH, stress-response proteins NST1 / 2, the CMGC protein kinase family, calmodulin (CaMK), voltage-gated potassium channels (KV), and Ca²⁺ channel proteins. LeUSPH and Ca²⁺ channel proteins were specifically detected only in the heat shock group, while NST1, CMGC, and KV showed significantly upregulated expression after heat shock.

[0109] Core components LeRho1, PKC1, MKK, MAPK1, and MAPK2 were identified in the CWI pathway, but BCK1 kinase was not detected. Protein abundance analysis showed that MKK was significantly upregulated after heat shock, MAPK1 was only detected at room temperature, while MAPK2 was stable at both room temperature and heat shock. These results differ from the single MAPK component stl2 in yeast; two homologous proteins, MAPK1 and MAPK2, are present in the shiitake mushroom strain.

[0110] Cell wall synthesis-related proteins include β-1,3-glucan synthase (FKS1), glucose 1,3-β-glucanase D, α-1,3-glucan synthase (ags1), L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoacetylglucosamine mutase 1 / 2, UDP-N-acetylglucosamine pyrophosphorylase, chitin synthase family (Chitinsynthase 1 / 3 / 7), chitin synthase regulatory factor 4, CHAPS domain proteins, and mannan endonuclease-1,6-α-mannosylase (DCW1). Among these, the abundance of α-1,3-glucan synthase, phosphoacetylglucosamine mutase 2, chitin synthase family, and DCW1 significantly increased after heat shock; while glucose 1,3-β-glucanase D was not detected after heat shock treatment, suggesting that it may be involved in maintaining cell wall homeostasis under normal temperature conditions. Heat shock treatment can alter the composition and abundance of LeRho1-GFP interacting proteins, especially in the cell wall synthesis pathway where chitin synthase has multiple isoforms. Among them, Chitinsynthase1 may dominate the chitin synthesis compensation mechanism under heat stress.

[0111] Example 5: Effect of LeUSPA / LeUSPH on the heat resistance of shiitake mushrooms (1) Construction of LeUSPA / LeUSPH-mCHERRY fusion expression vector and LeUSPA / LeUSPH gene RNAi silencing vector To further elucidate the association between LeUSPA / LeUSPH and LeRho1, this study constructed a red fluorescent (mCherry) expression vector containing LeUSPA or LeUSPH and Carbix resistance. The sequence of the inserted LeUSPA gene is shown in SEQ ID NO. 7, and the sequence of the LeUSPH gene is shown in SEQ ID NO. 8. The same Kozak sequence was used upstream of the recombinant DNA fragment. To reduce the impact of the large spatial conformation of the mCherry protein on the cellular localization and activity of LeUSPA / LeUSPH proteins, a linker sequence (e.g., SEQ ID NO. 9: -GGCGGTGGTGGCTCTGGTGGCGGTGGCTCC-) was added between them to construct the pCAMBIA-SDHB-LeGPDi-LeUSPA-mCherry vector. pCAMBIA-SDHB-LeGPDi-LeUSPH-mCherry vector.

[0112] For the vector pCAMBIA-SDHB-LeGPDi-LeUSPA-mCherry or The bacteria were transformed into Agrobacterium tumefaciens EHA105 using the transformation method, and a single positive clone was selected for colony PCR verification. Figure 13 (Lower middle section), after being identified as a positive clone, it is stored at -80℃.

[0113] To elucidate the association between LeUSPA / LeUSPH and heat tolerance in shiitake mushrooms, this study also constructed a LeUSPA / LeUSPH gene silencing vector. Due to the uncertainty of mRNA expression by bidirectional promoters, we constructed a hairpin-structured RNAi silencing vector, pCAMBIA-E-LeGPDi-RiIntron-LeUSPA (…). Figure 14 ) and pCAMBIA-E-LeGPDi-RiIntron-LeUSPH vector ( Figure 15 The inserted 527bp CDS sequence for silencing LeUSPA is shown in SEQ ID NO. 10, and the inserted 692bp CDS sequence for silencing LeUSPH is shown in SEQ ID NO. 11. The constructed vector was transformed into *E. coli*, and colony PCR, verification, and sequencing were performed. The electrophoresis band sizes were 1kb and 1.1kb, respectively. The electrophoresis bands and sequencing results indicate that the gene silencing vector was successfully constructed. The plasmid was transformed into *Agrobacterium* EHA105, and a single positive clone was selected for colony PCR verification. After confirming a positive clone, it was stored at -80℃.

[0114] (2) Screening and validation of LeUSPA / LeUSPH-mCherry red fluorescent protein fusion expression transformants and LeUSPA / LeUSPH silencing transformants of strain YS3334 The red fluorescent fusion expression vector LeUSPA / LeUSPH and the RNAi silencing vector were transformed into the YS3334-G22 transformant and the YS3334 wild-type shiitake mushroom strain using Agrobacterium-mediated transformation. After a single infection, the infected mycelial blocks were screened on MYG medium resistant to both hygromycin and carboxin. Twenty transformants were obtained with the LeUSPA-mCherry vector, and thirteen transformants were obtained with the LeUSPH-mCherry vector. Fluorescence microscopy revealed that all transformants exhibited significant red and green fluorescent signals. (Image: [Image not provided]) Figure 16 (A to C represent co-expression of LeRho1-eGFP and LeUSPA-mCherry; D to F represent co-expression of LeRho1-GFP and LeUSPH-mCherry.)

[0115] After a single infection, the gene silencing vectors LeUSPA / LeUSPH and the hairpin-structured empty vector were used. The infected mycelial blocks were screened on hygromycin-resistant MYG medium, yielding 167 and 142 potential transformants, respectively. Forty transformants from each vector were selected for DNA extraction, PCR verification, and RNA extraction to detect target gene expression levels. Electrophoresis showed 24 positive transformants for gene silencing with LeUSPA, 24 positive transformants for gene silencing with LeUSPH, and 7 interfering empty vector strains. Figure 17 Eight LeUSPA gene-silencing transformants and six LeUSPH gene-silencing transformants were selected for RT-qPCR detection of target gene downregulation. All transformants showed varying degrees of downregulation. LeUSPA-1 gene expression was downregulated by 90% compared to the control (CK). LeUSPA-3, LeUSPA-5, LeUSPA-6, LeUSPA-10, and LeUSPA-11 were downregulated by 44%, 34%, 42%, 51%, and 63%, respectively. LeUSPH-1, LeUSPH-2, LeUSPH-3, LeUSPH-4, and LeUSPH-7 gene expression was downregulated by 30%, 50%, 63%, 49%, and 58%, respectively, compared to the CK.

[0116] Example 6: Effect of LeUSPA / LeUSPH gene silencing on the heat resistance of shiitake mushroom strain YS3334 mycelium To investigate the effects of LeUSPA and LeUSPH gene silencing on the heat-resistant shiitake mushroom strain YS3334, 5-day-old YS3334-CK (empty vector control pCAMBIA-SDHB-LeGPDi-mCherry), PeRi-LeUSPA-1, PeRi-LeUSPA-3, PeRi-LeUSPA-10, PeRi-LeUSPA-11, PeRi-LeUSPH-2, PeRi-LeUSPH-4, and PeRi-LeUSPH-7 mycelial blocks were selected. Mycelial blocks were inoculated onto MYG agar plates and pre-cultured at 25°C in the dark for 5 days. After pre-culture, the two vertical diameters of the colonies were measured using the cross-hatching method and recorded as D1A and D1B. The colonies were then heat-shocked at 38°C for 24 hours and recovered for 5 days. After the recovery period, the colony diameter was measured again using the cross-hatching method, and the diameters D2A (major axis) and D2B (orthogonal axis) were recorded for each sample. Calculate the growth increment: DA = D2A - D1A and DB = D2B - D1B. Calculate the relative growth rate using the simplified formula: QQ (%) = (ΔD / D1) × 100, where for the horizontal axis, ΔD = DA and D1 = D1A; for the vertical axis, ΔD = DB and D1 = D1B.

[0117] After 5 days of recovery culture, the relative mycelial growth rate was calculated using the formula [(recovery days) / pre-culture days] × 100%. Among them, the mycelial growth rate of LeUSPA-1 decreased to 24% of the normal temperature growth rate after heat shock treatment, which was 12.5% ​​lower than the empty vector control strain PeRiCK-2. PeRi-LeUSPA-1 showed a significantly enhanced mycelial recovery ability after heat shock. The mycelial growth rates of PeRi-LeUSPA-3, PeRi-LeUSPA-10, PeRi-LeUSPA-11, PeRi-LeUSPH-2, PeRi-LeUSPH-4, and PeRi-LeUSPH-7 decreased by 22.8%, 30.6%, 25.1%, 25.3%, 35.4%, and 39.5%, respectively, all showing significant differences. Figure 18 The experimental results showed that silencing the LeUSPA or LeUSPH genes could improve the heat resistance of shiitake mushrooms, indicating that the LeUSPA and LeUSPH genes negatively regulate the heat resistance of shiitake mushroom strains.

[0118] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A LeRho1 gene expression vector, characterized in that, The expression vector is a recombinant expression vector into which a target gene encoding LeRho1 protein is inserted, and the amino acid sequence of the LeRho1 protein is shown as SEQ ID NO.

1.

2. The LeRhol gene expression vector as set forth in claim 1, wherein, The nucleotide sequence of the target gene LeRho1 is shown as SEQ ID NO.

2.

3. The LeRhol gene expression vector as set forth in claim 2, wherein, The recombinant expression vector further integrates a Kozak sequence specific to Lentinula edodes, and the Kozak sequence is shown as "-GCCATC-" in SEQ ID NO. 5, which is directly connected to the translation initiation site "ATG" of the target gene LeRho1.

4. The LeRhol gene expression vector according to any one of claims 1 to 3, wherein, It also carries a resistance gene, a gene encoding a tag protein, or a gene encoding a fluorescent protein; the gene encoding a tag protein or a gene encoding a fluorescent protein is connected downstream of the target gene LeRho1.

5. The LeRhol gene expression vector of claim 4, wherein, It is a recombinant plasmid for expressing a fusion protein, and the fusion protein is LeRho1-tag protein or LeRho1-fluorescent protein; the tag protein includes Flag tag, HA tag, Myc tag; and the fluorescent protein includes green fluorescent protein GFP, eGFP, red fluorescent protein mCherry.

6. The LeRhol gene expression vector as set forth in claim 4, wherein, It is a recombinant plasmid carrying the target gene LeRho1, a resistance gene, and a fluorescent protein gene, and the promoter driving the resistance gene is a LeEF1α promoter, and the sequence of the LeEF1α promoter is shown as SEQ ID NO.

6.

7. The LeRho 1 gene expression vector as set forth in claim 6, wherein, The recombinant plasmid carrying the target gene LeRho1, a resistance gene, and a fluorescent protein gene is prepared by the following method: The CaMV35S promoter of the pCAMBIA1300 vector is replaced with the LeEF1α promoter of Lentinula edodes to construct a pCAMBIA1300-E backbone vector; the pCAMBIA1300-E backbone vector itself carries a resistance gene connected downstream of the LeEF1α promoter; and the resistance gene includes the resistance gene Hyg; The Kozak sequence shown as SEQ ID NO. 5 is connected to the translation initiation site "ATG" of the target gene LeRho1, and the target gene LeRho1 and the fluorescent protein gene are inserted into the pCAMBIA1300-E backbone vector by EcoRI / BamHI double digestion to obtain a recombinant plasmid.

8. A method for improving heat tolerance of a Lentinula edodes strain and resistance to Trichoderma, characterized by, It includes the steps of transforming the LeRho1 gene expression vector into a Lentinula edodes strain to prepare transformants, and screening positive transformants.

9. A method for improving heat tolerance of a Lentinula edodes strain, characterized by, It includes the steps of transforming the gene silencing vector for silencing LeUSPA and / or LeUSPH into a Lentinula edodes strain to prepare transformants, and screening positive transformants.

10. The gene silencing vector is a hairpin RNAi silencing vector, which includes an interference fragment and a loop connecting the interference fragment, and the loop connecting the interference fragment is an intron sequence of a highly expressed gene of Lentinula edodes, shown as SEQ ID NO. 12; The gene silencing vector for silencing LeUSPA includes an interference fragment shown as the sequence of SEQ ID NO. 10; The gene silencing vector for silencing LeUSPH includes an interference fragment shown as the sequence of SEQ ID NO. 11.

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