Method for creating a cordyceps militaris strain with enhanced resistance to white muscadine disease using crisper / cas9 technology

Abstract

This invention belongs to the field of genetic breeding technology for edible and medicinal fungi, and relates to a Cordyceps militaris strain with enhanced resistance to white spot disease and its creation method. This invention provides a safe overexpression site CmSH1 (SEQ ID NO:1) in the Cordyceps militaris genome, the gene Cmhyd1 (SEQ ID NO:2) encoding a hydrophobic protein in Cordyceps militaris, a Cordyceps militaris strain with enhanced resistance to white spot disease, a method for creating the Cordyceps militaris strain with enhanced resistance to white spot disease using CRISPR / Cas9 gene editing technology, and a corresponding vector. The Cordyceps militaris strain of this invention can enhance resistance to white spot disease, and the fruiting body develops normally. Its creation method avoids food safety issues caused by the integration of transgenic elements into the genome.

Description

Technical Field

[0001] This invention belongs to the field of genetic breeding technology of edible and medicinal fungi, specifically involving the safe overexpression site CmSH1 of the Cordyceps militaris (L.) Fr. genome, the hydrophobic protein gene Cmhyd1, and Cordyceps militaris strains with enhanced resistance to white spot disease, as well as the method for creating Cordyceps militaris strains with enhanced resistance to white spot disease through CRISPR / Cas9 gene editing technology. Background Technology

[0002] White hair disease of Cordyceps militaris is a fungal disease caused by Calcarisporium cordycipiticola, which affects the quality and yield of Cordyceps militaris and is the number one killer in the cultivation process. Moreover, the disease generally occurs in the later stage of Cordyceps militaris growth and development, infecting the fruiting body and producing a large number of conidia, which is very difficult to control in production (Liu Qing, Wan Jiaxin, Zhang Yuchen, Dong Caihong. Study on biological characteristics of the pathogenic fungus Calcarisporium cordycipiticola of Cordyceps militaris [J]. Acta Mycosystema Sinica, 2018, 37(08):1054-1062.DOI:10.13346 / j.mycosystema.180034.). With the rapid development of molecular biology, genomics, genetics and gene editing technology, the molecular mechanism of Cordyceps militaris disease resistance has been continuously deepened, and many genes or proteins have been found to participate in the Cordyceps militaris disease resistance response process. Due to the limitations of traditional breeding methods, it is particularly important to use molecular means to select disease-resistant Cordyceps militaris strains. If genes related to resistance to white hair disease in Cordyceps militaris are discovered through genomics, transcriptomics, and related molecular techniques, and then edited using the CRISPR / Cas9 system to produce Cordyceps militaris strains that overexpress the resistance gene, then a convenient and efficient alternative to traditional Cordyceps militaris breeding methods can be provided. Summary of the Invention

[0003] In view of this, the purpose of this invention is to find target genes in the Cordyceps militaris genome that are safe overexpression sites related to enhanced resistance to white spot disease, and then to create Cordyceps militaris strains with enhanced resistance to white spot disease by using CRISPR / Cas9 (Clustered regularly interspaced shortpalindromic repeats / Cas9) gene editing technology to specifically overexpress the target genes, thereby providing new strains for the safe production of Cordyceps militaris and providing genetic material for the targeted improvement of new varieties.

[0004] On the one hand, the present invention provides a DNA molecule that is a safe overexpression site CmSH1 in the Cordyceps militaris genome, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0005] On the other hand, the present invention also provides a DNA molecule that is the Cordyceps militaris hydrophobic protein encoding gene Cmhyd1, and its nucleotide sequence is shown in SEQ ID NO:2.

[0006] In a third aspect, the present invention provides a Cordyceps militaris strain with enhanced resistance to white spot disease, wherein the strain overexpresses the target gene Cmhyd1 (SEQ ID NO:2) at the safe overexpression site CmSH1 (SEQ ID NO:1). This strain is currently deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC No. 40324 and deposit date of September 22, 2022.

[0007] In a fourth aspect, the present invention provides a method for creating Cordyceps militaris strains with enhanced resistance to white spot disease using CRISPR / Cas9 gene editing technology, the method comprising the following steps:

[0008] S1. Identify safe overexpression sites in the Cordyceps militaris genome;

[0009] S2. Identify the target genes in the Cordyceps militaris genome that enhance resistance to white spot disease;

[0010] S3. Determine the sgRNA at the safe overexpression site and construct a CRISPR-Cas9 site-directed overexpression intermediate vector expressing the sgRNA;

[0011] S4. Determine and amplify the upstream and downstream homologous arm nucleotide sequences of the safe overexpression site;

[0012] S5. Identify and amplify the complete expression cassette of the target gene;

[0013] S6. Connect the upstream and downstream homologous arm nucleotide sequences described in S4 and the complete expression cassette described in S5 to the CRISPR-Cas9 site-directed overexpression intermediate vector described in S3 to construct the target gene site-directed overexpression vector; and

[0014] S7. Transform the vector obtained in S6 into a pupae grassland bioplast to obtain a transformant that overexpresses the target gene.

[0015] In an embodiment of the fourth aspect of the present invention, the safe overexpression site is CmSH1, the nucleotide sequence of which is shown in SEQ ID NO:1. In an embodiment of the fourth aspect of the present invention, the upstream and downstream homologous arm nucleotide sequences of the safe overexpression site are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively. In an embodiment of the fourth aspect of the present invention, the nucleotide sequence of the sgRNA of CmSH1 is shown in SEQ ID NO:3.

[0016] In an embodiment of the fourth aspect of the present invention, the target gene is Cmhyd1, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0017] In an embodiment of the fourth aspect of the present invention, the nucleotide sequence of the complete expression cassette is shown in SEQ ID NO:9.

[0018] In an embodiment of the fourth aspect of the present invention, the method further includes the following steps:

[0019] S8. At the genomic DNA level, identify the transformants that site-specifically overexpress the target gene from the transformants obtained in S7, i.e., positive transformants; S9. At the cDNA level, verify the positive transformants obtained in S8 and confirm that the target gene is highly expressed in the positive transformants; S10. Passage the positive transformants obtained in S9 to obtain Cordyceps militaris strains with vector loss, no foreign gene insertion, and overexpression of the target gene; and S11. Infect the fruiting bodies developed by the Cordyceps militaris strain obtained in S10 with Cordyceps spores to verify whether the fruiting bodies overexpress the target gene and enhance resistance to white spot disease.

[0020] In a fifth aspect, the present invention provides a recombinant vector or expression cassette comprising the Cordyceps militaris hydrophobic protein encoding gene Cmhyd1 (SEQ ID NO:2).

[0021] In a sixth aspect, the present invention provides a CRISPR / Cas9 gene editing vector comprising the sgRNA of the safe overexpression site CmSH1 (SEQ ID NO:3), the upstream and downstream homologous arm nucleotide sequences of CmSH1 (SEQ ID NO:5, SEQ ID NO:6), and the Cordyceps militaris hydrophobic protein encoding gene Cmhyd1 (SEQ ID NO:2).

[0022] In an embodiment of the sixth aspect of the present invention, the skeletal carrier of the carrier may be a pAMA1-Cas9-hyg carrier.

[0023] In a seventh aspect of the invention, the invention relates to the application of DNA molecules of the first and second aspects, recombinant vectors or expression cassettes of the fifth aspect, or CRISPR / Cas9 gene editing vectors of the sixth aspect, in regulating resistance to Cordyceps militaris white spot disease.

[0024] In an eighth aspect of the invention, the invention relates to the use of the safe overexpression site CmSH1 in gene complementation and overexpression.

[0025] In a ninth aspect, the present invention provides the use of a hydrophobic protein from Cordyceps militaris in enhancing resistance to and / or preventing white spot disease, the hydrophobic protein being encoded by the gene Cmhyd1.

[0026] In a tenth aspect, the present invention provides the use of a hydrophobic protein from Cordyceps militaris in the preparation of formulations that enhance resistance to and / or prevent white spot disease, the hydrophobic protein being encoded by the gene Cmhyd1.

[0027] Compared with existing factory-produced Cordyceps militaris strains, this invention has significant advantages. This invention utilizes CRISPR / Cas9 gene editing technology to overexpress the potential disease-resistant gene Cmhyd1 in the Cordyceps militaris genome at the safe overexpression site CmSH1, thereby creating a Cordyceps militaris strain with enhanced resistance to white spot disease without affecting the commercial viability of the fruiting bodies. This not only helps reduce the harm of white spot disease to the Cordyceps militaris industry but also avoids food safety issues caused by the integration of transgenic elements into the genome. It provides a new approach and experimental basis for disease-resistant breeding of Cordyceps militaris, and compared with traditional breeding methods, it is faster and more targeted. Attached Figure Description

[0028] Figure 1 The red fluorescent protein expression status of wild-type Cordyceps militaris strain (WT) and CmSH1 site-directed red fluorescent protein expression strains (CmSH1-RFP-15, 66 and 74) is shown.

[0029] Figure 2 The fruiting body growth of wild-type Cordyceps militaris strain (WT) and CmSH1-expressing red fluorescent protein strains (CmSH1-RFP-15, 66 and 74) is shown.

[0030] Figure 3 The results of identifying the Cmhyd1 site-directed overexpression vector pAMA1-CmSH1-sgRNA-up-Cmhyd1-down are shown. A: PCR verification of the pAMA1-CmSH1-sgRNA-up-Cmhyd1-down vector, 1: DL2000 labeling, 2-4: pAMA1-CmSH1-sgRNA-up-Cmhyd1-down, 5: water, 6: pAMA1-Cas9-hyg; B: Restriction digestion verification of pAMA1-CmSH1-sgRNA-up-Cmhyd1-down, 1: BsrGI single digestion, 2: BsrGI and SspI double digestion, 3: 15kb labeling, 4: DL2000 labeling.

[0031] Figure 4The PCR detection of the Cmhyd1 site-directed overexpression strain (CmSH1-Cmhyd1oe) is shown, where A: strategy of overexpressing Cmhyd1 at the CmSH1 site using Cas9; B: PCR detection of CmSH1-Cmhyd1oe, M: DL2000, 1: CmSH1-Cmhyd1oe, 2: pAMA1-CmSH1-sgRNA-up-Cmhyd1-down, 3: WT, 4: water; C: detection of Cmhyd1 expression level in mycelia of wild-type Cordyceps militaris strain (WT) and Cmhyd1 site-directed overexpression strain (CmSH1-Cmhyd1oe).

[0032] Figure 5 Hygromycin susceptibility assays were performed on wild-type Cordyceps militaris strain (WT) and Cmhyd1 site-directed overexpression strain (CmSH1-Cmhyd1oe).

[0033] Figure 6 The fruiting body growth of wild-type Cordyceps militaris strain (WT) and Cmhyd1 site-directed overexpression strain (CmSH1-Cmhyd1oe) is shown.

[0034] Figure 7 The fruiting body characteristics of wild-type Cordyceps militaris strain (WT) and Cmhyd1 site-directed overexpression strain (CmSH1-Cmhyd1oe) are shown.

[0035] Figure 8 The disease incidence of wild-type Cordyceps militaris strain (WT) and Cmhyd1 site-directed overexpression strain (CmSH1-Cmhyd1oe) 10 days after inoculation with Cordyceps militaris 10 days later was shown.

[0036] Figure 9 The results show the number of diseased fruiting bodies and the area of ​​lesions 10 days after inoculation of wild-type Cordyceps militaris strain (WT) and Cmhyd1 site-directed overexpression strain (CmSH1-Cmhyd1oe) with Cordyceps sphaerophores.

[0037] Figure 10 The expression levels of Cmhyd1 in the fruiting bodies of wild-type Cordyceps militaris strain (WT) and Cmhyd1 site-directed overexpression strain (CmSH1-Cmhyd1oe) were shown 7 days after infection with Cordyceps dentata. Among them, WTi: wild-type Cordyceps militaris strain infected with Cordyceps dentata 7 days after infection, and CmSH1-Cmhyd1oeei: Cmhyd1 site-directed overexpression strain infected with Cordyceps dentata 7 days after infection. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art should understand that the embodiments described below are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Currently, gene overexpression and complementation in Cordyceps militaris generally employ random integration. However, this random integration can lead to positional effects, resulting in inconsistent expression levels. A solution to this problem is to select a transcriptionally stable and active genomic region and then target and integrate a gene into that region. This transcriptionally stable and active region is called a "genomic safe harbor," a concept originally derived from higher eukaryotes, where the region is considered to possess transcriptional activity without causing any identifiable phenotypic effects. Such regions have been identified and widely used in Cryptococcus neoformans and Aspergillus fumigatus.

[0040] CRISPR / Cas9 gene editing technology can improve bacterial strains through targeted genome editing. Compared with traditional breeding methods such as hybridization, mutagenesis, and domestication, this technology has advantages such as high targeting, short operation cycle, simplicity, high efficiency, and no introduction of exogenous DNA fragments. The vector element AMA1 (a self-replicating nucleotide sequence in filamentous fungi) ensures that the vector replicates independently of the chromosome. Therefore, the vector almost never inserts into the fungal genome. Under conditions without antibiotic selection pressure, vector loss in transformants is easily achieved, thus realizing truly traceless editing without exogenous gene insertion and avoiding the safety issues associated with transgenic technology.

[0041] Therefore, if a "safe harbor" can be identified in the Cordyceps militaris genome, and a target gene related to resistance to white spot disease can be found, then CRISPR / Cas9 editing technology can be used to perform site-specific overexpression of the target gene at the safe harbor, i.e., the safe site, to obtain Cordyceps militaris strains with enhanced resistance to white spot disease. This invention surprisingly discovers that the Cmhyd1 gene in the Cordyceps militaris genome encodes a hydrophobic protein associated with resistance to white spot disease, and further confirms that CmSH1 is a safe overexpression site in the Cordyceps militaris genome, thus leading to this invention.

[0042] Therefore, this invention provides a safe overexpression site CmSH1 in the Cordyceps militaris genome, the nucleotide sequence of which is shown in SEQ ID NO:1. The mycelial growth rate and light-induced color change (carotenoid accumulation) of the CmSH1-expressed Cordyceps militaris strain were no different from those of the wild-type strain. CmSH1-expressed red fluorescent protein produced strong red fluorescence in the mycelia, and the strain could develop normally into fruiting bodies on wheat culture medium, indistinguishable from the wild-type. This indicates that CmSH1-expressed overexpression has no effect on the cultivation traits of Cordyceps militaris, and therefore can be used as a safe overexpression site in Cordyceps militaris.

[0043] On the other hand, the present invention provides a DNA molecule that is the Cordyceps militaris hydrophobic protein encoding gene Cmhyd1, and its nucleotide sequence is shown in SEQ ID NO:2.

[0044] In a third aspect, the present invention provides a Cordyceps militaris strain with enhanced resistance to white spot disease, wherein the strain specifically overexpresses the Cmhyd1 gene at the safe overexpression site CmSH1. The mycelial growth rate and light-induced color change (carotenoid accumulation) of this Cordyceps militaris strain are not significantly different from those of the wild-type strain, and it can normally develop into fruiting bodies on wheat culture medium, with enhanced resistance to white spot disease.

[0045] In a fourth aspect, the present invention provides a method for creating Cordyceps militaris strains with enhanced resistance to white spot disease using CRISPR / Cas9 gene editing technology. The method includes the following steps: S1, identifying a safe overexpression site in the Cordyceps militaris genome; S2, identifying a target gene in the Cordyceps militaris genome that enhances resistance to white spot disease; S3, identifying the sgRNA of the safe overexpression site and constructing a CRISPR-Cas9 site-directed overexpression intermediate vector expressing the sgRNA; S4, identifying and amplifying the upstream and downstream homologous arm nucleotide sequences of the safe overexpression site; S5, identifying and amplifying the complete expression cassette of the target gene; S6, ligating the upstream and downstream homologous arm nucleotide sequences described in S4 and the complete expression cassette described in S5 to the CRISPR-Cas9 site-directed overexpression intermediate vector described in S3 to construct a site-directed overexpression vector for the target gene; and S7, transforming the vector obtained in S6 into Cordyceps militaris protoplasts to obtain a transformant overexpressing the target gene.

[0046] In an embodiment of the present invention, CmSH1 in the Cordyceps militaris genome was identified as a safe overexpression site through genomic and multiple transcriptome analyses and red fluorescent protein site-specific expression, and can be used for Cordyceps militaris gene complementation and overexpression. CmSH1 is the spacer region located between the CCM_00870 and CCM_00871 genes of Cordyceps militaris, and its nucleotide sequence is shown in SEQ ID NO:1.

[0047] In an embodiment of the present invention, through interaction transcriptome analysis and exogenous expression of CmHYD1 in Pichiapastoris, the present invention determined that the gene encoding a hydrophobic protein in the Cordyceps militaris genome is associated with the resistance of Cordyceps militaris to white spot disease, and is called Cmhyd1, which can be used as a target gene. Its nucleotide sequence is shown in SEQ ID NO:2.

[0048] sgRNA (single guide RNA) is an important component of the CRISPR gene knock-in / knockout system. It binds to the Cas9 enzyme and guides the Cas9 enzyme to target and cleave genomic DNA. In the embodiments of this invention, the sgRNA nucleotide sequence is 20 bp upstream of the CmSH1 protospacer-associated motif (PAM), i.e., 5′-N20-NGG-3′, where NGG represents the PAM nucleotide sequence and N20 represents the 20 bp recognition nucleotide sequence.

[0049] In a preferred embodiment of the present invention, the sgRNA nucleotide sequence is shown in SEQ ID NO:3.

[0050] In a preferred embodiment of the present invention, a site-directed overexpression intermediate vector, such as pAMA1-CmSH1-sgRNA, can be constructed by amplifying the sgRNA expression cassette (SEQ ID NO:4) and ligating it into a vector such as pAMA1-Cas9-hyg.

[0051] Using CRISPR / Cas9 gene editing technology, a double-strand break (DSB) site is formed in the target gene. The fungus's own DSB repair mechanism is utilized to introduce homologous arm nucleotide sequences at both ends to initiate homologous recombination repair. The upstream homologous arm nucleotide sequence is located upstream of the CmSH1 region, and the downstream homologous arm nucleotide sequence is located downstream of the CmSH1 region. The upstream and downstream homologous arm nucleotide sequences are tightly linked to the overexpressed target gene, thereby causing the target gene to be accurately inserted at the CmSH1 site.

[0052] In an embodiment of the present invention, the upstream and downstream homologous arm nucleotide sequences of the safe overexpression site CmSH1 are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0053] A complete expression cassette typically includes a promoter, a CDS (coding DNA sequence), and a terminator, enabling successful expression of a functional protein. In a specific embodiment of the present invention, the complete expression cassette comprises the promoter shown in SEQ ID NO:7, the CDS shown in SEQ ID NO:2 (i.e., the Cmhyd1 gene), and the terminator shown in SEQ ID NO:8.

[0054] In an embodiment of the present invention, a target gene site-directed overexpression vector can be constructed by linking the upstream and downstream homologous arm nucleotide sequences of the safe overexpression site, as well as a complete expression cassette containing a promoter, target gene, and terminator, to a CRISPE-Cas9 site-directed overexpression intermediate vector.

[0055] In an embodiment of the present invention, a transformant that overexpresses the target gene at a safe overexpression site is obtained by transforming a target gene-directed overexpression vector into a materia pulvinata bioplast. The transformation can be PEG-mediated, or performed via electroporation or gene gun methods.

[0056] In an embodiment of the present invention, the method for creating a Cordyceps militaris strain with enhanced resistance to white spot disease using CRISPR / Cas9 gene editing technology may further include: S8, at the genomic DNA level, identifying a transformant that specifically overexpresses the target gene from the transformants obtained in S7, i.e., a positive transformant; S9, at the cDNA level, verifying the positive transformant obtained in S8 to confirm that the target gene is highly expressed in the positive transformant; S10, subculturing the positive transformant obtained in S9 to obtain a Cordyceps militaris strain with vector loss, no foreign gene insertion, and overexpression of the target gene; and S11, infecting the fruiting bodies developed by the Cordyceps militaris strain obtained in S10 with Cordyceps spores to verify whether the fruiting bodies overexpress the target gene and enhance resistance to white spot disease.

[0057] After gene knockout using homologous recombination, a gene replacement experiment is necessary to better demonstrate gene function and confirm whether other genes have been knocked out, causing corresponding phenotypic changes. If the phenotype recovers or partially recovers after replacement, it proves that the phenotype is related to the knocked-out gene. Currently, gene replacement in Cordyceps militaris generally uses random integration, but this random integration may lead to positional effects, causing inconsistent expression levels and significantly increasing the workload for screening. The solution to this problem is to find suitable site-specific replacement sites for targeted gene replacement.

[0058] The *Cordyceps militaris* strain expressing red fluorescent protein at a specific site using CmSH1 showed no difference in mycelial growth rate and light-induced color change (carotenoid accumulation) compared to the wild-type strain. CmSH1-expressed red fluorescent protein produced strong red fluorescence in the mycelia, and the strain could develop into fruiting bodies normally on wheat culture medium, indistinguishable from the wild-type. This indicates that CmSH1 expression at a specific site has no effect on the cultivation traits of *Cordyceps militaris*, and therefore can serve as a safe overexpression site in *Cordyceps militaris*, as well as a potential site for gene complementation. Therefore, this invention provides the application of CmSH1 in gene complementation and overexpression.

[0059] This invention utilizes CRISPR / Cas9 technology to create a double-strand break (DSB) site in the target gene. Leveraging the fungal's own DSB repair mechanism, homologous arm nucleotide sequences are introduced at both ends to initiate homologous recombination repair. The upstream homologous arm nucleotide sequence is located upstream of the CmSH1 region, and the downstream homologous arm nucleotide sequence is located downstream of the CmSH1 region, with the target gene Cmhyd1 inserted in between for overexpression. The upstream homologous arm, the overexpressed target gene, and the downstream homologous arm nucleotide sequences are tightly linked, thereby achieving site-specific overexpression of the target gene without the insertion of resistance genes.

[0060] Example

[0061] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0063] The main reagents included: restriction endonucleases from NEB; high-fidelity DNA polymerase from TOYOBO; common DNA polymerase, one-step cloning and reverse transcription kits, and quantitative PCR reagents from Nanjing Novizan Biotechnology Co., Ltd.; RNA extraction kits from OMEGA; plasmid extraction kits and DNA recovery kits were purchased from Tiangen Biotech (Beijing) Co., Ltd.; ampicillin and hygromycin reagents were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. All other chemical reagents used in the examples were imported or domestically produced analytical grade reagents; primer synthesis and sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0064] The wild-type Cordyceps militaris strain used in the following examples is CGMCC 3.16323, and the Cordyceps dentata strain is CGMCC 5.2193. The vector pAMA1-Cas9-hyg used was kindly provided by the research group of Professor Liu Gang at the Institute of Microbiology, Chinese Academy of Sciences. The specific implementation method is illustrated by overexpression of Cmhyd1 at the CmSH1 site.

[0065] Example 1: Identification of the safe overexpression site CmSH1 in the Cordyceps militaris genome

[0066] Based on the published genomes of Cordyceps militaris CM01 (Zheng et al., Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional Chinese medicine. Genome Biology, 2012, R116.), ATCC 34164 (Kramer et al., Chromosome-level assembly and secondary metabolite potential of the parasitic fungus Cordyceps militaris. BMC Genomics, 2017 18(1): 912.), and multiple transcriptome sequencing analyses (Zhang et al., Dynamic genome-wide transcription profiling and direct target genes of CmWC-1 reveal hierarchical light signal transduction in Cordyceps militaris. Journal of Fungi, 2022, 8(6): 624.), and according to the criteria of "terminator regions of two genes, spacer length of 3000-7000 bp, and expression in the spacer region", CmSH1 (SEQ ID NO: 1) in the Cordyceps militaris genome was screened. ID NO:1) is a potential safe overexpression site. CmSH1 is a sequence segment in the intergenic space between CCM_00870 and CCM_00871 genes in the Cordyceps militaris CM01 (CGMCC3.14242) genome. Protoplast transformation mediated by PEG showed expression of red fluorescent protein at this site. All three screened transformants expressed strong red fluorescence, indicating that this site has strong transcriptional activity. Figure 1 Furthermore, the growth rate, light-induced color change, and fruiting body development of the three transformants were identical to those of the wild-type strain. Figure 2Therefore, CmSH1 was determined to be a safe overexpression site in the Cordyceps militaris genome.

[0067] Example 2: Identification of the target gene Cmhyd1 in the Cordyceps militaris genome that enhances resistance to white spot disease.

[0068] Transcriptome sequencing was performed on Cordyceps militaris fruiting bodies infected with Cordyceps dentata for 4 and 8 days, with healthy fruiting bodies serving as controls. Differential expression analysis of the transcriptome sequencing results revealed that the Cordyceps militaris hydrophobic protein gene Cmhyd1 (CCM_03537) was highly expressed at both 4 and 8 days after infection with Cordyceps dentata, especially at 4 days. Therefore, the hydrophobic protein gene Cmhyd1 (SEQ ID NO:2) was preliminarily identified as a target gene for enhancing resistance to white hair disease in Cordyceps militaris. Pichia pastoris strain GS115 was transformed with the recombinant vector pPICZαA-Cmhyd1 and its expression was induced. The resulting fermentation supernatant (concentration approximately 0.1 mg / mL) containing hydrophobic proteins was purified and used as the experimental group. The empty vector pPICZαA was also transformed with the Pichia pastoris strain GS115 and its expression was induced. The resulting fermentation supernatant, without hydrophobic proteins, was purified and used as the negative control (for methods of expressing and purifying hydrophobic proteins, empty vector pPICZαA, recombinant vector pPICZαA-Cmhyd1, and Pichia pastoris strain GS115, see Li Xiao, Study on the Function and Regulatory Mechanism of Hydrophobic Protein Family Genes in Cordyceps militaris, 2021 Doctoral Dissertation, University of Chinese Academy of Sciences). The fermentation supernatants were sprayed onto Cordyceps militaris fruiting bodies infected with Cordyceps dentata for one day. The results showed that spraying with the fermentation supernatant containing hydrophobic proteins improved the resistance of Cordyceps militaris to white spot disease, further confirming that Cmhyd1 plays an important role in enhancing resistance to white spot disease.

[0069] Example 3: Construction and detection of CRISPR / Cas9 gene editing intermediate vector for site-directed overexpression of Cmhyd1 by CmSH1

[0070] To further confirm the role of Cmhyd1 in enhancing resistance to white spot disease, Cmhyd1 was overexpressed using the safe overexpression site CmSH1 obtained from the Cordyceps militaris genome. To construct a vector for site-specific overexpression of Cmhyd1 using CmSH1, an intermediate vector expressing sgRNA, pAMA1-CmSH1-sgRNA, was needed. CmSH1 was selected as the target site, and the coding sequence of the sgRNA recognition region was positions 1566-1585 of the CmSH1 sequence. The sgRNA was designed online using the Eukaryotic Pathogen CRISPR guideRNA / DNA Design Tool website (http: / / grna.ctegd.uga.edu / ), with all parameters set to default. Primers containing target information were designed. The sgRNA nucleotide sequence is shown in SEQ ID NO:3.

[0071] The intermediate carrier was prepared according to the following steps:

[0072] 3.1 PCR amplification: Using pUC57-sgRNANc5SrRNA (described in the following literature: Wang Xuping, Construction of a high-efficiency gene editing system of Cordyceps militaris. Master's thesis of University of Chinese Academy of Sciences, 2021) as a template, PCR amplification was performed according to the instructions of TOYOBO's high-fidelity DNA polymerase 2×KODMasterMix, using Bste-5srRNA-F (SEQ ID NO:10) / CmSH1-sgRNA-R (SEQ ID NO:11) and CmSH1-sgRNA-F (SEQ ID NO:12) / Bste-gRNA-scaf-R (SEQ ID NO:13) as primers. The two PCR products were then excised and recovered.

[0073] 3.2 Enzyme digestion: The pAMA1-Cas9-hyg vector (described in the following literature: Wang Xuping, Construction of a high-efficiency gene editing system for Cordyceps militaris. Master's thesis, University of Chinese Academy of Sciences, 2021) was digested with BstEII and then gel-cleaved. Following the instructions of the Novizan one-step cloning kit, it was combined with the two PCR products recovered from gel digestion in step 3.1 and ligated into a three-fragment one-step cloning process to obtain the ligation product, which is the intermediate vector pAMA1-CmSH1-sgRNA. This vector expresses sgRNA.

[0074] The restriction endonuclease BstEⅡ digestion reaction system is as follows:

[0075] Carrier: 2μg

[0076] Restriction endonuclease: 2 μL

[0077] 10×NEBuffer 3.1: 10μL

[0078] Nuclease-free water: to 100μL

[0079] Enzyme digestion overnight at 60℃.

[0080] 3.3 Identification

[0081] 10 μL of the ligation product obtained in step 3.2, i.e., the intermediate vector pAMA1-CmSH1-sgRNA, was transformed into *E. coli* DH5α and cultured on LB medium containing 50 μg / mL ampicillin for screening. Single clones were identified by colony PCR using bsteck-F (SEQ ID NO:14) / bsteck-R (SEQ ID NO:15). Positive clones were selected and inoculated into 10 mL of LB liquid medium containing 50 μg / mL ampicillin. After incubation at 37°C with shaking overnight, the plasmid was extracted and then verified by PCR, enzyme digestion, and sequencing. The PCR, enzyme digestion, and sequencing results showed that the plasmid extracted from the positive clone was the vector pAMA1-CmSH1-sgRNA, which expresses sgRNA. The coding sequence of the sgRNA recognition region is positions 1566-1585 of SEQ ID NO:1.

[0082] The PCR identification reaction system for Escherichia coli DH5α colonies is as follows:

[0083] 2×Rapid Taq Master Mix: 5μL;

[0084] Primer bsteck-F (10 μmol / L): 0.2 μL;

[0085] Primer bsteck-R (10 μmol / L): 0.2 μL;

[0086] Bacterial suspension (capillaries from LB / Amp+ medium were dipped into 10 μL ddH2O): 4.6 μL.

[0087] The PCR amplification reaction procedure is as follows:

[0088] Pre-denaturation at 95℃ for 60 seconds, denaturation at 95℃ for 15 seconds, annealing at annealing temperature (Tm) for 15 seconds, extension at 72℃ for 5 seconds / Kb, 35 cycles, extension at 72℃ for 10 minutes, and storage at 4℃.

[0089] Example 4: Construction and detection of CRISPR / Cas9 gene editing vector for site-directed overexpression of Cmhyd1 by CmSH1

[0090] Based on the intermediate vector pAMA1-CmSH1-sgRNA, a CRISPR / Cas9 gene editing vector for site-specific overexpression of Cmhyd1, namely pAMA1-CmSH1-sgRNA-up-Cmhyd1-down, was constructed. This vector expresses sgRNA and overexpresses Cmhyd1 at the CmSH1 site. Homologous arms (SEQ ID NO:5, SEQ ID NO:6) were selected approximately 1000 bp above and below CmSH1 (SEQ ID NO:1). The hydrophobic protein encoding gene Cmhyd1 (SEQ ID NO:2) from wild-type Cordyceps militaris was used as the overexpression gene, and the promoter (SEQ ID NO:7) and terminator (SEQ ID NO:8) of Cmhyd1 itself were selected.

[0091] The carrier was prepared according to the following steps:

[0092] 4.1 PCR Amplification: Using wild-type Cordyceps militaris genomic DNA as a template, primers CmSH1_up_F (SEQ ID NO:16) / CmSH1_up_R (SEQ ID NO:17), Cmhyd1_P+CDS+T_F (SEQ ID NO:18) / Cmhyd1_P+CDS+T_R (SEQ ID NO:19), and CmSH1_down_F (SEQ ID NO:20) / CmSH1_down_R (SEQ ID NO:21) were used to amplify the upstream homologous arm of CmSH1 (SEQ ID NO:5), the complete expression cassette of Cmhyd1 (SEQ ID NO:9), and the downstream homologous arm of CmSH1 (SEQ ID NO:6) according to the instructions of TOYOBO's high-fidelity DNA polymerase 2×KODMasterMix. The three PCR products were then excised and recovered from the gel.

[0093] 4.2 Enzyme digestion: The pAMA1-CmSH1-sgRNA vector (constructed in step 3.1) was double-digested with SspI-HF and BsrGI and then recovered from the gel. Following the instructions of the Novizan one-step cloning kit, the vector was ligated into four fragments with the three PCR products recovered from the gel in step 4.1 to obtain the ligation product, namely pAMA1-CmSH1-sgRNA-up-Cmhyd1-down. This vector expresses sgRNA and Cmhyd1.

[0094] 4.3 Identification

[0095] Take 10 μL of the ligation product obtained in step 4.2, namely pAMA1-CmSH1-sgRNA-up-Cmhyd1-down, transform it into E. coli DH5α, and culture it on LB medium containing 50 μg / mL ampicillin for screening. Single clones were identified by colony PCR using primers 1774F (SEQ ID NO:22) / 1774R (SEQ ID NO:23). Positive clones were selected and inoculated into 10 mL of LB liquid medium containing 50 μg / mL ampicillin. After incubation at 37°C with shaking overnight, the plasmid was extracted, and then verified by PCR, enzyme digestion, and sequencing. PCR, enzyme digestion, and sequencing results showed that the plasmid extracted from the positive clone was the recombinant CRISPR vector pAMA1-CmSH1-sgRNA-up-Cmhyd1-down. Figure 3 This vector expresses sgRNA and contains the upstream and downstream homologous arms of CmSH1 and the complete expression cassette of Cmhyd1. Figure 4 A).

[0096] Example 5: Construction and identification of Cmhyd1 site-directed overexpression CRISPR-Cas9 strain

[0097] 5.1 Construction of Cmhyd1-directed overexpression CRISPR-Cas9 strain

[0098] After successful construction of the vector pAMA1-CmSH1-sgRNA-up-Cmhyd1-down, it needs to be transformed into wild-type Cordyceps militaris strains. Following the method described in Wang Xuping's (Construction of a High-Efficiency Gene Editing System for Cordyceps militaris. 2021 Master's Thesis, University of Chinese Academy of Sciences), wild-type Cordyceps militaris bioplasts were prepared and transformed. Single colonies germinating on resuscitation medium supplemented with 500 μg / mL hygromycin were picked and transferred to a new selection medium (PPDA + 500 μg / mL hygromycin) for secondary selection. Transformants were screened using primers 1774F (SEQ ID NO:22) / 1774R (SEQ ID NO:23) (p2) and CmSH1-1723F (SEQ ID NO:24) / CmSH1-1723R (SEQ ID NO:25) (p1). The positions of the two pairs of selection primers (p1 and p2) are shown below. Figure 4 As shown in Figure A. Using the two pairs of primers (p1 and p2) mentioned above, the Cmhyd1 site-directed overexpression strain CmSH1-Cmhyd1oe was screened, in which Cmhyd1 had been integrated into the CmSH1 site ( Figure 4 B). cDNA detection revealed that, compared to wild-type Cordyceps militaris strains, the Cmhyd1 gene was highly expressed in the mycelium of the CmSH1-Cmhyd1oe strain with site-directed overexpression of Cmhyd1. Figure 4 C).

[0099] 5.2 Loss of pAMA1-CmSH1-sgRNA-up-Cmhyd1-down vector

[0100] Conidia of the Cmhyd1 site-directed overexpression strain CmSH1-Cmhyd1oe were collected using a sterile toothpick and inoculated onto the resuscitation medium without hygromycin for subculturing until the hygromycin-resistant vector pAMA1-CmSH1-sgRNA-up-Cmhyd1-down was lost, thus obtaining the traceless site-directed overexpression strain CmSH1-Cmhyd1oe (CGMCC No. 40324) without the resistance selection tag. This traceless site-directed overexpression strain CmSH1-Cmhyd1oe is consistent with the wild-type Cordyceps militaris strain and does not grow on PPDA medium supplemented with 500 μg / mL hygromycin. Figure 5 ).

[0101] 5.3. Detection of fruiting body characteristics, Cmhyd1 expression level, and pathogenicity of the Cmhyd1-free site-specific hyperexpressing strain CmSH1-Cmhyd1oe

[0102] Fruiting body cultivation and pathogenicity testing were conducted on wild-type Cordyceps militaris strain and CmSH1-Cmhyd1oe strain with scarless site-directed overexpression of Cmhyd1. At the same time, the expression level of the hydrophobic protein gene Cmhyd1 in the fruiting bodies of wild-type Cordyceps militaris strain and CmSH1-Cmhyd1oe strain with scarless site-directed overexpression of Cmhyd1 was detected before and after infection with Cordyceps dentata.

[0103] WT and CmSH1-Cmhyd1oe mycelial blocks were inoculated into seed culture medium (20g glucose, 5g peptone, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, and tap water to a final volume of 1L) and cultured at 150rpm in the dark for 3 days to prepare liquid spawn. 5mL of the above liquid spawn was inoculated onto wheat culture medium (20g hulled wheat; 28mL nutrient solution: 20g glucose, 10g peptone, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 1g ammonium citrate, 20mg vitamin B1; tap water to a final volume of 1L) and placed in a 20℃ intelligent mushroom cultivation box (Beijing Zhitai Kangxing Biotechnology Co., Ltd.). The development of Cordyceps militaris fruiting bodies in the wheat culture medium was observed.

[0104] The results showed that site-directed overexpression of the hydrophobic protein gene Cmhyd1 in Cordyceps militaris did not affect the formation of the Cordyceps militaris matrix, but it did cause some changes in the morphology of the fruiting bodies. Compared with the wild-type Cordyceps militaris strain, the fruiting bodies of the Cmhyd1-overexpressing strain CmSH1-Cmhyd1oe were slightly shorter, with slightly enlarged heads, and thicker fruiting bodies, but the number of fruiting bodies was slightly reduced. The wet and dry weights were no different from the wild type, and this did not affect its marketability. (See [link to relevant documentation]). Figure 6 and Figure 7 .

[0105] Statistical analysis of the number of diseased fruiting bodies and the area of ​​lesions revealed that the CmSH1-Cmhyd1oe strain had significantly fewer diseased fruiting bodies and lesion areas than the wild-type Cordyceps militaris strain. Figure 8 and Figure 9 As shown, infection with Cordyceps militaris induces high expression of Cmhyd1, and the expression level of CmSH1-Cmhyd1oe strain after infection with Cordyceps militaris is significantly higher than that of the wild type, such as... Figure 10 As shown.

[0106] Based on this invention, some modifications or improvements can be made, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of this invention are within the scope of protection claimed by this invention.

Claims

1. A DNA molecule, characterized in that, It is the safe overexpression site CmSH1 in the Cordyceps militaris genome, and its nucleotide sequence is shown in SEQ ID NO:

1.

2. A strain of Cordyceps militaris with enhanced resistance to white mold, characterized in that, The Cordyceps militaris strain overexpresses the target gene Cmhyd1 at a safe overexpression site CmSH1. The nucleotide sequence of the safe overexpression site CmSH1 is shown in SEQ ID NO: 1, and the nucleotide sequence of the target gene Cmhyd1 is shown in SEQ ID NO:

2. The strain has the accession number CGMCC No. 40324.

3. A method for creating Cordyceps militaris strains with enhanced resistance to white spot disease using CRISPR / Cas9 gene editing technology, comprising the following steps: S1. Identify safe overexpression sites in the Cordyceps militaris genome; S2. Identify the target genes in the Cordyceps militaris genome that enhance resistance to white spot disease; S3. Determine the sgRNA at the safe overexpression site and construct a CRISPR-Cas9 site-directed overexpression intermediate vector expressing the sgRNA; S4. Determine and amplify the upstream and downstream homologous arm nucleotide sequences of the safe overexpression site; S5. Identify and amplify the complete expression cassette of the target gene; S6. Connect the upstream and downstream homologous arm nucleotide sequences described in S4 and the complete expression cassette described in S5 to the CRISPR-Cas9 site-directed overexpression intermediate vector described in S3 to construct the target gene site-directed overexpression vector; and S7. Transform the vector obtained in S6 into pupae grassland bioplasts to obtain transformants that overexpress the target gene. The safe overexpression site is CmSH1, whose nucleotide sequence is shown in SEQ ID NO:

1. The nucleotide sequence of the sgRNA of CmSH1 is shown in SEQ ID NO:

3. Furthermore, the target gene is Cmhyd1, whose nucleotide sequence is shown in SEQ ID NO:

2.

4. The method of claim 3, wherein, The upstream and downstream homologous arm nucleotide sequences of the safe overexpression site are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

5. The method according to claim 3, characterized in that, The nucleotide sequence of the complete expression cassette is shown in SEQ ID NO:

9.

6. The method according to claim 3, characterized in that, The method further includes the following steps: S8. At the genomic DNA level, identify the transformants that site-specifically overexpress the target gene from the transformants obtained in S7, i.e., positive transformants; S9. At the cDNA level, verify the positive transformants obtained in S8 and confirm that the target gene is highly expressed in the positive transformants. S10. The positive transformants obtained in S9 were passaged to obtain Cordyceps militaris strains with vector loss, no exogenous gene insertion, and overexpression of the target gene; and S11. Infect the fruiting bodies developed from the Cordyceps militaris strain obtained in S10 with Calcisporium cordycipiticola to verify whether the fruiting bodies overexpress the target gene and enhance resistance to white spot disease.

7. A recombinant vector or expression cassette characterized in that, Contains the DNA molecule as described in claim 1.

8. A CRISPR / Cas9 gene editing vector characterized in that, The vector comprises the sgRNA of CmSH1 as described in claim 1, the nucleotide sequences of the upstream and downstream homologous arms of CmSH1, and the Cordyceps militaris hydrophobic protein encoding gene Cmhyd1, wherein the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 3, and the nucleotide sequence of Cmhyd1 is shown in SEQ ID NO:

2.

9. The CRISPR / Cas9 gene editing vector of claim 8, wherein, The upstream and downstream homologous arm nucleotide sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

10. The CRISPR / Cas9 gene editing vector as described in claim 8, wherein the backbone vector of the vector is the pAMA1-Cas9-hyg vector.

11. The use of the DNA molecule of claim 1, the recombinant vector or expression cassette of claim 7, or the CRISPR / Cas9 gene editing vector of claim 8 in enhancing resistance to Cordyceps militaris white spot disease.

12. Application of the safe overexpression site CmSH1, whose nucleotide sequence is shown in SEQ ID NO: 1, in the overexpression of the Cordyceps militaris hydrophobic protein encoding gene Cmhyd1, wherein the nucleotide sequence of the Cordyceps militaris hydrophobic protein encoding gene Cmhyd1 is shown in SEQ ID NO:

2.

13. Application of the Cordyceps militaris hydrophobic protein encoding gene Cmhyd1 in enhancing resistance to Cordyceps militaris white hair disease / controlling Cordyceps militaris white hair disease, wherein the nucleotide sequence of the Cordyceps militaris hydrophobic protein encoding gene Cmhyd1 is shown in SEQ ID NO:

2.

14. Application of the Cordyceps militaris hydrophobic protein encoding gene Cmhyd1 in the preparation of formulations that enhance resistance to / control Cordyceps militaris white hair disease, wherein the nucleotide sequence of the Cordyceps militaris hydrophobic protein encoding gene Cmhyd1 is shown in SEQ ID NO:2.

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