Application of oxidoreductase 1 in regulating resistance of morel mushrooms to white mold

Abstract

This invention discloses the application of oxidoreductase 1 in regulating the resistance of morel mushrooms to white mold. This invention belongs to the field of biotechnology. The protein MsOXR1 of this invention, or substances that regulate the activity or content of said protein, or regulate the expression of the gene encoding said protein, can be applied to: 1) regulating the resistance of morel mushrooms to white mold; 2) preparing products that regulate the resistance of morel mushrooms to white mold; 3) cultivating morel mushrooms with altered resistance to white mold; 4) preparing products of morel mushrooms with altered resistance to white mold; 5) morel mushroom breeding. The function of MsOXR1 was studied by constructing overexpression and RNA interference strains using genetic transformation technology. The results showed that MsOXR1 overexpression can enhance the resistance of morel mushroom strains to *Pseudomonas longissima*, indicating that this gene can positively regulate the disease resistance of morel mushrooms and accelerate the morel mushroom breeding process.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of oxidoreductase 1 in regulating the resistance of morel fungi to white mold. Background Technology

[0002] Morel mushrooms ( Morel Morel mushrooms (spp.) belong to the subphylum Ascomycota, class Discomycetes, order Discomyles, and family Discomycetes. The cap of the morel mushroom has a wrinkled, net-like surface, very similar to a sheep's stomach. With the development of the health industry and people's pursuit of health, morel mushrooms have gained widespread popularity among consumers. More and more researchers have begun to study the nutritional components and functions of morel mushrooms. Studies have shown that morel mushrooms are not only rich in protein, amino acids, and vitamins, but also contain bioactive substances such as polysaccharides, polyphenols, and sterols, possessing antioxidant, antitumor, hepatoprotective, and antimicrobial activities. Furthermore, research reports indicate that ergosterol derivatives and polysaccharides isolated from morel mushrooms have in vitro and in vivo antioxidant and NF-κB inhibitory properties. Therefore, the development of the edible and medicinal value of morel mushrooms is continuously promoted, and the area of ​​artificial cultivation and annual yield are constantly increasing to meet the growing market demand. However, morel mushrooms are extremely sensitive to the environment and susceptible to diseases; therefore, the stable production of morel mushrooms has become a hot topic of concern. Currently, with the expansion of morel cultivation, fungal diseases have become one of the most important diseases affecting morel mushrooms, seriously impacting their yield, quality, and economic benefits. Several fungal diseases have been reported in morel cultivation, such as white mold, cobweb disease, and brown rot.

[0003] White mold is one of the most common and serious fungal diseases in morel mushrooms. It can occur at all stages of morel development, such as primordia, young mushrooms, and fruiting bodies, and frequently during post-harvest transportation. Studies have shown that the pathogens of morel white mold are *Pseudomonas longissima* and *Penicillium spp.* Early studies identified *Pseudomonas longissima* as the pathogen collected and isolated in Taigu, Shanxi, China. Furthermore, research has shown that *Pseudomonas longissima* infection leads to ascus shrinkage and rupture, cell wall relaxation, and significant membrane rupture in morel mushrooms, accompanied by severe cytoplasmic leakage. High temperature and high humidity are the main factors contributing to the rapid development of white mold during morel growth. Currently, research on white mold mainly focuses on the identification of pathogenic microorganisms, but reports on the response of morel mushrooms to pathogenic infections are scarce.

[0004] MsOXR1, an oxidoreductase from *Morchella esculenta*, belongs to the redox pathway and is a member of the flavin disulfide reductase (FDR) family. It is a FAD-dependent enzyme that reduces specific oxidized substrates, disulfides, via electron transfer from NAD(P)H. Reported enzymes in the flavin disulfide reductase family include (dihydro)acetamide dehydrogenase (LipDH), glutamate-thioreductase (GR), tryptophan reductase (TR), thioredoxin reductase (TrxR), alkyl peroxide reductase F subunit (AhpF), mercury reductase (MerA), and thioredoxin / glutathione reductase (TGR). However, the specific substrates and products of OXR1 remain unclear. Summary of the Invention

[0005] The main problem this invention aims to solve is how to improve the resistance of morel mushrooms to white mold.

[0006] To address the problems existing in the prior art, the present invention provides the application of proteins or substances that regulate the activity or content of said proteins or regulate the expression of the gene encoding said proteins in regulating plant resistance to white mold.

[0007] The use of the protein or gene expression regulator or substance regulating the activity or content of said protein provided by this invention in any of the following:

[0008] 1) Application in regulating plant resistance to white mold; 2) Application in the preparation of products that regulate plant resistance to white mold; 3) Application in cultivating plants with altered resistance to white mold; 4) Application in the preparation and cultivation of products containing plants with altered resistance to white mold; 5) Applications in plant breeding; The protein is any of the following proteins: a1) A protein with the amino acid sequence SEQ ID No:2; a2) A protein having the same function as the amino acid sequence shown in SEQ ID No:2, but with one or more amino acid residues substituted and / or deleted and / or added. Proteins that have more than 75% identity with the amino acid sequence defined in (a3), (a1), or (a2) and have the same function; The fusion protein is obtained by attaching a tag to the end of any of the proteins defined in (a4), (a1), (a3).

[0009] The protein described in a1) above is named MsOXR1.

[0010] In this invention, the pathogen causing morel white mold disease is *Pseudomonas longissima* (…). Pseudodiplospora longispora ).

[0011] To facilitate the purification or detection of the protein in a1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No:2 in the sequence listing.

[0012] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0013] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0014] Those skilled in the art can readily mutate the nucleotide sequence encoding the MsOXR1 protein of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that possess 75% or more of the nucleotide sequence identity with the MsOXR1 protein isolated in this invention, provided they encode and function as MsOXR1, are derived from and equivalent to the nucleotide sequence of this invention.

[0015] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0016] In this article, identity refers to the similarity between amino acid sequences or nucleotide sequences. The identity of amino acid or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid or nucleotide sequences, then the identity value (%) can be obtained.

[0017] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0018] In this document, the above 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0019] In the above applications, the protein is derived from morel mushrooms (Morchella esculenta). Morel spp.).

[0020] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein MsOXR1.

[0021] In the above text, the substance that regulates gene expression can be a substance that performs at least one of the following six types of regulation: 1) Regulation occurring at the transcriptional level of the aforementioned gene; 2) Regulation that occurs after the gene is transcribed (i.e., regulation of the splicing or processing of the primary transcript of the gene). 3) Regulation of RNA transport of the gene (that is, regulation of the transport of mRNA of the gene from the nucleus to the cytoplasm). 4) Regulation of the translation of the aforementioned genes; 5) Regulation of mRNA degradation of the aforementioned gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0022] In this invention, the regulation can be up-regulation, enhancement, or increase. The regulation can also be suppression, reduction, or down-regulation.

[0023] In this article, the upregulation, enhancement, or increase of the expression level of the coding gene of the aforementioned protein in the recipient plant, and / or the enhancement, increase, or upregulation of the activity and / or content of the coding gene of the aforementioned protein, is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.

[0024] In this article, regulating the expression of the gene encoding the protein can also be achieved by inhibiting, reducing, or downregulating the expression of the gene. Inhibition, reduction, or downregulation of the gene expression can be achieved through gene knockout or gene silencing.

[0025] In the above applications, the substance regulating gene expression or the substance regulating protein activity or content can be a biological material related to the protein described above, and the biological material can be any of the following: c1) The nucleic acid molecule that encodes the protein described above; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-coding genes mentioned above; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

[0026] In the above applications, c1) the nucleic acid molecule can be any of the following DNA molecules: d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No:1; d2) The coding region sequence is the DNA molecule shown in SEQ ID No:3; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein described above; d4) A DNA molecule that hybridizes under strict conditions with a nucleotide sequence defined by d1) or d2) and encodes the protein described above.

[0027] In the above applications, the nucleic acid molecule described in e1) can be a DNA molecule with nucleotide sequences shown in SEQ ID No:4 and SEQ ID No:5.

[0028] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0029] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, they may be vector plasmids OE- Mnsod1 RNAi- Mnsod1 .

[0030] Existing plant expression vectors can be used to construct structures containing... MsOXR1 Recombinant gene expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0031] use MsOXR1 When constructing recombinant plant expression vectors, any type of enhancing promoter or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0032] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0033] Using any vector capable of guiding the expression of exogenous genes in plants, the present invention can be used to... MsOXR1 Introducing genes or gene fragments into plant cells or recipient plants can yield transgenic cell lines and transgenic plants with altered resistance to white mold. MsOXR1 Gene expression vectors can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0034] The present invention also provides a method for improving plant resistance to white mold, the method comprising step M, wherein step M is to enhance, increase or upregulate the activity and / or content of the aforementioned protein in the target plant, or / and enhance, increase or upregulate the expression level of the encoding gene of the aforementioned protein, thereby improving plant resistance to white mold.

[0035] The present invention also provides a method for reducing plant resistance to white mold, the method comprising step P, wherein step P is to inhibit or reduce or silence the activity and / or content of the aforementioned protein in the target plant, or / and, inhibit or reduce or silence the expression level of the gene encoding the aforementioned protein, thereby reducing plant resistance to white mold.

[0036] This invention provides a method for cultivating plants with enhanced resistance to white mold, comprising upregulating or enhancing or increasing the expression of the coding gene of the aforementioned protein and / or the content and / or activity of the aforementioned protein in the target plant, or / and upregulating or enhancing or increasing the activity and / or content of the coding gene of the aforementioned protein, to obtain plants with enhanced resistance to white mold.

[0037] In one embodiment of the present invention, the breeding method for cultivating plants with enhanced resistance to white mold includes the following steps: (1) Construct recombinant expression vectors that upregulate, enhance, or improve the coding genes of the proteins mentioned above; (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant to obtain a plant with stronger resistance to white mold than the recipient plant.

[0038] In this invention, the purpose of plant breeding may include cultivating plants with enhanced resistance to white mold.

[0039] In this invention, the improved resistance to white mold is specifically manifested in the following way: under the action of the white mold pathogen, compared with the wild type, MsOXR1 The overexpression strain showed significantly increased mycelial growth rate, enhanced morel mycelial cell viability, thickened and loosened cell walls, and a significant increase in intracellular cavitary structures compared to the wild type, thereby improving the morel's tolerance to white mold.

[0040] In this invention, the plant may be one of the following: N1) Discomycetes; N2) Discoideales; N3) Morchaceae family; N4) Morel (Morchella genus); N5) Morel mushroom.

[0041] This invention utilizes genetic transformation technology to construct overexpression and RNA interference strains for research. MsOXR1 The function displays the results. MsOXR1 The overexpressing strain exhibited significantly enhanced mycelial resistance under stimulation with *Pseudomonas longissima* fermentation broth, indicating that this gene can positively regulate the disease resistance of *Morchella*. Related studies have shown that... MxOXR1 Loss of gene function (including expression repression or gene knockout) can have significant effects at different biological levels. Attached Figure Description

[0042] Figure 1 Morel mushroom MsOXR1 Gene cloning.

[0043] Figure 2 for MsOXR1 Gene structure; Figure 3 for MsOXR1 Promoter analysis; Figure 4 Neighbor-joining phylogenetic tree and motifs for 16 OXR1 protein sequences from fungal species; Figure 5 Analysis of the interaction between NAD(P)H, FAD and MsOXR1.

[0044] Figure 6 For OE- MsOXR1 and RNAi- MsOXR1 Plasmid mapping; Figure 7 For OE- MsOXR1 and RNAi- MsOXR1 Identification of transformed strains; Figure 8 For OE- MsOXR1 The strain enhanced the tolerance of Morel mycelia to Pseudomonas longisporus by maintaining cell wall integrity. Different letters indicate significant differences between samples (according to Duncan's test). p <0.05).

[0045] Figure 9 For OE- MsOXR1 The strain enhanced the tolerance of Morel mycelium to the metabolites of Pseudomonas longisporus by maintaining cell viability. This included a. mycelial growth rate in PDA medium; b. MsOXR1 Overexpression of [a substance] enhances the resistance of Morel mycelia to white mold; c. Under the action of Pseudomonas longissima metabolites, MsOXR1 Overexpression of [a specific substance] can maintain the stability of the cell wall of morel hyphae. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0047] 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.

[0048] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0049] The overexpression plasmids and RNAi plasmids used in the following examples are described in: Ludan Hou, etc. Mnsod1 promotes the development of Oyster mushroom and enhances the tolerance of mycelia to heat stress. Microbial Cell Factories. 2022 Aug 8;21(1):155. doi: 10.1186 / s12934-022-01878-2. The biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention and may not be used for any other purpose.

[0050] Morel and Pseudomonas longissima in the following examples are described in: Shurong Wang, etc. Integrated Transcriptomics–Proteomics Analysis Reveals the Response Mechanism of Morel sextelata to Pseudodiplospora longispora Infection. Journal of Fungi, 2024, 10, 604. https: / / doi.org / 10.3390 / jof10090604. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention; it may not be used for any other purpose.

[0051] The Agrobacterium GV3101 competent cells used in the following examples have been described in: Wang Lining, Characterization and Functional Study of Catalase Gene in Pleurotus erythrorhizon, Chinese Academy of Agricultural Sciences, 2019, Beijing. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0052] The tested strain DH 5α Escherichia coli in the following examples was purchased from Xi'an Qingke Biotechnology Co., Ltd. The EZNAPlant RNA Kit was purchased from Omega Laboratories, USA; the cDNA reverse transcription kit was purchased from Nanjing Novizan; the gel DNA recovery kit was purchased from Hangzhou Xinjing; the high-purity plasmid miniprep kit was purchased from Hangzhou Xinjing; and the 2×TransStart® Fast PfuFlyPCR SuperMix was purchased from Beijing TransGen Biotech.

[0053] The ampicillin sodium, kanamycin sulfate, hygromycin (Hyg), cefotaxime sodium (Cef), acetylsyringone (As), rifampin (Rif), and all K2HPO4·3H2O, MES, NH4NO3, ZnSO4·7H2O, H3BO3, CuSO4·5H2O, MnSO4·H2O, Na2MoO4, glycerol, and CaCl2·2H2O used in the genetic transformation experiments were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0054] The CYM medium in the following examples consists of 10 g maltose, 20 g glucose, 2 g yeast extract, 2 g peptone, 0.5 g MgSO4·7H2O, and 4.6 g KH2PO4, diluted to 1000 mL of distilled water.

[0055] The induction medium (IM) in the following examples consists of: 10 mL K-buffer, 20 mL MN buffer, 5 mL 20% glucose (w / v), 10 mL 0.01% FeSO4 (w / v), 2.5 mL 20% (NH4)2SO4 (w / v), 1 mL 1% CaCl2 (w / v), 10 mL 50% Glycerol (w / v), 7.808 g MES (40 mmol / L, MW 195.2), and deionized water (ddH2O) to a final volume of 1000 mL. The pH is adjusted to 5.6 using H3PO4 or NaOH. The IM medium should be prepared fresh and used immediately. It should be protected from light during preparation and use and stored at 4°C.

[0056] The following examples used statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and the Duncan test was used. p <0.05 indicates a statistically significant difference between the experimental groups.

[0057] Example 1: Morel mushrooms MsOXR1 Gene cloning and bioinformatics analysis 1. MsOXR1 Cloning of genes Primers were designed using the genome sequence from the NCBI database (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_020137385.1 / ) as a reference sequence and Primer Premier 5 was used. MsOXR1 -F / MsOXR1 -R clone MsOXR1 The full length of the gene, MsOXR1 -F:5'-ATGTTGGTCATAAAGCAGTGG-3'; MsOXR1 -R:5'-TTAATTCTTTTCCTCACTCGA-3'.

[0058] Morel mycelial cDNA and gDNA were used as templates, and PCR cloning was performed according to the high-fidelity enzyme usage method. Reaction system: 2×TransStart ® FastPfu FlyPCR SuperMix 25 μL, primers MsOXR1 -F / MsOXR1 1 μL of each of -R, 2 μL of template, and 21 μL of sterile water. Reaction program: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 20 s, 55℃ annealing for 20 s, 72℃ extension for 60 s, 35 cycles; 72℃ extension for 10 min, and storage at 4℃.

[0059] 2. Morel mushrooms MsOXR1 Bioinformatics analysis of genes The whole genome of morel mushrooms was compared and analyzed using the NCBI website (https: / / www.ncbi.nlm.nih.gov / datasets / genome / ), and the results were downloaded. MsOXR1 The nucleic acid and amino acid sequences of the gene were obtained. The number of amino acids, theoretical isoelectric point, and molecular weight were predicted using the online software ExPASy (https: / / web.expasy.org / compute_pi / ?tdsourcetag=spctim_aiomsg). The secondary structure of the protein was predicted using SOPMA (https: / / npsa-prabi.ibcp.fr / cgi-bin / secpred_sopma.pl) and TMHMM Server v.2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / ). The tertiary structure model of the protein was predicted using SWISS-MODEL (https: / / swissmodel.expasy.org / interactive / ?ac=A0A3N4KPB6). Extraction was performed using TBtools. MsOXR1 The cis-regulatory elements of the 2000bp promoter region of a gene were predicted using PlantCARE software and visualized using TBtools software. TBtools software was then used to further analyze the gene promoter region. MsOXR1 The location of genes in chromosomes is analyzed and mapped.

[0060] The results are as follows Figure 1 As shown: Morel mushrooms MsOXR1 The gene consists of four exons and three introns. The nucleotide sequence of the genome is SEQ ID No:1 in the sequence listing, and the CDS coding sequence is 1368 bp in length. Figure 1 and 2 That is, the CDS encoding sequence is SEQ ID No:3 in the sequence listing, and the encoding amino acid sequence is SEQ ID No:2 of the MsOXR1 protein.

[0061] Using PlantCARE software MsOXR1 Cis-acting element analysis was performed on the promoter region of OXR1. The results showed that the OXR1 promoter region contains not only photoresponsive elements, but also methyl jasmonate (MeJA) responsive elements, cryoresponsive elements, abscisic acid responsive elements, meristem responsive elements, salicylic acid responsive elements, etc. Figure 3 ).

[0062] To determine the evolutionary relationship between MsOXR1 identified in *Morchella esculenta* and OXR1 homologous sequences in other fungal species, a detailed phylogenetic analysis and comparison of conserved motifs were conducted. Figure 4 The results show that the fungal OXR1 protein is typically distributed across two branches. MsOXR1 is the second largest protein in terms of molecular weight, and it is related to... Morchella snyderi KAI5846231.1 is the closest. Conserved motif analysis shows that motifs 7, 4, 8, 3, 1, 9, 6, 2, 10, and 5 are present in all OXR1 amino acid sequences. MsOXR1 and Morchella snyderi KAI5846231.1 contains both conserved motifs 11 and 12.

[0063] Based on bioinformatics analysis and published research reports, MsOXR1 has been preliminarily identified as belonging to the flavin disulfide reductase (FDR) family. However, X-ray crystal structure analysis of OXR1 is still lacking. Previous studies have clearly shown that FDRs are flavin adenine dinucleotide (FAD)-dependent enzymes that transfer electrons from nicotinamide adenine dinucleotide phosphate (NAD(P)H) to reduce specific oxidized substrates, disulfides. Therefore, in this study, molecular docking analysis revealed that NAD(P)H and FAD interact with OXR1, and that NAD(P)H can form eight hydrogen bonds with positions 182 (Arg), 221 (Val), 250 (Ser), 244 (Arg), 243 (Ser), 277 (Arg), and 278 (Val) of OXR1. FAD can form 8 hydrogen bonds with OXR1, which are associated with positions 103 (Glu), 148 (Ala), 177 (Ser), 62 (Gly), 351 (Asp), 361 (Val), and 224 (Glu). Figure 5 ).also, Figure 5 The lengths of the 16 hydrogen bonds and the amino acid residues involved in the hydrophobic interactions were also shown. It is speculated that NAD(P)H and FAD may interact with MsOXR1.

[0064] Further bioinformatics analysis of the MsOXR1 sequence was performed to determine its physicochemical properties (Table 1). MsOXR1 The gene encodes a polypeptide of 455 amino acids (amino acid sequence SEQ ID No:2), with a molecular weight of 449.14 kDa and a pI of 6.14. It is an acidic protein; the protein is unstable and is hydrophilic.

[0065] Table 1. Characteristics and physicochemical properties of MsOXR1

[0066] Example 2, Morel mushrooms MsOXR1Overexpression and RNAi plasmid construction 1) MsOXR1 Construction of overexpression plasmids Overexpression plasmid OE- Mnsod1 The graph was entered into the online database at https: / / www.takarabio.com / learning-centers / cloning / primer-design-and-other-tools, and then selected... Spe I and PSP Using OMⅠ as the double restriction site, primers OE- with homologous arms of the overexpression plasmid were designed. MsOXR1 -F / OE- MsOXR1 -R, where OE- MsOXR1 -F: 5'-GGTCAAAGTTACTAGTATGTTGGTCATAAAGCAGTGGTC-3'; OE- MsOXR1 -R: 5'-CAATTCTAGAGGGCCCTTAATTCTTTTCCTCACTCGACAC-3'; Using morel cDNA as a template, amplification was performed to obtain cDNA with homologous arms of the overexpression vector. MsOXR1 Gene fragments were purified by gel extraction. Remove and store at -80℃ containing plasmid OE- Mnsod1 After activation by streak plating, single colonies of the DH5α strain were picked and cultured in LB liquid medium at 37°C and 180 rpm with shaking, and plasmids were extracted. Select Spe I and PSP OMI serves as a restriction site for the original overexpression plasmid OE- Mnsod1 Double digestion was performed, and the digestion products were recovered and purified. The double digestion system was as follows: 1.5 μg overexpression plasmid; 1 μL SpeⅠ; PSP OMⅠ 1 μL; 10×Cutsmart Buffer 5 μL; sterile water to a final volume of 50 μL. Incubate at 37℃ for 3 h, then perform electrophoresis to detect the enzyme digestion results. The linearized vector was recovered from the gel.

[0067] Homologous recombination was performed using the Novizan reagent kit. The recombination system was as follows: OE- Mnsod1 Linearized vector 9 μL; gene fragment 1 μL; 5×CE II Buffer 4 μL; Exnase II 2 μL; sterile water 4 μL. Homologous recombination program: 37℃, 30 min; on ice for 2 min.

[0068] Transformation of E. coli DH5α with ligation product: Add 10 μL of ligation product to competent cells, gently tap to mix, place on ice for 30 min, heat shock at 37℃ for 45 s, add 700 μL of antibiotic-free LB medium, incubate at 37℃ and 180 rpm for 1 h, centrifuge the cells, spread on LB (Kan) solid medium and incubate overnight, pick single colonies for bacterial PCR verification and sequencing, preserve the bacterial cultures with correct sequencing results in glycerol and store at -80℃.

[0069] MsOXR1 The structure of the overexpression plasmid is described below: it is based on the original plasmid OE- Mnsod1 of Spe I and PSP A DNA fragment with the sequence SEQ ID No:3 was inserted between the two restriction sites of OMI to maintain the overexpression of the original plasmid OE- Mnsod1 The recombinant vector obtained by keeping other sequences unchanged.

[0070] 2) MsOXR1 Construction of RNAi plasmids Table 2, RNAi- MsOXR1 Primers for plasmid construction and primers for detecting transformed strains

[0071] a. RNAi- (The text abruptly ends here, likely due to an incomplete sentence or a formatting error.) Mnsod1 The graph is input into the online website https: / / www.takarabio.com / learning-centers / cloning / primer-design-and-other-tools. First, use... Bgl II and Spe I. Two restriction enzyme sites will cleave the plasmid RNAi- Mnsod1 The original sequence above was replaced with MsOXR1 A 618bp gene fragment (RNAi-Sence fragment: nucleotide sequence SEQ ID No:4) was used to design the first pair of primers for linking the RNAi plasmid (RNAi- MsOXR1- sence-F, RNAi- MsOXR1- The plasmids that were successfully ligated (see Table 2) were used as intermediate vectors and named RNAi- MsOXR1 -sence.

[0072] Then use Spe I and PSP The two restriction enzyme sites of OMⅠ will cleave the intermediate vector RNAi- MsOXR1The original sequence on the RNAi-anti fragment was replaced with the first 558 bp of the reverse complementary sequence of the 618 bp sequence (nucleotide sequence SEQ ID No:4) (RNAi-anti fragment: nucleotide sequence SEQ ID No:5), and a second pair of primers (RNAi- MsOXR1- anti-F, RNAi- MsOXR1- (anti-R) (Table 2).

[0073] b. Using *Morchella esculenta* cDNA as a template, amplify the results using the primers listed in Table 2. MsOXR1 The RNAi-sence (SEQ ID No:4) and RNAi-anti (SEQ ID No:5) fragments were recovered and purified. MsOXR1- The fragment obtained from sence-F / R amplification was named RNAi-sence (the fragment includes an intron at the end), and was used with RNAi- MsOXR1- The gene fragment that is anti-F / R amplified and is inversely complementary to the RNAi-sence fragment is named RNAi-anti (the inversely complementary fragment of RNAi-sence after removing the terminal introns).

[0074] c. Double digest the original RNAi plasmid with enzymes. The reaction system is as follows: original RNAi plasmid RNAi- Mnsod1 1.5 μg; Spe Ⅰ1 μL; 10×Cut smart Buffer5 μL; ddH2OTo 50 μL. 37℃, 2 h.

[0075] after, Bgl II 1 μL; 10×NEB Buffer 3.15 μL; 5 M NaCl 1 μL. Incubate at 37℃ for 2 h to obtain the linearized vector.

[0076] d. Homologous recombination was performed using the Novizan C112 kit. The recombination system was as follows: RNAi- Mnsod1 Linearized vector 9 μL; RNAi-sence gene fragment 1 μL; 5×CE II Buffer 4 μL; Exnase II 2 μL; ddH2O 4 μL. Incubate at 37℃ for 30 min, then cool on ice for 2 min.

[0077] e. The recombinant product was transformed into *E. coli* competent cells DH5α, single clones were selected and expanded, and the target fragment was verified by amplification and sequencing. The successfully ligated plasmid was named RNAi- MsOXR1- Sense, proceed to the next experiment.

[0078] RNAi- MsOXR1- The structure of the Sence vector is described below: it is based on the RNAi-starting vector. Mnsod1 of Spe I and Bgl II. A DNA fragment with the sequence SEQ ID No:4 is inserted between the two restriction sites, while keeping the other sequences of the original vector unchanged, to obtain a recombinant vector.

[0079] f. Connect the ligated plasmid RNAi- MsOXR1- Sence was subjected to double enzyme digestion. The enzyme digestion reaction system is as follows: RNAi- MsOXR1- 1.5 μg of Sence plasmid; Spe Ⅰ 1 μL; PSP 1 μL of OMI; 5 μL of 10×Cut smart buffer; ddH2O to make up to 50 μL. Incubate at 37℃ for 2 h to obtain the linearized vector.

[0080] g. Homologous recombination was performed using the Novizan reagent kit C112. The recombination system was as follows (20 μL): RNAi- MsOXR1- 9 μL of Sence linearized vector; 1 μL of RNAi-anti gene fragment; 4 μL of 5×CE II Buffer; 2 μL of Exnase II; 4 μL of ddH2O. Incubate at 37℃ for 30 min, then cool on ice for 2 min.

[0081] h. The recombinant product was transformed into *E. coli* competent cells DH5α using chemical transformation, and then... Bgl II and PSP Primers were designed on both sides of the two restriction enzyme sites of OMⅠ (results obtained from the Anti gene fragment are prone to false positives; the successfully ligated Sense fragment contains the entire Anti fragment, so primers needed to be redesigned). The amplified sequence was sequenced, and the sequencing results were compared. The plasmid with correct alignment was named RNAi- MsOXR1 Store the bacterial strain containing the corresponding plasmid in glycerol at -80°C.

[0082] RNAi- MsOXR1 The structure of the vector is described as follows: In the original vector RNAi- Mnsod1 of Bgl II and Spe I. Insert an RNAi-sence DNA fragment (SEQ ID No:4) between the two restriction enzyme sites; Spe I and PSP An RNAi-anti DNA fragment (SEQ ID No:5) is inserted between the two restriction enzyme sites of OMⅠ, maintaining the original RNAi- Mnsod1 The recombinant vector obtained by keeping other sequences unchanged.

[0083] 3) Preparation of Agrobacterium competent cells The laboratory-preserved GV3101 glycerol strain was activated by streak plating on LB solid medium containing Rif resistance (concentration 50 μg / mL) and incubated upside down in a 28℃ constant temperature incubator for 48-72 h. Select morphologically sound single colonies and inoculate them into 10 mL of LB (Rif) liquid medium. Incubate overnight (approximately 16-18 h) in a constant temperature shaker at 28°C with shaking at 180 r / min. The activated bacterial culture was transferred to 100 mL of fresh LB (Rif) liquid medium at a ratio of 1:100, and cultured under the same conditions with continuous shaking until the logarithmic growth phase (approximately 5-6 hours). The OD value of the bacterial culture was monitored using a spectrophotometer. 600 The value reaches 0.4-0.5; Immediately after the culture is completed, the cells are placed in an ice bath for 30 min. Then, the bacterial pellet is collected in a 50 mL centrifuge tube at 4°C and 5000 rpm for 10 min. After discarding the supernatant, add 10 mL of pre-cooled 0.15 M NaCl solution, vortex to fully suspend it, and repeat low-temperature centrifugation (4℃, 5000 rpm, 10 min) to complete the washing. Finally, the bacterial cells were resuspended in 5 mL of 20 mM CaCl2 solution, aliquoted into 100 μL per tube into pre-cooled EP tubes, and then transferred to an ultra-low temperature freezer at -80℃ for long-term storage after being instantly frozen in liquid nitrogen for 1 min.

[0084] 4) Overexpression and transformation of Agrobacterium competent cells with RNAi plasmids Will carry MsOXR1 E. coli overexpressing and containing RNAi recombinant plasmids were inoculated onto LB solid plates containing Kan resistance (final concentration 50 μg / mL), incubated upside down at 37°C for 12-16 h, and single colonies were picked for expansion culture and plasmids were extracted. The prepared Agrobacterium GV3101 competent cells were taken out of the -80℃ ultra-low temperature freezer, thawed, and immediately transferred to an ice box to maintain the low temperature. Add 10 μL of purified plasmid to 100 μL of thawed competent cells and mix by gently tapping. After equilibration in an ice bath for 5 minutes, the mixture was subjected to liquid nitrogen quick-freezing (1 minute), heat shock at 37°C (5 minutes), and ice bath treatment (5 minutes) in sequence. Add 700 μL of antibiotic-free LB medium to the tube and incubate at 28℃ and 180 rpm for 2-3 h with shaking. Collect bacterial cells by centrifugation at 5000 rpm for 2 min, resuspend them, and then spread 100 μL onto LB (Kan+Rif) plates and incubate upside down at 28℃ for 48 h. Positive clones were screened and expanded for culture. After identification by colony PCR and confirmation by sequencing, glycerol was stored at -80°C.

[0085] 5) Agrobacterium-mediated plasmid transformation of morel mushrooms I. Screening of Hygromycin Concentration Prepare CYM solid medium with different gradient concentrations of hygromycin, inoculate wild-type morel mushrooms, and culture at 18℃ for 30 days. Select an appropriate concentration of hygromycin based on the mycelial growth.

[0086] II. Preparation of Morel Mycelial Blocks a. Inoculate the activated morel mushroom spawn into PDA plates and incubate at 18°C ​​for 4-5 days; b. Use a 5 mm punch to punch holes in the plates with good growth, place the bacterial blocks in 100 mL of CYM liquid medium (prepare two bottles for each plasmid, with about 35 bacterial blocks in each bottle), and incubate at 18°C ​​for 1 day.

[0087] III. Culture and induction of Agrobacterium GV3101 (containing target plasmid) a. Agrobacterium GV3101 carrying the target plasmid was streaked onto resistant LB (Kan+Rif) solid medium and incubated upside down at 28°C for 2 days; b. Pick a single colony and incubate it in 10 mL of LB (Kan+Rif) liquid medium at 28°C and 180 rpm for 2 days; c. Take 500 μL of the above bacterial culture and inoculate it into 100 mL of LB (Kan+Rif) liquid medium. Incubate at 28℃ and 180 rpm for expansion. d. Collect bacterial cells using 50 mL sterile centrifuge tubes, 4500 rpm, 4℃, 15 min; e. Prepare IM liquid culture medium (prepare fresh and use immediately, keep away from light, store at 4℃ in the dark); f. Rinse the cells with a small amount of IM liquid medium, discard the supernatant, add 12 mL of IM liquid medium to the cells, and vortex to mix. h. Combine the bacterial cultures from both tubes into a 250 mL Erlenmeyer flask and induce in the dark at 28°C and 90 rpm for 5 h.

[0088] IV. Co-culture of Agrobacterium and mycelium a. Pour morel mycelium blocks (fluffy hyphae) that have been cultured in CYM medium for 1 day into 50 mL centrifuge tubes (two centrifuge tubes per bottle of medium), collect the mycelium blocks, and remove the CYM medium; b. Gently rinse the culture block with sterile water, filter out the liquid, and retain the culture block; c. Transfer the bacterial block to the induced Agrobacterium GV3101 containing the target plasmid and incubate at 25°C in the dark for 5 hours.

[0089] V. Prepare 1M solid culture medium, pour into plates, and cover with cellophane (the cellophane should be slightly smaller than the plate). VI. Transfer the co-cultured bacterial blocks to sterile filter paper, absorb off excess bacterial solution, and then place the bacterial blocks one by one on IM plates. Incubate at 18°C ​​in the dark for 3-4 days. VII. Transfer the bacterial culture block along with the cellophane to CYM (15 μg / mL Hyg + 300 μg / mL Cef) solid medium and incubate at 18°C ​​for 25-30 days. VIII. Take the edge of the germinating hyphae and transfer them to a newly prepared CYM plate (containing Hyg and Cef resistance) for secondary screening; IX. After secondary screening, the germinated hyphae were cultured in a PDA, the hyphae were collected, DNA was extracted, and amplified. hygge Fragment; X. Selection hygge Strains with correctly amplified fragments were cultured and RNA was extracted, which was then reverse transcribed into cDNA. The relative expression level of the target gene was further detected by qRT-PCR, and transformed strains were obtained through further screening.

[0090] 6) Detection of target gene expression level in transformed strains Wild-type and transformed strains were inoculated into PDA medium and cultured in the dark at 18°C ​​for 5 days. Mycelia were collected, total RNA was extracted, and reverse transcribed into cDNA. MsOXR1 The expression levels of overexpressing strains and RNAi strains were detected using qPCR primers (Table 2).

[0091] This embodiment is constructed MsOXR1 Overexpression (OE) and RNA interference (RNAi) vectors ( Figure 6 Through Agrobacterium-mediated genetic transformation, a system was constructed. MsOXR1 OE and RNAi transformed strains. Figure 7 The transformed strains are shown in Hygge The fragment amplification results were obtained, and OE- was successfully obtained through qPCR screening. MsOXR1 -4、OE- MsOXR1 -9. RNAi- MsOXR1 -8 and RNAi- MsOXR1-12 transformed strains were used for further research on the function of MsOXR1.

[0092] Example 3: Detection of disease resistance of transformed strains to fermentation filtrate of *Pseudomonas longissima* Sample to be tested: Wild-type morel mushrooms MsOXR1 Overexpression strain OE- MsOXR1 -4 and OE- MsOXR1 -9、 MsOXR1 Knockdown strain RNAi- MsOXR1 -8 and RNAi- MsOXR1 -12.

[0093] The sample to be tested was inoculated onto one side of a PDA plate, and a block of *Pseudomonas longissima* was inoculated onto the other side. The growth of morel mycelium was then observed.

[0094] 1. Treatment method for pathogenic bacteria fermentation broth The specific experimental steps are as follows: Pathogenic fungal hyphae with a diameter of 6 mm were inoculated into a 250 mL Erlenmeyer flask containing 150 mL of potato dextrose broth (PDB) and cultured with shaking at 25℃ and 150 rpm / min for 6 days. First, the fermentation stock solution was obtained using hyphal isolation technology, followed by secondary filtration using a sterile filter with a pore size of 0.22 μm to obtain the final sterile fermentation broth. After sterilizing the PDA solid medium and cooling it to approximately 55℃, 5 mL of sterile fermentation broth was added to every 100 mL of PDA culture medium, mixed thoroughly, and then poured into agar plates.

[0095] 2. The effect of pathogen fermentation broth on the integrity of mycelial cell membranes Using the slide insertion method, a sterile coverslip was inserted obliquely into the culture medium at a 45-degree angle with clean tweezers and incubated at 18°C ​​for 5 days. Once the hyphae had grown to two-thirds of the coverlip, the coverslip was removed. The micromorphological characteristics of the hyphae were observed and photographed using an optical microscope.

[0096] Similarly, hyphal samples for observing hyphal cell membrane integrity were prepared using the slide insertion method. A staining solution was prepared by thoroughly mixing 20 µL of fluorescein diacetate (FDA), 60 µL of propidium iodide (PI), and 920 µL of sterile water. 20 µL of the staining solution was dropped onto a glass slide, and a coverslip was carefully removed. The coverslip was then inverted onto the slide using tweezers and gently pressed down evenly. The sample was incubated at room temperature for 5 min to ensure complete staining before observation. FDA was added to ensure a final concentration of 100 µg / mL, and PI was added to ensure a final concentration of 60 µg / mL. The excitation wavelength of the PI-DNA complex was 535 nm, and the emission wavelength was 615 nm. The excitation wavelength indicated by FDA was 488 nm, and the emission wavelength was 530 nm. Images were obtained using a laser confocal microscope.

[0097] Figure 8 The results showed that in PDA medium containing pathogenic bacterial metabolites, compared with the wild-type (WT) strain, OE- MsOXR1 -4 and OE- MsOXR1 The mycelial growth rate of strain -9 was significantly increased, while RNAi- MsOXR1 -8 and RNAi- MsOXR1 The growth rate of strain -12 was significantly inhibited.

[0098] Further measurements of cell viability using FDA and PI probes revealed that, compared to WT strains, OE- MsOXR1 -4 and OE- MsOXR1 The hyphae of strain -9 exhibited significantly enhanced cellular activity under the influence of pathogenic metabolites, and a distinct green fluorescence could be observed; conversely, RNAi- MsOXR1 -8 and RNAi- MsOXR1 The hyphae of strain -12 only showed red filamentous fluorescence ( Figure 8 This indicates that, MsOXR1 The overexpressing strain was able to maintain the viability of morel mycelial cells under the action of Pseudomonas longisporum metabolites.

[0099] Cell walls play a crucial role in fungal responses to biotic and abiotic stresses. Figure 9 The results of the confrontation between different strains and the pathogen of white mold were presented in the middle ac, and the OE- strain was compared with the wild type (WT) strain. MsOXR1 -4 and OE- MsOXR1 -9 strain showed significantly enhanced growth; conversely, RNAi- MsOXR1 -8 and RNAi- MsOXR1 The growth rate of strain -12 was significantly reduced under treatment with the pathogen causing white mold.

[0100] Further observation using transmission electron microscopy (TEM) revealed that, under the influence of the white mold pathogen, the mycelial cell walls of the WT strain became loose, and multiple cavitary structures appeared within the cells; compared to the WT strain, the OE- MsOXR1 -4 and OE- MsOXR1 -9 strain has a clear cell wall structure and no hollow structures formed within the cell; conversely, RNAi- MsOXR1 -8 and RNAi- MsOXR1 The cell walls of strain -12 are thickened and loose, and the intracellular cavity structure is significantly increased compared to WT.

[0101] In summary, under the influence of pathogenic bacteria, MsOXR1The overexpressing strain may maintain the cell viability of morel mycelium by regulating cell wall integrity, thereby improving the tolerance of morels to the pathogen of white mold.

[0102] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a protein, or a substance that regulates the activity or content of said protein, or a substance that regulates the expression of the gene encoding said protein, in any of the following: 1) Application in regulating resistance to white mold in morel mushrooms; 2) Application in the preparation of products that regulate resistance to morel white mold; 3) Application in cultivating morel mushrooms with altered resistance to white mold; 4) Application in the preparation of morel mushrooms with altered resistance to white mold; 5) Applications in morel mushroom breeding; The protein is any of the following proteins: a1) A protein with the amino acid sequence SEQ ID No:2; a2) A protein having the same function as the amino acid sequence shown in SEQ ID No:2, but with one or more amino acid residues substituted and / or deleted and / or added. a3) Proteins that share more than 75% identity with the amino acid sequence defined by a1) or a2) and have the same function; a4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in a1)-a3).

2. Use according to claim 1, characterized in that: The protein is derived from morel mushrooms.

3. Use according to claim 1 or 2, characterized in that: The substance is any one of the following: c1) A nucleic acid molecule encoding the protein of claim 1 or 2; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) Transgenic morel mycelium containing the nucleic acid molecule described in c1), or transgenic morel mycelium containing the expression cassette described in c2); c6) Transgenic morel tissue containing the nucleic acid molecules described in c1), or transgenic morel tissue containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding gene; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); e5) Transgenic morel mycelium containing the nucleic acid molecule described in e1), or transgenic morel mycelium containing the expression cassette described in e2); e6) A transgenic morel tissue containing the nucleic acid molecule described in e1), or a transgenic morel tissue containing the expression cassette described in e2).

4. The application according to claim 3, characterized in that: c1) The nucleic acid molecule is any of the following DNA molecules. d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No:1; d2) The coding region sequence is the DNA molecule shown in SEQ ID No:3; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein of claim 1; d4) Hybridizes under stringent conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein of claim 1.

5. A method for improving resistance to white mold in morel mushrooms, characterized in that: The method includes enhancing, increasing, or upregulating the activity and / or content of the protein described in claim 1 or 2 in the target morel mushroom, and / or enhancing, increasing, or upregulating the expression level of the gene encoding the protein described in claim 1 or 2, to improve the resistance of morel mushrooms to white mold.

6. A method for reducing resistance to morel white mold, characterized in that: The method is to inhibit, reduce, or silence the activity and / or content of the protein described in claim 1 or 2 in the target morel mushroom, or / and, inhibit, reduce, or silence the expression level of the gene encoding the protein described in claim 1 or 2, in order to reduce the resistance of morel mushrooms to white mold.

7. A breeding method for morel mushrooms with enhanced resistance to white mold, characterized in that: This includes enhancing, increasing, or upregulating the expression level of the gene encoding the protein described in claim 1 or 2 in the recipient morel, and / or the activity and / or content of the protein, to obtain morel with enhanced resistance to white mold, wherein the morel with enhanced resistance to white mold has higher resistance to white mold than the recipient morel.

8. The method according to claim 7, characterized in that: Includes the following steps: (1) Constructing a recombinant expression vector that enhances, improves, or upregulates the coding gene of the protein described in claim 1 or 2; (2) The recombinant expression vector constructed in step (1) is transferred into the recipient morel to obtain morel with higher resistance to white mold disease than the recipient morel.

9. The protein as described in claim 1 or 2 and / or the biomaterial as described in claim 3 or 4.

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