Application of substance for regulating and controlling protein coding gene expression of flammulina velutipes

Abstract

The invention discloses an application of a substance for regulating and controlling protein coding gene expression of flammulina velutipes. The technical problem to be solved by the invention is how to regulate and control agronomic characters (such as improvement of sporocarp yield, stipe length, hypha branching distance and / or growth rate) of flammulina velutipes. The invention provides an application of a substance for regulating and controlling the expression of a protein coding gene of flammulina velutipes in regulating and controlling agronomic traits of flammulina velutipes. The protein is a protein with an amino acid sequence as shown in a sequence 2. Experiments prove that the encoding gene of the flammulina velutipes protein is an important regulation and control gene for growth and development of flammulina velutipes and affects mycelial growth, mycelial density, stipe length and sporocarp yield of flammulina velutipes. The method can be used for breeding good varieties of the flammulina velutipes, and the flammulina velutipes with long stems and high yield can be conveniently screened.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of a substance for regulating the expression of a gene encoding a protein of Flammulina velutipes. Background Art

[0002] Flammulina velutipes, also known as golden mushroom, mushroom, mushroom, hairy money mushroom, etc., belongs to the phylum Basidomycota, class Agaricomycetes, order Agaricales, family Physalacriaceae. Flammulina velutipes is one of the most popular edible mushrooms in the world, with delicious taste and high nutritional value. The crude protein content of Flammulina velutipes is 24.31g·100 -1 g -1 , the ratio of essential amino acid content to total amino acid content is higher than the ideal protein standard, and it is a source of high-quality protein. Flammulina velutipes not only contains carotene, VB1, VB2, VC and other vitamins, but also rich in phosphorus, potassium, calcium and other minerals. In addition, Flammulina velutipes is also a medicinal fungus with multiple functions. Its important active ingredients mainly include ribosome inactivating protein, Flammulina velutipes toxin, fungal immunomodulatory protein, polysaccharides, fatty acids, ergosterol, phenols, terpenes, etc., which play an important role in anti-allergy, anti-virus, anti-fatigue, and memory improvement.

[0003] The problem of Flammulina velutipes strains is mainly reflected in the maintenance of the existing strains and the selection of new varieties. Frequent expansion of strains will cause strain degradation, affecting the quality and yield of Flammulina velutipes products. At present, the main cultivated varieties in China are still foreign varieties, and there is a serious shortage of varieties with independent intellectual property rights. Infringement of domestic seed industry has become the norm, the seed source is unclear, the varieties are chaotic, the enthusiasm for technology research and development is poor, and small workshops are the main production. There are very few specialized strain enterprises. Traditional breeding mainly includes selective breeding, hybrid breeding, and mutation breeding. For example, through natural selection of the parent Jiangshan white mushroom (F21) year by year, a new variety Jiangshan white mushroom (F21-2) with high yield, high quality and strong stress resistance was bred; through multi-spore hybridization with white Flammulina velutipes strain QFC and yellow Flammulina velutipes strain Q100 as parents, a light yellow Flammulina velutipes variety suitable for factory cultivation was obtained; through space mutagenesis treatment of Flammulina velutipes strain F3-1, a characteristic high-quality germplasm resource with high content of active substances was successfully screened. Most of the above traditional breeding methods are time-consuming, have low mutagenesis efficiency, and are highly random. However, the use of bioinformatics and other technologies to deeply explore the genomics of edible fungi has made initial progress in breeding through genetic engineering. Four key genes for terpenoid synthesis were identified by sequencing the Flammulina velutipes genome; by constructing a guanine nucleotide exchange factor interference plasmid of the small molecule G protein Rho family, functional genes that respond to abiotic stresses such as salt, high temperature, low temperature, oxidation, and acid-base and regulate fruiting body development were obtained; based on the Flammulina velutipes genome, a gene Fvclp1 involved in the Flammulina velutipes mating type A factor pathway was annotated, and this gene positively regulates fruiting body development. In addition, interfering with the HMG-box transcription factor FvHmg1 involved in the mating type pathway can also promote fruiting body growth. In summary, genetic engineering breeding can accurately select high-yield strains with strong stress resistance, and has been widely used in the excellent breeding of Flammulina velutipes. Therefore, how to increase the yield of Flammulina velutipes through genetic engineering is a technical problem faced by people in this field. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to regulate the agronomic traits of Flammulina velutipes (such as improving fruiting body yield, stipe length, hyphae branching distance and / or growth rate).

[0005] In order to solve the above technical problems, the present invention provides the following applications.

[0006] Use of a substance for regulating the expression of a protein-coding gene in any of the following;

[0007] A1) Application in regulating the yield of Flammulina velutipes fruiting bodies and / or application in preparing products for regulating the yield of Flammulina velutipes fruiting bodies;

[0008] A2) application in regulating the length of Flammulina velutipes and / or application in preparing a product for regulating the length of Flammulina velutipes;

[0009] A3) Application in regulating the branching distance of Flammulina velutipes hyphae and / or application in preparing a product for regulating the branching distance of Flammulina velutipes hyphae;

[0010] A4) application in reducing the growth rate of Flammulina velutipes mycelium and / or application in preparing a product for reducing the growth rate of Flammulina velutipes;

[0011] The protein is any of the following proteins:

[0012] B1) The amino acid sequence is the protein shown in SEQ ID NO: 2;

[0013] B2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in B1) and having 80% or more identity with the protein described in B1) and having the same function.

[0014] In the present application, the yield may be the fruiting body yield.

[0015] In the present application, the regulation may be up-regulation, improvement or increase.

[0016] In the present application, the regulation may be down-regulation, reduction or decrease.

[0017] In the above application, the application is the application of the substance for increasing the expression of protein encoding gene in any of the following;

[0018] 1) Application in increasing the yield of Flammulina velutipes fruiting bodies and / or application in preparing products for increasing the yield of Flammulina velutipes fruiting bodies;

[0019] 2) Application in increasing the length of Flammulina velutipes and / or application in preparing products for increasing the length of Flammulina velutipes;

[0020] 3) Application in increasing the branching distance of Flammulina velutipes hyphae and / or application in preparing a product for increasing the branching distance of Flammulina velutipes hyphae;

[0021] 4) Application in reducing the growth rate of Flammulina velutipes mycelium and / or application in preparing a product for reducing the growth rate of Flammulina velutipes;

[0022] The protein is any of the following proteins:

[0023] 1) The amino acid sequence is the protein shown in SEQ ID NO: 2;

[0024] 2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in 1) and having 80% or more identity with the protein described in 1) and having the same function.

[0025] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0026] In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0027] In the above proteins, the above 80% identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0028] In the above protein, sequence 2 (SEQ ID No. 2) consists of 238 amino acid residues. It is named protein FfGH79. Its encoding gene is FfGH79 gene.

[0029] In the above application, the protein is derived from Flammulina velutipes.

[0030] In the above, the Flammulina velutipes may be Flammulina velutipes monokaryotic strain 6-3.

[0031] In the above, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, 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 the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0032] In the above application, the substance is a substance that increases the expression of protein-coding genes; the substance is any one of the following:

[0033] A1) a nucleic acid molecule encoding the protein;

[0034] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0035] A3) a recombinant vector containing the nucleic acid molecule described in A1), or a recombinant vector containing the expression cassette described in A2);

[0036] A4) A recombinant microorganism containing the nucleic acid molecule described in A1), or a recombinant microorganism containing the expression cassette described in A2), or a recombinant microorganism containing the recombinant vector described in A3).

[0037] In the above application, the substance is a substance that reduces the expression of protein-coding genes, and the substance is any one of the following:

[0038] B1), a nucleic acid molecule that inhibits, reduces or downregulates the expression of the gene encoding the protein;

[0039] B2), expressing the coding gene of the nucleic acid molecule described in B1);

[0040] B3), an expression cassette containing the gene described in B2);

[0041] B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3);

[0042] B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

[0043] Herein, the vector is well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (ie, cosmid), Ti plasmid or viral vector.

[0044] In the above application, the nucleic acid molecule described in A1) is a DNA molecule whose coding sequence is sequence 1 and / or a DNA molecule whose nucleotide sequence is sequence 3;

[0045] and / or;

[0046] B1) The nucleic acid molecule is a DNA molecule whose nucleotide sequence is sequence 4.

[0047] In the above application, sequence 1 is the coding sequence of the FfGH79 gene, which is also the cDNA gene of FfGH79; sequence 3 is the genomic gene of FfGH79.

[0048] In order to solve the above technical problems, the present invention also provides a method for improving the agronomic traits of Flammulina velutipes, the method comprising up-regulating or enhancing or increasing the expression level of the gene of the above protein in the recipient Flammulina velutipes to obtain the target Flammulina velutipes, the agronomic traits of the target Flammulina velutipes being better than those of the recipient Flammulina velutipes.

[0049] In the above method, the agronomic trait may be fruiting body yield, stipe length, hyphae branching distance and / or growth rate.

[0050] In the present application, the Flammulina belongs to the phylum Basidomycota, Agaricomycetes, the order Agaricales, the family Physalacriaceae, and the genus Flammulina. The Flammulina genus may be Flammulina monokaryotic strain 6-3.

[0051] In the above method, the step of upregulating, enhancing or increasing the expression level of the gene of the above protein in the recipient Flammulina velutipes comprises the step of introducing the above substance into the recipient Flammulina velutipes.

[0052] In any of the above applications or methods, the Flammulina velutipes is Flammulina velutipes variety 6-3.

[0053] The above proteins.

[0054] The above-mentioned substances that regulate the expression of protein encoding genes.

[0055] Product, the product is the above-mentioned protein or the above-mentioned substance that regulates the expression of protein encoding gene.

[0056] Beneficial Effects

[0057] The present invention overexpresses the gene of the protein in Flammulina velutipes and conducts RNAi experiments to find that the gene is an important regulatory gene for the growth and development of Flammulina velutipes, affecting the growth of Flammulina velutipes hyphae, hyphae density, stipe length and / or fruiting body yield. High expression of the gene increases the distance of hyphae branching and reduces hyphae density on the one hand, and shortens the fruiting cycle on the other hand, promotes stipe elongation, and increases fruiting body yield. However, the gene interference expression has the opposite result. Therefore, the expression of the gene in Flammulina velutipes is an important factor affecting the growth and development of Flammulina velutipes fruiting bodies. The gene can be applied to the breeding of excellent varieties of Flammulina velutipes, so as to facilitate the screening of Flammulina velutipes with long stipes and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a diagram of the mycelial growth of Flammulina velutipes starting strains 6-3 and GH79 on PDA plates, identification of insertion sites, analysis of FfGH79 gene transcription levels, and results of the FfGH79 gene structure.

[0059] Figure 2 The construction strategy of FfGH79 gene overexpression (FfGH79-OE) and interference (FfGH79-Ri) vectors.

[0060] Figure 3 This is the result of PCR verification of the target fragment of Flammulina velutipes starting strain 6-3, FfGH79 overexpression transformant strain and FfGH79 interference expression transformant strain, as well as analysis of the transcription level of the FfGH79 gene.

[0061] Figure 4 This is a graph showing the mycelial growth and mycelial growth rate of Flammulina velutipes starting strain 6-3, GH79, FfGH79 overexpression transformant strain and FfGH79 interference expression transformant strain on PDA plates.

[0062] Figure 5 These are the results of mycelial microscopic morphology and mycelial branching distance of Flammulina velutipes starting strain 6-3, GH79, FfGH79 overexpression transformant strain and FfGH79 interference expression transformant strain.

[0063] Figure 6 This is a lock-shaped joint diagram of the Flammulina velutipes 6-3 hybrid strain, GH79 hybrid strain, FfGH79 overexpression transformant hybrid strain and FfGH79 interference expression transformant hybrid strain, as well as the results of the FfGH79 gene transcription level analysis.

[0064] Figure 7 This is a graph showing the fruiting conditions and biomass statistics of the Flammulina velutipes 6-3 hybrid strain, GH79 hybrid strain, FfGH79 overexpression transformant hybrid strain, and FfGH79 interference expression transformant hybrid strain. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0066] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0067] The following examples were plotted using GraphPad 8.0.2 software, and the significance of the difference between the two groups was compared based on Student's t-test. The difference between the two groups was expressed as a probability value (*, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.001). SPSS software was used for Spearman correlation coefficient analysis.

[0068] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0069] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear, cyclic or branched, it may contain modified amino acids, particularly conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes modified amino acid polymers such as those that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, isoprenylation, racemization, selenoylation, transfer-RNA mediated amino addition such as arginylation, ubiquitination, or any other operation such as conjugation with a labeling component. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptide mimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0070] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as subsequently modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. In this article, amino acids may be represented by the common three-letter codes or single-letter codes recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by their generally accepted single-letter codes.

[0071] The term "gene" refers to a segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer regions) that are involved in the transcription / translation of the gene product and the regulation of said transcription / translation, as well as intervening sequences (introns) between individual coding regions (exons).

[0072] The term "template" refers to any nucleic acid molecule that can be used for amplification according to the present invention. Non-natural double-stranded RNA or DNA can be made into double-stranded DNA and used as double-stranded DNA. Any double-stranded DNA or product containing a variety of different double-stranded DNA molecules can be used as template DNA to amplify one or more loci of interest contained in the template DNA.

[0073] The term "primer" refers to an oligonucleotide that can be used in an amplification method such as the polymerase chain reaction (PCR) for amplifying a nucleotide sequence based on a polynucleotide sequence that corresponds to a specific genomic sequence. At least one PCR primer used to amplify a polynucleotide sequence is sequence-specific for the sequence.

[0074] The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that binds to another polynucleotide (commonly referred to as a "target polynucleotide") through complementary base pairing. The probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. The probe can be labeled directly or indirectly.

[0075] The term "amplification reaction" refers to a process for copying a nucleic acid one or more times. In embodiments, the amplification method includes, but is not limited to, polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-β phage amplification, strand displacement amplification, or overlap extension splicing polymerase chain reaction. In some embodiments, a single molecule nucleic acid is amplified, for example, by digital PCR.

[0076] The term "amplification product" refers to a nucleic acid product produced by a nucleic acid amplification technique.

[0077] The term "kit" refers to any delivery system used to deliver substances. In reaction assays, such delivery systems include systems that store, transport or deliver reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, instructions for performing the assay, etc.) from one location to another. For example, a kit comprises one or more housings (e.g., boxes) containing the relevant reaction reagents and / or support materials.

[0078] The terms "include", "including", "have", "contain" and the like are all open terms, meaning including but not limited to.

[0079] The term "nucleic acid" refers to a polymer that exists in a single or double-stranded form and comprises at least two deoxynucleotides or nucleotides. Unless specifically limited, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise noted, a specific nucleic acid sequence also implicitly encompasses variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences and sequences that are clearly noted. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassolet al. (1992); Rossolin et al., Mol. Cell. Probes 8:91-98 (1994)). "Nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked by the phosphate group. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that substitute new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and halogenated alkyls. The DNA may be present as antisense, plasmid DNA, portions of plasmid DNA, pre-compressed DNA, products of polymerase chain reaction (PCR), vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. The term nucleic acid is used interchangeably with gene, cDNA, mRNA encoded by a gene, and interfering RNA molecules.

[0080] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0081] When the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in MolecμLar Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the local homology algorithm of Pearson et al. (1988) P. The similarity search method of Proc. Natl. Acad. Sci. 85: 2444; Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altsch μL et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAS T-2 (Altsch μL et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESGHIT, BLAST Altsch μL et al., supra, FASTA, and GHASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0082] The public can obtain the biological materials of Flammulina velutipes variety 6-3, Flammulina velutipes variety GH79 and Flammulina velutipes variety 6-21 in the following examples from Beijing Academy of Agriculture and Forestry. The biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0083] The Flammulina velutipes variety 6-21 (also known as 6-21, Flammulina velutipes 6-21 or Flammulina velutipes monokaryotic strain 6-21, monokaryotic strain 6-21 or 6-21 monokaryotic strain) is recorded in the following document: "QTL Positioning Analysis of Mycelial Growth Rate of Flammulina velutipes Monosporic Strain, Journal of Mycology, 2019", and the name in the document is the monokaryotic strain "6-21" or "6-21".

[0084] The Flammulina velutipes variety 6-3 (also known as 6-3, Flammulina velutipes 6-3 or Flammulina velutipes monokaryotic strain 6-3, monokaryotic strain 6-3 or 6-3 monokaryotic strain) is recorded in the following document: "QTL Positioning Analysis of Mycelial Growth Rate of Flammulina velutipes Monosporic Strain, Journal of Mycology, 2019", and the name in the document is monokaryotic strain "6-3" or "6-3".

[0085] The Flammulina velutipes variety GH79 is the transformant TF21 in the last row of Table 3 on page 987 of the following document: Matrix design for detecting exogenous DNA insertion sites in Flammulina velutipes transformants, 22 June 2020, 39(6): 983-992 Mycosystema, ISSN 1672-6472 CN11-5180 / Q DOI: 10.13346 / j.mycosystema.200031.

[0086] Example 1 Discovery and Identification of FfGH79 Gene

[0087] Based on the inventor's previous research on the mutant library of Flammulina velutipes, a mutant GH79 with significant phenotypic differences and stable passage compared with the wild-type strain 6-3 was obtained. The insertion site of the exogenous fragment in the 6-3 genome was detected based on the whole gene resequencing technology. PFfGH79-F and PFfGH79-R were used to amplify the upstream and downstream fragments of the insertion site and the FfGH79 gene sequence. The primers used are shown in Table 1. Zoom lite software was used to align FfGH79 with transcriptome reads (the number of mismatched bases was set to 40), determine the position of the intron, and use the GSDS2.0 (http: / / gsds.gao lab.org / index.php) online website to draw the gene structure diagram. PlantCARE (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) online tool was used to predict the cis-acting elements of the promoter sequence of the FfGH79 gene. The conserved domains of FfGH79 gene were predicted using Conserved Domains (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) in NCBI database, and the function of FfGH79 protein was analyzed using InterProScan (https: / / www.ebi.ac.uk / interpro / ).

[0088] Based on the phenotypic analysis of transformants in the Flammulina velutipes mutant library, a transformant GH79 with sparse mycelial morphology and slower growth rate compared with the wild-type strain 6-3 was obtained. Figure 1 As shown in A. The insertion site results showed that 5619-7669bp in the plasmid replaced Chr09:756583-756603bp in the genome. The insertion site sequence was amplified using specific primers PFfGH79-F / PFfGH79-R. The results showed that GH79 amplified the band of the integrated plasmid DNA fragment ( Figure 1 (B).

[0089] Using the 6-3 genome annotation file as a reference, the genome replacement sequence of GH79 was compared, and the results showed that the insertion site was located 21-41bp upstream of the FfGH79 gene. PlantCARE was used to perform cis-acting element prediction on the FfGH79 promoter region, and the results showed that the promoter region contained two core promoter elements TATA-box, a common cis-acting element CAAT-box, and a light response element TCCC-motif, and the inserted fragment destroyed part of the light response element region. RT-qPCR was further used to detect the expression level of the gene FfGH79 downstream of the insertion site in the transformant GH79, and the results are as follows: Figure 1 As shown in C, the expression level of transformant GH79 is 9.7 times that of 6-3. It is speculated that the transcriptional regulatory sequence in the insertion site may affect the expression of FfGH79 gene, thereby regulating the growth and development of Flammulina velutipes.

[0090] The FfGH79 gene structure was determined using Zoom lite software. The results showed that FfGH79 is 971 bp in length, consisting of 5 exons and 4 introns, encoding 238 amino acids ( Figure 1 (D) The FfGH79 protein sequence was submitted to NCBI for comparison, and the results showed that there was no homologous protein matching it, and no conserved domain was identified in the protein. The sequence was further submitted to the InterProScan database for comparison, and the results showed that the FfGH79 protein had no protein family members and its function was unknown.

[0091] Example 2 Construction of FfGH79 overexpression and interference vector

[0092] Flammulina 6-3 contains the FfGH79 gene, the nucleotide sequence of its coding chain is sequence 3, positions 1-189 are the first exon, positions 190-245 are the first intron, positions 246-263 are the second exon, positions 264-349 are the second intron, positions 350-497 are the third exon, positions 498-553 are the third intron, positions 554-688 are the fourth exon, positions 689-744 are the fourth intron, and positions 745-971 are the fifth exon. The coding sequence of the FfGH79 gene is sequence 1, and the encoded amino acid sequence is the protein of sequence 2.

[0093] The monokaryotic strain 6-3 of Flammulina velutipes was cultured in a PDA medium (200 g potato, 20 glucose, 20 agar powder) at 25° C. When the strain grew to 7 days, the DNA of the monokaryotic strain 6-3 of Flammulina velutipes was extracted using the CTAB method to obtain the monokaryotic strain 6-3 DNA of Flammulina velutipes (also known as 6-3 DNA).

[0094] The target fragment carrying the homologous sequence at the end of the linearized vector was amplified using the monokaryotic strain 6-3 DNA as a template to obtain the amplified fragment. The primer sequences are shown in Table 1.

[0095] The backbone vector pBHg-eGFP (GenBank: MZ420392.1, Oct 8, 2022) was linearized by restriction digestion using SpeI and ApaI enzymes (TaKaRa Bio, Japan) to obtain a linearized vector after restriction digestion. IIOne Step Cloning Kit (Cat. No.: C112-01 / 02, Vazyme, Nanjing, China) was used to connect the target amplified fragment (amplified by the primer pair FfGHS79-OE-F-SpeI / FfS79-OE-R-ApaI) with the linearized vector to construct the FfGH79 gene overexpression recombinant vector pBHg-FfGH79-OE. The FfGH79 gene overexpression recombinant vector pBHg-FfGH79-OE is a recombinant vector obtained by replacing the fragment between the restriction endonuclease SpeI and Apa I recognition sites in the backbone vector pBHg-eGFP with the DNA fragment of the FfGH79 gene (sequence 3), while keeping the other sequences of the backbone vector pBHg-eGFP unchanged. The recombinant vector was named the FfGH79 gene overexpression recombinant vector pBHg-FfGH79-OE.

[0096] Sequence 1 is as follows:

[0097] ATGAAAATGGACTCTATTCCGCTGGCGCCGCTTTTTGGCATGTATTACCAGGAAATGCCAGAGGCGCACGCATATACGCATATCGCACAACTTTTAAATCGCCTTGAAAGCTATTCTGAGGGCTTAGACGACTTGTATCAACTTTTGTGTGCATATTCGGCGTCCTCTCCTGATGTTAGTCCTGTATCACTGTTAGAGCTCGTTCAGATTGCAAGAGACACACTGCTTCGATCTGGTGTTAAGCTGCATGCTGCTGTTCCTTGTCGACACGATATGACACCACACCGACGTTCTTATTTTTCTCTCCTGTACGCTTTGAAAAACAAAAGACCCTTATTTCCCGCTGCTTTGCAAGACCAACTCTCTCGATTCGTTCTTCCGCTCGCTAGCGCTGCTTTGTTACGACGGTCTGTGCGAACCTCAAACCGCCAGCGTTGCGCCACAAGCACGACTCCTCGGCGTATTGAAGACGATAGGTATCTCATGCCACCGCCTGACATGTTTCCCCCACGTTCCTCGCTGGTATGCAGACAAAGGAAGCCGATGGTCTTCCCGCGCGAAGTCATTCAGAGATACATTGCTTCAGAGGCGGAGGATTCGGACGTAGAAGAAGAAGAGAAGGACGATACCTCTCGACGATTCGTCATGCAGCCCTTGACGAAACGGTTTGGCATTTGGTGTGGTAGTTCGACCTCGATGTTTCGGATGCGAGATTGA。

[0098] The FfGH79 gene sequence is as shown in Sequence 3, specifically as follows:

[0099] ATGAAAATGGACTCTATTCCGCTGGCGCCGCTTTTTGGCATGTATTACCAGGAAATGCCAGAGGCGCACGCATATACGCATATCGCACAACTTTTAAATCGCCTTGAAAGCTATTCTGAGGGCTTAGACGACTTGTATCAACTTTTGTGTGCATATTCGGCGTCCTCTCCTGATGTTAGTCCTGTATCAGTAAGTTGGTCTCCCTTTGTCGTCGAGAGAATATGGGCTGATCGTGGCATCTACAGCTGTTAGAGCTCGTTCAGGTGATTATCTGCTCCCTAACCTTATTCCGCTTTTCCTTTGTTTGTTTTTATAGGAACGTAAAGCTGACTGTACTTTTGCCACGCAGATTGCAAGAGACACACTGCTTCGATCTGGTGTTAAGCTGCATGCTGCTGTTCCTTGTCGACACGATATGACACCACACCGACGTTCTTATTTTTCTCTCCTGTACGCTTTGAAAAACAAAAGACCCTTATTTCCCGCTGCTTTGCAAGGTAATCATTGTGTCGTGTATCCTTCCCCTATTCAGTCTACTGACGATGAATCGCAGACCAACTCTCTCGATTCGTTCTTCCGCTCGCTAGCGCTGCTTTGTTACGACGGTCTGTGCGAACCTCAAACCGCCAGCGTTGCGCCACAAGCACGACTCCTCGGCGTATTGAAGACGATAGGTATCTCATGCCACGTAAGTTTAGCTCTATCTTCCCTATATATTGGACGATACTGAGCTCATAATTCCAGCGCCTGACATGTTTCCCCCACGTTCCTCGCTGGTATGCAGACAAAGGAAGCCGATGGTCTTCCCGCGCGAAGTCATTCAGAGATACATTGCTTCAGAGGCGGAGGATTCGGACGTAGAAGAAGAAGAGAAGGACGATACCTCTCGACGATTCGTCATGCAGCCCTTGACGAAACGGTTTGGCATTTGGTGTGGTAGTTCGACCTCGATGTTTCGGATGCGAGATTGA。

[0100] The amino acid sequence of the protein encoded by the FfGH79 gene is shown in SEQ ID NO: 2, and is as follows:

[0101] MKMDSIPLAPLFGMYYQEMPEAHAYTHIAQLLNRLESYSEGLDDLYQLLCAYSASSPDVSPVSLLELVQIARDTLLRSGVKLHAAVPCRHDMTPHRRSYFSLLYALKNKRPLFPAALQD QLSRFVLPLASAALLRRSVRTSNRQRCATSTTPRRIEDDRYLMPPPDMFPPRSSLVCRQRKPMVFPREVIQRYIASEAEDSDVEEEEKDDTSRRFVMQPLTKRFGIWCGSTSTSMFRMRD.

[0102] The backbone vector pBHg-eGFP was linearized by restriction enzyme digestion with BglII and Apa I (TaKaRa Bio, Japan) to obtain the linearized vector. The target amplified fragment (amplified by the primer pair FfS79-RNAi-F-BglII / FfS79-RNAi-R and the primer pair FfS79-RNAi-F / FfS79-RNAi-R-ApaI) was connected with the linearized vector by using the Ultra One Step Cloning Kit (Cat. No. C115-01, Vazyme, Nanjing, China) to construct the FfGH79 gene interference recombinant vector pBHg-FfGH79-RNAi. The FfGH79 gene interference recombinant vector pBHg-FfGH79-RNAi is a recombinant vector obtained by replacing the fragment between the restriction endonuclease BglII and Apa I recognition sites in the backbone vector pBHg-eGFP with a hairpin structure sequence consisting of the fourth intron and the fifth exon (nucleotide sequence is sequence 4), while keeping the other sequences of the backbone vector pBHg-eGFP unchanged. The recombinant vector is named the FfGH79 gene interference recombinant vector pBHg-FfGH79-RNAi. pBHg-FfGH79-RNAi contains a shRNA gene for silencing the FfGH79 gene, and the nucleotide sequence of the shRNA gene is sequence 4. Positions 1-227 of sequence 4 are the reverse complementary sequence of the fifth exon; positions 228-283 of sequence 4 are introns, corresponding to positions 689-744 of sequence 3; positions 284-510 of sequence 4 are the fifth exon, corresponding to positions 491-717 of sequence 1 and positions 745-971 of sequence 3.

[0103] Sequence 4 is as follows:

[0104] TCAATCTCGCATCCGAAACATCGAGGTCGAACTACCACACCAAATGCCAAACCGTTTCGTCAAGGGCTGCATGACGAATCGTCGAGAGGTATCGTCCTTCTCTTCTTCTTCTACGTCCGAATCCTCC GCCTCTGAAGCAATGTATCTCTGAATGACTTCGCGCGGGAAGACCATCGGCTTCCTTTGTCTGCATACCAGCGAGGAACGTGGGGGAAACATGTCAGGCGGTAAGTTTAGCTCTATCTTCCCTATATA TTGGACGATACTGAGCTCATAATTCCAGCGCCTGACATGTTTCCCCCACGTTCCTCGCTGGTATGCAGACAAAGGAAGCCGATGGTCTTCCCGCGCGAAGTCATTCAGAGATACATTGCTTCAGAGGC GGAGGATTCGGACGTAGAAGAAGAAGAGAAGGACGATACCTCTCGACGATTCGTCATGCAGCCCTTGACGAAACGGTTTGGCATTTGGTGTGGTAGTTCGACCTCGATGTTTCGGATGCGAGATTGA.

[0105] The vector construction strategy is shown in Figure 2 .

[0106] Table 1

[0107]

[0108]

[0109] Example 3: Verification of pseudo-transformants and expression levels

[0110] The constructed overexpression vector (FfGH79 gene overexpression recombinant vector pBHg-FfGH79-OE) and interference vector (FfGH79 gene interference recombinant vector pBHg-FfGH79-RNAi) were transformed into Escherichia coli DH5α (TIANGEN, Beijing, China) by heat shock transformation method. Single clones were picked after overnight culture and verified by colony PCR using the primers in Table 1.

[0111] The verified positive single clones were cultured in broth medium with 100 μg / ml kanamycin (Solarbio, Beijing, China). The pBHg-FfGH79-OE and pBHg-FfGH79-RNAi plasmids were extracted using a plasmid mini-extraction kit (TIANGEN, Beijing, China). The above two plasmids were transferred into Agrobacterium separately according to the instructions of Agrobacterium tumefaciens GV3101 (Bomed Bio, Beijing, China). After 3 days of cultivation, colony PCR was performed on the single clones using the primers in Table 1. Recombinant Agrobacterium pBHg-FfGH79-OE / GV3101 containing pBHg-FfGH79-OE plasmid and recombinant Agrobacterium pBHg-FfGH79-RNAi / GV3101 containing pBHg-FfGH79-RNAi plasmid were obtained respectively.

[0112] In the fungal transformation experiment, recombinant Agrobacterium pBHg-FfGH79-OE / GV3101 and recombinant Agrobacterium pBHg-FfGH79-RNAi / GV3101 were used as donor strains and cultured in broth medium. After centrifugation, the bacterial solution was mixed with mycelial blocks of Flammulina velutipes cultivar 6-3 in IM induction medium (K2HPO4 2.05 g; KH2PO4 1.45 g; NaCl 0.15 g; MgSO4 0.5 g; CaCl2·2H2O 0.067 g; (NH4)2SO4 0.0025 g; glucose 1.8 g; glycerol 5 g; FeSO4·7H2O 0.0025 g; MES 0.853g; AS200μM water 1L) co-cultured, after 6h, the bacterial block was transferred to IM solid medium with cellophane and cultured for 3-5d, and when the bacterial block germinated again, it was washed with sterile water containing cephalosporin (200μmol / L), and transferred to CYM plate containing hygromycin (30μg / mL) and continued to be cultured for 15-25d, and the pseudo-transformants were obtained by preliminary screening. Then, the pseudo-transformants were transferred to PDA medium containing hygromycin (60μg / mL), and repeated screening was performed to obtain pseudo-transformants with stable inheritance.

[0113] DNA extraction and PCR verification of pseudo-transformants:

[0114] The total DNA was extracted by modified CTAB, and the target sequence in the overexpression plasmid (FfGH79 gene overexpression recombinant vector pBHg-FfGH79-OE) was amplified by primers VOE-F / VOE-R, and the forward / reverse target sequence of the interference plasmid (FfGH79 gene interference recombinant vector pBHg-FfGH79-RNAi) was amplified by primers VRNAI-fF / VRNAI-fR and VRNAI-rF / VRNAI-rR in Table 1. The band size of the amplified product was detected by agarose gel electrophoresis. The verified correct pseudo-transformants were transferred to a PDA plate with cellophane, cultured at 25°C for 7 days, and three biological replicates were performed for each sample. After sampling, they were stored in a -80°C refrigerator for gene expression detection.

[0115] RNA extraction and RT-qPCR detection of relative gene expression:

[0116] The total RNA of the samples was extracted using the OMEGAE.ZNAPlant RNAKit kit (OmegaBio-Tek, USA), and the RNA that passed the quality test was used All-in-One First-Strand cDNA SynthesisSuperMix for qPCR Kit (TransGen Biotech, Beijing) was used to reverse transcribe into cDNA. Primer5.0 software was used to design quantitative primers (QFfS79-F / QFfS79-R), and the sequence information is shown in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (QFfGAPDH) was used as the internal reference (QFfGAPDH-F / QFfGAPDH-R) gene, and Flammulina velutipes 6-3 was used as the control for quantitative detection. -ΔΔCt The relative expression level of the target gene was calculated by this method.

[0117] The results showed that after multiple screenings with increased hygromycin concentrations, a total of 5 FfGH79 gene overexpression transformants and 7 FfGH79 gene interference transformants were obtained. The 5 FfGH79 gene overexpression transformants obtained by transforming Flammulina velutipes variety 6-3 with recombinant Agrobacterium pBHg-FfGH79-OE / GV3101 were named OE#4, OE#9, OE#25, OE#31 and OE#49 (also known as Flammulina velutipes OE#4, OE#9, OE#25, OE#31 and OE#49, respectively), and the 7 FfGH79 gene interference transformants obtained by transforming Flammulina velutipes variety 6-3 with recombinant Agrobacterium pBHg-FfGH79-RNAi / GV3101 were named RNAi#1, RNAi#13, RNAi#32, RNAi#33, RNAi#34, RNAi#37 and RNAi#39 (also known as Flammulina Enoki R#1, Flammulina Enoki R#13, Flammulina Enoki R#32, Flammulina Enoki R#33, Flammulina Enoki R#34, Flammulina Enoki R#37, Flammulina Enoki R#39 or also known as Flammulina Enoki RNAi#1, Flammulina Enoki RNAi#13, Flammulina Enoki RNAi#32, Flammulina Enoki RNAi#33, Flammulina Enoki RNAi#34, Flammulina Enoki RNAi#37, Flammulina Enoki RNAi#39 or also known as R#1, R#13, R#32, R#33, R#34, R#37, R#39).

[0118] The specific primers VOE-F and VOE-R in Table 1 were used to amplify the target gene in the FfGH79 gene overexpression transformant (including the DNA fragment whose nucleotide sequence is the 1st to 971st position in Sequence 3), the specific primers VRNAI-fF / VRNAI-fR in Table 1 were used to amplify the forward target gene in the FfGH79 gene interference transformant (including the DNA fragment whose nucleotide sequence is the 1st to 283rd position in Sequence 4), and the specific primers VRNAI-rF / VRNAI-rR in Table 1 were used to amplify the reverse target gene in the FfGH79 gene interference transformant (including the DNA fragment whose nucleotide sequence is the 284th to 510th position in Sequence 4). The results showed that all transformants were able to amplify the target bands consistent with the expectations ( Figure 3 3 out of 3 A, 3 out of 3 B, 3 out of 3 C; Figure 3 WT is 6-3, OE#4, OE#9, OE#25, OE#31, OE#49 and R#1, R#13, R#32, R#33, R#34, R#37, R#39 correspond to the above-mentioned overexpression transformants and interference transformants, respectively, and water is double distilled water). The results showed that all transformants were positive transformants. RT-qPCR was further used to detect the expression of the FfGH79 gene in the above positive transformants, excluding 2 transformants without overexpression effect and 4 transformants without interference effect. The expression levels of the remaining transformants are as follows Figure 3As shown in D (6-3 and GH79 correspond to Flammulina velutipes varieties 6-3 and Flammulina velutipes varieties GH79, respectively): the expression levels of FfGH79 gene in OE#4, OE#9, and OE#49 were 43.25, 3.25, and 4.58 times that of 6-3, respectively, and the expression levels in RNAi#1, RNAi#32, and RNAi#34 were reduced by 54%, 62%, and 22% compared with 6-3, respectively. The above results prove that the 6 transformants have successfully achieved overexpression and interference of FfGH79 gene and are used for the following studies.

[0119] Example 4 Effect of FfGH79 on the Phenotype of Flammulina velutipes Mycelium

[0120] The mycelial morphology and growth rate of Flammulina velutipes variety 6-3, Flammulina velutipes variety GH79, OE#4, OE#9, OE#49, RNAi#1, RNAi#32 and RNAi#34 in Example 3 were detected on a PDA plate. The specific method is as follows:

[0121] The activated strains (Flammulina velutipes variety 6-3, Flammulina velutipes variety GH79, OE#4, OE#9, OE#49, RNAi#1, RNAi#32 and RNAi#34) were transferred to the center of a 90 mm PDA plate and cultured at 25°C in the dark. When the fastest growing mycelium grew to 1.5 cm from the center of the bacterial block, a "cross" was drawn at the bottom of the plate with the bacterial block as the center. A starting line was drawn at the intersection of the end of the mycelium and the "cross". After 3 days of continuous culture, a termination line was drawn. The distance between the starting line and the termination line was measured with a vernier caliper, the daily growth rate of the mycelium was calculated, and the mycelium morphology was recorded by taking photos. Each strain was biologically replicated 5 times.

[0122] Mycelial morphology: Mycelial morphology results are as follows Figure 4 As shown in the hyphae morphology diagram in Figure A, the colony size of the three overexpression transformants is significantly smaller than that of the wild-type strain 6-3, with fewer aerial hyphae and sparse hyphae density, which is similar to the colony morphology of GH79. However, although the colonies of the three interference transformants are also significantly smaller than those of the wild-type strain 6-3, they have more aerial hyphae and denser hyphae.

[0123] Growth rate: The growth rate was measured by the cross-line method. Daily growth rate = growth distance / number of growth days.

[0124] The results are as follows Figure 4As shown in B, the mycelial growth rates of 6-3, GH79, OE#4, OE#9, and OE#49 were 5.75, 3.10, 1.78, 4.52, and 3.98 mm / d, respectively. The growth rates of GH79 and the three overexpression transformants were significantly lower than those of 6-3. However, the interference transformants also showed a decreasing trend, and the mycelial growth rates of RNAi#1, RNAi#32, and RNAi#34 were reduced by 38%, 34%, and 73%, respectively, compared with 6-3. It is speculated that the expression level of FfGH79 may be related to mycelial growth. Figure 4 In the figure, 6-3 and GH79 correspond to the Flammulina velutipes varieties 6-3 and GH79, respectively, and OE#4, OE#9, OE#49 and RNAi#1, RNAi#32, and RNAi#34 correspond to the above-mentioned overexpression transformants and interference transformants, respectively.

[0125] Example 5 Effect of FfGH79 on the Phenotype of Flammulina velutipes Mycelium Edge

[0126] To further verify the role of the FfGH79 gene in hyphae density, the morphology of the hyphae (6-3, GH79, OE#4, OE#9, OE#49, RNAi#1, RNAi#32, and RNAi#34) edge and hyphae branch distance were detected. The specific methods are as follows:

[0127] Mycelial edge morphology:

[0128] The activated strains (Flammulina velutipes variety 6-3, Flammulina velutipes variety GH79, OE#4, OE#9, OE#49, RNAi#1, RNAi#32 and RNAi#34) were transferred to the center of a 90 mm PDA plate and cultured at 25°C in the dark. When the activated mycelium grew to 80% of the area of ​​the PDA plate, a stereo fluorescence microscope LEICADFC550 (Leica Microsystems, Germany) was used to observe the microscopic morphology of the mycelium edge of each strain and take pictures for record.

[0129] Mycelial branch distance:

[0130] The activated strains (Flammulina velutipes variety 6-3, Flammulina velutipes variety GH79, OE#4, OE#9, OE#49, RNAi#1, RNAi#32 and RNAi#34) were transferred to the center of a 90 mm PDA plate and cultured at 25°C in the dark. After 4 days of culture, the hyphae covered half of the coverslip and were stained with a fluorescent brightener (sigma, USA). The hyphae microscopic morphology was recorded using a fluorescence microscope BX51 (Olympus, Japan). ImageJ software was used to count the hyphae spacing (the length between each two consecutive branches). At least 50 biological replicates were performed for each strain.

[0131] Microscopic observation of mycelium Figure 5 As shown in A, the hyphae edges of the three overexpression transformants are sparser than those of 6-3 and are similar in morphology to GH79. Among them, the sparseness of OE#4 and OE#9 is significantly different, while OE#49 has no significant difference. At the same time, the hyphae edges of the three interference transformants are denser than those of 6-3, and the difference of RNAi#34 is extremely significant. ImageJ software was used to measure the hyphae branch distance. The hyphae branch distance measurement results are shown in Figure 1. Figure 5 As shown in B, the hyphae branching distances of 6-3, GH79, OE#4, OE#9, and OE#49 were 68.35, 81.61, 84.13, 95.81, and 70.24um, respectively. Among them, the branching distances of GH79, OE#4, and OE#9 were larger than those of 6-3, while the branching distance of OE#49 was slightly larger than that of 6-3 and there was no significant difference. The hyphae branching distances of RNAi#1, RNAi#32, and RNAi#34 were 0.77, 0.77, and 0.58 times that of 6-3, respectively, and the three interference transformants all had significantly reduced differences. The correlation results showed that the correlation coefficient between the expression level of the FfGH79 gene and the hyphae branching distance was 0.786, reaching a high positive correlation. The above results speculate that the FfGH79 gene negatively regulates hyphae density. Figure 5 In the figure, 6-3 and GH79 correspond to the Flammulina velutipes varieties 6-3 and GH79, respectively, and OE#4, OE#9, OE#49 and RNAi#1, RNAi#32, and RNAi#34 correspond to the above-mentioned overexpression transformants and interference transformants, respectively.

[0132] Example 6 Obtaining transformant hybrid strains

[0133] The monokaryotic strain 6-21 was hybridized with 6-3, Flammulina velutipes variety GH79 and each transformant strain (OE#4, OE#9, OE#49 and RNAi#1, RNAi#32, RNAi#34) by single hybridization. The specific method is as follows:

[0134] Inoculate the 6-3 monokaryotic strain at a position 1-1.5 cm from the center of the culture dish (90 mm in size), and then inoculate the 6-21 monokaryotic strain at a position about 3 cm from the center of the culture dish. After the mycelia of the two monokaryotic strains grow to contact and contact for about half a month, pick the mycelia on the far right side of the culture dish for activation, and use a fluorescent microscope to check the lock-like union of the hybrid strains. If there is a lock-like union, it indicates that the hybridization is successful and the 6-3 hybrid strain is obtained, and the next step of the fruiting experiment can be carried out.

[0135] The monokaryotic strain 6-21 was hybridized with the Flammulina velutipes variety GH79 and each transformant strain (OE#4, OE#9, OE#49 and RNAi#1, RNAi#32, RNAi#34), respectively, using the same hybridization method as described above, to obtain the corresponding transformant hybrid mycelia (i.e., GH79 hybrid strain, OE#4 hybrid strain, OE#9 hybrid strain, OE#49 hybrid strain and RNAi#1 hybrid strain, RNAi#32 hybrid strain and RNAi#34 hybrid strain), respectively.

[0136] The 6-3 hybrid strain is a strain obtained by hybridizing the monokaryotic strain 6-21 with the Flammulina velutipes variety 6-3. The GH79 hybrid strain is a strain obtained by hybridizing the monokaryotic strain 6-21 with the Flammulina velutipes variety GH79. The OE#4 hybrid strain is a strain obtained by hybridizing the monokaryotic strain 6-21 with the Flammulina velutipes variety OE#4 in Example 3. The OE#9 hybrid strain is a strain obtained by hybridizing the monokaryotic strain 6-21 with the Flammulina velutipes variety OE#9 in Example 3. The OE#49 hybrid strain is a strain obtained by hybridizing the monokaryotic strain 6-21 with the Flammulina velutipes variety OE#49 in Example 3. The RNAi#1 hybrid strain is a strain obtained by hybridizing the monokaryotic strain 6-21 with the Flammulina velutipes variety RNAi#1 in Example 3. The RNAi#32 hybrid strain is a strain obtained by hybridizing the monokaryotic strain 6-21 with the Flammulina velutipes variety RNAi#32 in Example 3. The RNAi#34 hybrid strain is a strain obtained by hybridizing the monokaryotic strain 6-21 with the Flammulina velutipes variety RNAi#34 in Example 3.

[0137] qRT-PCR detection method of FfGH79 gene expression:

[0138] The binuclear hybrid mycelia with lock-like union characteristics (6-3 hybrid strain, GH79 hybrid strain, OE#4 hybrid strain, OE#9 hybrid strain, OE#49 hybrid strain and RNAi#1 hybrid strain, RNAi#32 hybrid strain and RNAi#34 hybrid strain) were activated, and the expression level of the FfGH79 gene in the transformant hybrid strains was quantitatively verified using the 6-3 hybrid mycelia as a control. The quantitative detection method refers to Example 3.

[0139] Hybridization of transformant hybrid strains and qRT-PCR detection results of FfGH79 gene expression:

[0140] The lock-like union of hybrid strains was observed under a fluorescence microscope. The binucleate hyphae of 6-3, GH79 and all transformants formed obvious lock-like union structures. The lock-like union microscopic examination is shown in the figure below. Figure 6 Middle A( Figure 6In A, the 6-3 hybrid, GH79 hybrid, OE#4 hybrid, OE#9 hybrid, OE#49 hybrid and RNAi#1 hybrid, RNAi#32 hybrid and RNAi#34 hybrid correspond to the hybrid strains of monokaryotic strains 6-3, GH79, OE#4, OE#9, OE#49 and RNAi#1, RNAi#32 and RNAi#34 with monokaryotic strain 6-21, i.e., 6-3 hybrid strain, GH79 hybrid strain, OE#4 hybrid strain, OE#9 hybrid strain, OE#49 hybrid strain and RNAi#1 hybrid strain, RNAi#32 hybrid strain and RNAi#34 hybrid strain), all have lock-like union, and the results in the figure show that all transformants are successfully hybridized. The binucleate strains that showed lock-like union under microscopic examination were quantitatively verified, and the results showed that the OE-4 hybrid strain and the RNAi#1 hybrid strain had lost the overexpression and interference effect on the target gene ( Figure 6 Therefore, 6-3, GH79, OE#9, OE#49, RNAi#32, and RNAi#34 hybrid strains were selected for subsequent fruiting experiments. Figure 6 In B, 6-3 hybrid, OE#4 hybrid, OE#9 hybrid, OE#49 hybrid, GH79 hybrid, RNAi#1 hybrid, RNAi#32 hybrid, and RNAi#34 hybrid correspond to the hybrid strains of each variety with 6-21.

[0141] Example 7 Effect of FfGH79 on the Fruiting Body Development of Flammulina velutipes

[0142] In order to explore the function of the FfGH79 gene in the regulation of fruiting body development, the hybrid strains of Example 6 (6-3 hybrid strain, GH79 hybrid strain, OE#9 hybrid strain, OE#49 hybrid strain, RNAi#32 hybrid strain and RNAi#34 hybrid strain) were placed in a mushroom house for culture and the agronomic traits of their fruiting bodies were measured. The specific method is as follows:

[0143] 1. Inoculation

[0144] The 6-3 hybrid strain, GH79 hybrid strain, OE#9 hybrid strain, OE#49 hybrid strain, RNAi#32 hybrid strain and RNAi#34 hybrid strain) were respectively transferred into liquid culture medium (15 g corn flour, 10 g soybean flour, 10 g glucose, 11 grains of VB1, 1 grains of VB2, 2000 ml of water), and the conical flasks were placed in a shaker at 25°C and 160 rpm for shaking culture for 7 days to obtain liquid spawn of Flammulina velutipes of each strain. The liquid strains of Flammulina velutipes of the above strains were transferred to cultivation bags containing 1000g of cultivation materials (35% cottonseed hulls, 33% sawdust, 25% bran, 5% corn flour, 1% gypsum, 1% sugar, and 600g water), and the specific method was as follows: the polypropylene plastic angled bags with specifications of 18*36*0.005cm were loaded by mechanical loading, and after loading, they were manually punched. The bags that met the requirements should be sealed in time with plastic sleeves and plastic air-permeable valves, and the tightness should be moderate. After sealing, they were pushed into the autoclave for sterilization in time, and sterilized at 121℃ for 3h. After sterilization, they were pushed into the buffer room for cooling, and when the temperature dropped to about 20℃, the above hybrid strains were inoculated, and the inoculation amount per bag was about 10g, and 36 bags were inoculated for each strain.

[0145] 2. Spawning management: The inoculated bags are transferred to the sprouting room by conveyor belt. Cultivate in the dark, control the temperature at about 23℃, and the air humidity at 65%-70%. Pay attention to ventilation.

[0146] 3. Management of mushroom production: After the mycelium is fully grown, the scratching operation is carried out, that is, after removing the air valve and plastic ring, the cultivation bag is rolled from top to bottom until it is level with the surface of the cultivation material, and then the old mushroom skin is removed with a shovel. The management of the scratching method is mainly divided into four processes: scratching, bud induction, antibacterial, and fruiting body growth. When the fruiting body grows 3cm to 4cm from the bottle mouth, it is necessary to put on the paper (plastic) tube in time. The method of bag planting and bagging is to directly roll up the bag that was rolled down during the scratching and straighten it. The harvesting standard is that the cap is not open and the length of the stem is 13-16cm.

[0147] 4. Agronomic trait detection

[0148] Stipe length: The stipe length of each strain during the elongation period was measured using a vernier caliper. Each strain was cultivated in 36 bags, and 3 stipes in each bag were selected for testing.

[0149] Mycelial yield: When the fastest growing strain has filled the cultivation bag (49 days from the start of inoculation), the fruiting bodies are uniformly collected, weighed and the yield is recorded, with 36 bags for each strain.

[0150] Fruiting body phenotype analysis:

[0151] Observe and record the fruiting body morphology of each strain during its growth and development.

[0152] There was no significant difference in the growth rate of each transformant hybrid strain on the culture medium compared with the 6-3 hybrid strain, and all of them grew full of fungus bags after 25 days. The primordium of the hybrid strain appeared 4 days after scratching (37 days after inoculation), and the primordium phenotype was recorded at 39 days of cultivation. There was no significant difference in the number of primordia between the 6-3 hybrid strain and the GH79 hybrid strain and the OE#9 hybrid strain and the OE#49 hybrid strain, but the number of primordia was larger than that of the RNAi#32 hybrid strain and the RNAi#34 hybrid strain ( Figure 7 In the figure, 6-3 hybrid, GH79 hybrid, OE#9 hybrid, OE#49 hybrid, RNAi#32 hybrid and RNAi#34 hybrid correspond to the hybrid strains of 6-3, GH79, OE#9, OE#49, RNAi#32 and RNAi#34 with 6-21, i.e. 6-3 hybrid strain, GH79 hybrid strain, OE#9 hybrid strain, OE#49 hybrid strain, RNAi#32 hybrid strain and RNAi#34 hybrid strain). During the fruiting body development process, the cap morphology of each transformant hybrid strain had no significant difference compared with the 6-3 hybrid strain, however, the stipe length was significantly different, i.e. the stipe length of the 6-3 hybrid strain was significantly lower than that of the GH79 hybrid strain and the two overexpression transformant hybrid strains, but significantly higher than that of the two interference transformant hybrid strains. The stipe length was further measured (stipe length as shown in Figure 7 As shown in the middle bar graph, 6-3 hybrid, GH79 hybrid, OE#9 hybrid, OE#49 hybrid, RNAi#32 hybrid and RNAi#34 hybrid correspond to the hybrid strains of 6-3, GH79, OE#9, OE#49, RNAi#32 and RNAi#34 with 6-21, i.e., 6-3 hybrid strain, GH79 hybrid strain, OE#9 hybrid strain, OE#49 hybrid strain, RNAi#32 hybrid strain and RNAi#34 hybrid strain). Figure 7 The results of the stipe length bar chart showed that at 47 days, the stipe length of the 6-3 hybrid strain had reached 64.01 mm, and the stipes of the GH79 hybrid strain, OE#9 hybrid strain and OE#49 hybrid strain were all higher than those of the 6-3 hybrid strain, which were 69.00 mm, 70.26 mm and 74.05 mm, respectively. However, the stipes of the RNAi#32 hybrid strain and the RNAi#34 hybrid strain were significantly lower than those of the 6-3 hybrid strain, and the stipe lengths of the RNAi#32 hybrid strain and the RNAi#34 hybrid strain decreased by 22.14% and 39.86%, respectively; at 49 days, the stipe length also showed the same trend. In addition, the yield of the transformant hybrid strain was also significantly affected compared with the 6-3 hybrid strain. The fruiting body yield results of the transformant hybrid strain are shown in Figure 2. Figure 7As shown in the bar chart of fruiting body yield (6-3 hybrid, GH79 hybrid, OE#9 hybrid, OE#49 hybrid, RNAi#32 hybrid and RNAi#34 hybrid correspond to the hybrid strains of 6-3, GH79, OE#9, OE#49, RNAi#32 and RNAi#34 with 6-21, i.e. 6-3 hybrid strain, GH79 hybrid strain, OE#9 hybrid strain, OE#49 hybrid strain, RNAi#32 hybrid strain and RNAi#34 hybrid strain), the yields of GH79 hybrid strain, OE#9 hybrid strain and OE#49 hybrid strain were 1.14, 1.17 and 1.17 times that of 6-3 hybrid strain, respectively, while the yields of RNAi#32 hybrid strain and RNAi#34 hybrid strain were lower than those of 6-3 hybrid strain, especially RNAi#34 hybrid strain, which decreased by 42.34%. The correlation was analyzed using SPSS software, and the results showed that the correlation coefficients between the expression of FfGH79 gene and the length of the stipe during the elongation period and the fruiting body yield were 0.943 and 0.943, respectively, both of which were positively correlated with length. The above results suggest that the FfGH79 gene is involved in the regulation of the number of primordia and stipe elongation during the development of Flammulina velutipes fruiting bodies.

[0153] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of a substance for regulating the expression of a protein encoding gene in any of the following; A1) Application in regulating the yield of Flammulina velutipes fruiting bodies and / or application in preparing products for regulating the yield of Flammulina velutipes fruiting bodies; A2) application in regulating the length of Flammulina velutipes and / or application in preparing a product for regulating the length of Flammulina velutipes; A3) Application in regulating the branching distance of Flammulina velutipes hyphae and / or application in preparing a product for regulating the branching distance of Flammulina velutipes hyphae; A4) application in reducing the growth rate of Flammulina velutipes mycelium and / or application in preparing a product for reducing the growth rate of Flammulina velutipes; The protein is any of the following proteins: B1) The amino acid sequence is the protein shown in SEQ ID NO: 2; B2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in B1) and having 80% or more identity with the protein described in B1) and having the same function.

2. The use according to claim 1, characterized in that: The regulation is up-regulation, improvement or increase.

3. The use according to claim 1, characterized in that: The regulation is down-regulation, reduction or decrease.

4. The use according to claim 1 or 2, characterized in that: The substance is a substance that increases the expression of protein-coding genes; the substance is any one of the following: A1) a nucleic acid molecule encoding the protein; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1), or a recombinant vector containing the expression cassette described in A2); A4) A recombinant microorganism containing the nucleic acid molecule described in A1), or a recombinant microorganism containing the expression cassette described in A2), or a recombinant microorganism containing the recombinant vector described in A3).

5. The use according to claim 1 or 3, characterized in that: The substance is a substance that reduces the expression of protein-coding genes, and the substance is any one of the following: B1), a nucleic acid molecule that inhibits, reduces or downregulates the expression of the gene encoding the protein; B2), expressing the coding gene of the nucleic acid molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

6. The use according to claim 4 or 5, characterized in that: A1) The nucleic acid molecule is a DNA molecule whose coding sequence is sequence 1 and / or whose nucleotide sequence is sequence 3; and / or; B1) The nucleic acid molecule is a DNA molecule whose nucleotide sequence is sequence 4.

7. A method for improving the agronomic traits of Flammulina velutipes, characterized in that: The method comprises up-regulating or enhancing or increasing the expression level of the gene of the protein of claim 1 in the recipient Flammulina velutipes to obtain the target Flammulina velutipes, wherein the agronomic traits of the target Flammulina velutipes are better than those of the recipient Flammulina velutipes.

8. The method according to claim 7, characterized in that The agronomic traits are fruiting body yield, stipe length, hyphae branching distance and / or growth rate.

9. The method according to claim 7 or 8, characterized in that The up-regulation, enhancement or increase of the expression level of the gene of the protein described in claim 1 in the recipient Flammulina velutipes comprises the step of introducing the substance described in claim 4 into the recipient Flammulina velutipes.

10. A product, characterized in that The product is the protein described in claim 1 or the substance for regulating the expression of protein encoding gene described in claim 4.

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