CN108558992B

CN108558992B - Transcription factor PDD1 for regulating and controlling growth of needle mushroom fruiting body and coding gene and application thereof

Info

Publication number: CN108558992B
Application number: CN201810028494.2A
Authority: CN
Inventors: 李少杰; 吴塔菊; 孙宪昀; 张振颖
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2018-01-12
Filing date: 2018-01-12
Publication date: 2020-01-31
Anticipated expiration: 2038-01-12
Also published as: CN108558992A

Abstract

The invention discloses transcription factors PDD1 for regulating fruit body development, coding genes and application thereof.A coding gene sequence of the transcription factor PDD1 is shown as a sequence 1 in a sequence table, and an amino acid sequence of the transcription factor is shown as a sequence 2 in the sequence table. PDD1 overexpression and RNAi experiments in flammulina velutipes show that PDD1 is a positive regulation factor necessary in the growth process of flammulina velutipes hyphae and the development process of the fruit body, influences the primordial stage and the mature stage of the flammulina velutipes, the quantity, the height and the wet weight of the fruit body, and flammulina velutipes mutant strains (PDD 1) with short fruiting period and high yield are also disclosed^OE#31) The method has the characteristic of entering the maturation stage in advance, can improve the yield of the flammulina velutipes, shorten the production period of the flammulina velutipes, reduce the energy consumption and the labor cost, and has huge application prospects.

Description

Transcription factor PDD1 for regulating and controlling growth of needle mushroom fruiting body and coding gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to transcription factors PDD1 for positively regulating and controlling hypha growth and fruiting body development of flammulina velutipes (Flammulinavelutipes), and a strain PDD1 overexpression mutant strain (PDD 1) with short fruiting period and high yield^OE#31)。

Background

Flammulinavelipes (Flammulinavelipes), also known as dried mushrooms, unpurified mushrooms, Stropharia rugoso-annulata and the like, are typical basidioid edible fungi and are widely cultivated in countries all over the world, particularly Asia countries, the Flammulina velutipes contains rich protein, dietary fibers and saccharides, in addition, rich vitamins B1, B2, B3, vitamins C, D and E. the Flammulina velutipes are used as traditional edible fungi, not only have rich nutritional values, but also have important medicinal values.

In recent years, with large-area planting of flammulina velutipes, a plurality of researches on yield increasing technologies and methods of the flammulina velutipes are carried out successively, the researches mainly comprise optimization of nutrition conditions and genetic breeding, the optimization of the nutrition conditions comprises improvement of a cultivation mode, optimization of a cultivation material formula and addition of yield-increasing substances, for example, narrow bag cultivation, red light induced fruiting, horizontal two-head fruiting and other cultivation modes are adopted to promote growth of fruiting bodies, or more suitable carbon sources and nitrogen sources are selected, rice husks are used for promoting hypha growth instead of corn cobs, or substances such as triacontanol, special-effect plant nutrient (SPNE), soybean flour and the like are added externally, and exciton entities are generated, however, the methods focus on changing an external environment to adapt to growth of strains, the yield increasing capability is limited, and the cost needs to be considered, the genetic breeding is changed to adapt to the common environment, the current methods applied to the genetic breeding of the flammulina velutipes mainly comprise natural needle breeding, mutagenesis, cross breeding and genetic engineering breeding, for example, a needle strain breeding with a natural needle breeding, a sanming , a western strain, a needle strain with a high-producing gene coding gene, a gene coding gene, a gene.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate the growth of hypha and the development of fruiting bodies of the flammulina velutipes and cultivate the flammulina velutipes with high yield and short fruiting period.

In order to solve the technical problems, the invention firstly provides proteins capable of regulating growth of flammulina velutipes hyphae and development of fruiting bodies, wherein the name of the protein capable of regulating growth of the flammulina velutipes hyphae and development of the fruiting bodies is PDD1, and the PDD1 protein is the protein of the following a), b), c) or d):

a) the amino acid sequence is a protein shown in a sequence 2;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2;

c) the protein with the same function is obtained by replacing and/or deleting and/or adding or several amino acid residues of the amino acid sequence shown in the sequence 2;

d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 2 and having the same function.

In order to facilitate the purification of the protein in a), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (in general)Is 5 pieces)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of c) above, wherein the substitution and/or deletion and/or addition of or several amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in the c) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The gene encoding the protein of c) above can be obtained by deleting or several amino acid residues from the DNA sequence shown in SEQ ID No. 1, and/or performing a missense mutation of or several base pairs, and/or attaching a coding sequence of the tag shown in Table 1 to the 5 'end and/or 3' end thereof.

In the above d), "homology" includes an amino acid sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more homology with the amino acid sequence represented by the sequence 2 of the present invention.

In order to solve the technical problems, the invention also provides a biomaterial related to PDD1 protein.

The biomaterial related to the PDD1 protein provided by the invention is any of the following A1) to A8):

A1) a nucleic acid molecule encoding a PDD1 protein;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising the recombinant vector of a 4).

In the above biological material, the nucleic acid molecule of A1) is a gene represented by the following 1) or 2) or 3):

1) the coding sequence is a cDNA molecule or a genome DNA molecule shown in a sequence 1;

2) a cDNA molecule or a genome DNA molecule which has 75 percent identity or more than 75 percent identity with percent of the nucleotide sequence defined by 1) and codes PDD1 protein;

3) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and codes PDD1 protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Those nucleotides that have been artificially modified to have a identity of or more to 75% of the nucleotide sequence encoding PDD1 protein, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention, as long as they encode PDD1 protein and have the same function.

As used herein, the term "identity " refers to sequence similarity to a native nucleic acid sequence, "identity " includes nucleotide sequences that have 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater identity to the nucleotide sequence of a protein that consists of the amino acid sequence represented by coding sequence 2 of the present invention identity can be evaluated visually or using computer software the identity between two or more sequences can be expressed as a percentage (%) which can be used to evaluate identity between related sequences.

The above 75% or more than 75% of the same sex property as may be 80%, 85%, 90% or more than 95% of the same sex property as .

In the above biological materials, the expression cassette containing a nucleic acid molecule encoding PDD1 according to a2) refers to DNA capable of expressing PDD1 in a host cell, which may include not only a promoter for promoting transcription of PDD1 but also a terminator for terminating transcription of PDD 1. further , the expression cassette may further include an enhancer sequence.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

In order to solve the technical problems, the invention also provides a new application of the PDD1 protein or the related biological materials.

The invention provides application of the PDD1 protein or the related biological material in any of (b1) - (b6) as follows:

(b1) regulating and controlling the growth of the flammulina velutipes hyphae;

(b2) regulating the development of needle mushroom fruiting bodies;

(b3) the biomass and/or the yield of the flammulina velutipes are/is improved;

(b4) shortening the fruiting period of the needle mushrooms;

(b5) cultivating transgenic flammulina velutipes with high yield and short fruiting period;

(b6) and (5) breeding needle mushrooms.

In the above application, the regulation is facilitated, and the improvement of the biomass and/or yield of the flammulina velutipes is embodied in of any one of (c1) - (c3) as follows:

(c1) increasing the number of needle mushroom fruiting bodies;

(c2) increasing the height of needle mushroom fruiting bodies;

(c3) increasing the wet weight of needle mushroom fruiting bodies;

the shortening of the fruiting period of the flammulina velutipes is realized by promoting the primordial stage and/or the mature stage of the flammulina velutipes to be advanced.

The invention respectively constructs pdd1 overexpression mutant strains (pdd 1) in needle mushroom wild types^OE) And pdd1 mutant strains with reduced expression (pdd 1)^RNAi). Hypha growth experiments are carried out on the flammulina velutipes wild type, pdd1 overexpression mutant strains and pdd1 knockdown expression mutant strains, phenotypes are analyzed, and the fact that hypha growth of the pdd1 knockdown expression mutant strains on a synthetic culture medium and a cultivation material is slower than that of the wild type is found. Pdd1 the hypha growth of the over-expression mutant strain on the cultivation material is faster than that of the wild type strain, and the hypha growth period is shortened. Pdd1 shows that it has positive regulation and control effect on the growth of Flammulina velutipes mycelium.

The method simultaneously performs fruiting experiments on the wild type flammulina velutipes, the pdd1 overexpression mutant strain and the pdd1 knockdown expression mutant strain, and observes and photographs the flammulina velutipes in different time periods. Analysis of the fruiting phenotype of all strains revealed that the wild type strain produced primordia (first grade fruiting body of Flammulina velutipes) 7 days after the stimulation of fruiting treatment, whereas the pdd1 overexpression mutant strain produced larger area of primordia 1 day earlier than the wild type. The fruiting culture is continued, pdd1 overexpression mutant strains are found to enter a mature harvest period 4 days earlier than wild strains, and in pdd1 knock-down expression mutant strains, no primordium is generated in the same fruiting time as the wild strains after pdd1 is knocked down in a small amplitude, and a very small amount of primordium can be generated in the fruiting time; however, when pdd1 was knocked down to a large extent, primordia could not be produced even if the fruiting time was prolonged. The expression level of pdd1 is proved to significantly affect the generation of the primordium of the flammulina velutipes and the development of the fruiting body, and pdd1 is a positive regulatory factor necessary in the process of the fruiting body development of the flammulina velutipes.

In order to solve the technical problems, the invention finally provides a method for culturing transgenic needle mushrooms with high yield and short fruiting period.

The method for cultivating the transgenic flammulina velutipes with high yield and short fruiting period, provided by the invention, comprises the steps of improving the expression quantity and/or activity of PDD1 protein in the recipient flammulina velutipes to obtain the transgenic flammulina velutipes; the yield of the transgenic needle mushroom is higher than that of the receptor needle mushroom and/or the fruiting period of the transgenic needle mushroom is shorter than that of the receptor needle mushroom.

In the method, the yield of the transgenic needle mushroom is higher than that of the recipient needle mushroom in any of (d1) - (d3), (d1) the number of the fruit bodies of the transgenic needle mushroom is more than that of the recipient needle mushroom, (d2) the height of the fruit bodies of the transgenic needle mushroom is higher than that of the recipient needle mushroom, (d3) the wet weight of the fruit bodies of the transgenic needle mushroom is larger than that of the recipient needle mushroom, and the fruiting period of the transgenic needle mushroom is shorter than that of the recipient needle mushroom in the primordial period and/or the mature period of the transgenic needle mushroom is earlier than that of the recipient needle mushroom.

Specifically, compared with a recipient flammulina velutipes, the transgenic flammulina velutipes with high yield and short fruiting period provided by the invention generate primordia 1 day ahead and enter a mature period 4 days ahead, the number of sporophores is increased by 19.85 +/-4.64%, the height of the sporophores is increased by 40.36 +/-7.25%, the total wet weight is increased by 62.47 +/-8.88%, the primordia period and the mature period are both advanced, and the total biomass is remarkably increased.

In the method, the method for improving the expression quantity and/or activity of the PDD1 protein in the recipient flammulina velutipes is to over-express the PDD1 protein in the recipient flammulina velutipes.

In the method, the overexpression method is to introduce a gene encoding the PDD1 protein into recipient flammulina velutipes.

In the above method, the nucleotide sequence of the gene encoding the PDD1 protein is a DNA molecule represented by SEQ ID No. 1.

In a specific embodiment of the invention, the gene encoding PDD1 protein is introduced into the recipient plant via a recombinant expression vector comprising a PDD1 gene expression cassette. The recombinant expression vector is 11261bp in size obtained by fusing a gpd promoter fragment (Pgpd-OE), an pdd1OE fragment and a terminator fragment (TtrpC-OE) with a recombinant kit at an XmnI enzyme cutting site and a plasmid pBHg-BCA 1.

In the above method, the recipient Flammulina velutipes may be Flammulina velutipes strain FL19 (yellow variety).

The invention discovers transcription factor PDD1 and coding gene PDD1 thereof in flammulina velutipes for the first time, and discovers that PDD1 is a necessary positive regulation and control factor in the growth of hyphae and the development of fruiting bodies of the flammulina velutipes, influences the primordial stage and the mature stage of the flammulina velutipes, the quantity, the height and the wet weight of the fruiting bodies, and also provides flammulina velutipes mutant strains (PDD 1) with short fruiting period and high yield^OE#31) The method has the characteristic of entering the original basal period and the mature period in advance, can improve the yield of the flammulina velutipes, shorten the production period of the flammulina velutipes, reduce energy consumption and labor cost, and has huge application prospect in the breeding of good strains of the flammulina velutipes.

Drawings

FIG. 1 is a graph showing the transcription levels of genes encoding HMG-box structural proteins at different stages of the fruiting body development of Flammulina velutipes.

FIG. 2 is a structural diagram of the transcription factor PDD 1.

FIG. 3 is a graph of pdd1 transcript level analysis results in wild-type, pdd1 overexpressing mutant strain, and pdd1 knock-down expressing mutant strain.

FIG. 4 is a graph of the hyphal growth results of wild type, pdd1 overexpressing mutant strain, and pdd1 knock-down expressing mutant strain on CYM plates.

FIG. 5 is a graph of the hyphal growth results of wild-type, pdd1 overexpressing mutant strains and pdd1 knock-down expressing mutant strains on the compost.

FIG. 6 is a graph of fruiting results of wild type, pdd1 overexpression mutant strain and pdd1 knock-down expression mutant strain on the compost.

FIG. 7 is a graph of biomass statistics for fruiting wild type, pdd1 overexpression mutant strain and pdd1 knock-down expression mutant strain.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The formulations of the media and solutions referred to in the following examples are as follows:

LB culture medium: 1% tryptone, 0.5% yeast extract, 1% NaCl, adding a proper amount of distilled water for dissolving, adjusting the pH to 7.0, fixing the volume, and sterilizing by high-pressure steam.

CYM medium: 1% maltose, 2% glucose, 0.2% tryptone, 0.2% yeast extract, adding a proper amount of distilled water for dissolving, naturally adjusting the pH to a constant volume, and sterilizing by high-pressure steam.

And (3) fruiting cultivation material culture medium: 30% of sawdust, 43.5% of cottonseed hulls, 25% of bran, 1% of light calcium carbonate, 0.5% of lime and 60% of water are added with water, fully and uniformly mixed and soaked for 4-5 hours, then the mixture is subpackaged into tissue culture bottles with the volume of 350mL, 325g of cultivation materials are packaged in each bottle, and the mixture is sterilized by high-pressure steam for 3 hours.

Agrobacterium transformation Induction Medium (IM): 2.05g K₂HPO₄，0.15g NaCl，0.5g MgSO₄·7H₂O，0.067g CaCl₂·2H₂O，0.0025g FeSO₄·7H₂O，0.5g(NH₄)₂SO₄1.8g glucose, 5mL glycerol, 8.53g2- (N-Morpholino) ethanesulfofonic Acid (pH5.3, filter sterilized), 200. mu.M acetosyringone (filter sterilized, added on plating).

2×CTAB buffer：2％CTAB，100mM Tris-HCl(pH8.0)，20mM EDTA(pH8.0)，1.4MNaCl，1％PVP(polyvinyl pyrrolidone)。

Soil DNA extraction buffer(SDEB)：100mM NaCl，50mM EDTA，0.25M Tris-HCl，5％SDS。

The plasmid pBHg-BCA1 in the following examples was derived from the Junior research center of agriculture and forestry university of Fujian, and is described in "Lu, Y.P., et al," A Jacalin-Related linkage Regulated the Format of American Mycelium and friendly Body in Flammulina velutipes. int J Mol Sci,2016.17(12) ".

The plasmid pCSN44 in the examples below was derived from the American Fungal genetic resource Center (Fungal genetics stock Center) and is described in the literature "Chen, X., et al, De-expression of CSP-1 active genetic resources to anti-genetic adhesives Rep,2016.6: p.19447".

The needle mushroom wild types in the following examples are all needle mushroom strain FL19 (yellow), which originates from the center of the cultural research of the university of agriculture and forestry, Fujian.

Example 1, PDD1 Gene and transcription factor PDD1 obtaining and sequence analysis thereof

, PDD1 gene nucleotide sequence and amino acid sequence of transcription factor PDD1

The coding genes of 8 genes with both nuclear localization sequences and HMG-box structural proteins are found in needle mushroom genomes, the transcription levels of the 8 genes at different development stages of sporocarp are analyzed by RNA-seq and quantitative PCR, genes are gradually up-regulated from an primordial period to an elongation period and a maturation period (figure 1) and are possibly play important functions in needle mushroom sporocarp development, the genes are named PDD1(primordium degradation defect1), the nucleotide sequences of the genes are the sequences 1 and PDD1, the transcription factor PDD1 is coded by the gene, and the amino acid sequence of the transcription factor PDD1 is the sequence 2.

Second, sequence analysis

Using a 2000bp extension of each of the upstream and downstream sequences of pdd1 gene as a reference sequence, and Zillions of OligosMapped (ZOOM) software to locate reads of the transcriptome in the reference sequence, the analysis revealed that the pdd1 gene had a total length of 1204bp from the start codon to the stop codon and contained 1 intron of 49 bp.

The amino acid sequence of the transcription factor PDD1 was analyzed by SMART (http:// SMART. embl-heidelberg. de /) and the software DNAMAN the results showed that the transcription factor PDD1 was proteins having HMG-box domain and nuclear localization sequence (FIG. 2), molecular weight 42472.68Da, isoelectric point 5.64.

Example 2, construction of pdd1 overexpression mutant strains and pdd1 knock-down expression mutant strains

and pdd1 overexpression vector construction

(1) Carrying out PCR amplification by using primers Pgpd-F and Pgpd-OE-R and using the genomic DNA of a wild flammulina velutipes strain as a template to obtain a flammulina velutipes gpd promoter fragment (Pgpd-OE), wherein the gpd promoter comprises th introns and exons of a gpd gene, and the size of a target sequence is 920 bp., and the primer sequences are as follows:

Pgpd-F:5’-CAGATCCCCCGAATTATTCGAGCTCGGTACAGTCGTG-3’；

Pgpd-OE-R:5’-AGAAAGAGTGGACCTGTAAAATGGTGAGCAAGAC-3’。

the PCR reaction program is: 30s at 98 ℃; 10s at 98 ℃, 90s at 55 ℃, 60s at 72 ℃ (25 cycles); 10min at 72 ℃; 4 ℃ is prepared.

(2) Carrying out PCR amplification by using primers pdd1-F and pdd1-R and genome DNA of a needle mushroom wild strain as a template to obtain a pdd1OE fragment, wherein the size of a target sequence is 1203bp, and the sequences of the primers are as follows:

pdd1-F:5’-TTTACAGGTCCACTCTTTCTCAGCCTACTACGACCA-3’；

pdd1-R:5’-AAGTGGATCCTCAAAATGCAAGGCCACTACCT-3’。

the PCR reaction program is: 30s at 98 ℃; 98 ℃ for 10s, 59 ℃ for 90s, 72 ℃ for 60s (25 cycles); 10min at 72 ℃; 4 ℃ is prepared.

(3) Primers TtrpC-OE-F and TtrpC-R were used to amplify by PCR using plasmid pCSN44 as a template to obtain a trpC terminator fragment (TtrpC-OE) with a target sequence size of 720 bp. The primer sequences are as follows:

TtrpC-OE-F:5’-TGCATTTTGAGGATCCACTTAACGTTACTGAAATCA-3’；

TtrpC-R:5’-AATTAACGCCGAATTCATGCCTGCAGGTCGAGAAAG-3’。

the PCR reaction program is: 30s at 98 ℃; 98 ℃ for 10s, 58 ℃ for 90s, 72 ℃ for 60s (25 cycles); 10min at 72 ℃; 4 ℃ is prepared.

(4) Extracting a pBHg-BCA1 plasmid by using a common plasmid miniextraction kit, digesting the plasmid by using restriction enzyme XmnI, and recovering a digestion product by using an agarose gel DNA recovery kit to obtain the pBHg-BCA1 after digestion and recovery.

(5) Pgpd-OE, pdd1OE and TtrpC-OE fragments are ligated into the digested and recovered pBHg-BCA1 (according to the kit instructions) by using a fragment recombination kit (Nanjing Nozao Toxon Biotech Co., Ltd., Cat: C113-01), Escherichia coli DH5 α is transformed into competence, then single colonies are picked up, the single colonies are subjected to colony PCR (polymerase chain reaction) verification by using verification primers Pgpd-detect-F and TtrpC-detect-R, the target sequence is 1558bp, and a pdd1 overexpression vector pBHg-BCA 1-pd1OE is obtained, wherein the primer sequence is as follows:

Pgpd-detect-F:5’-AACCGCCATCTTCCACACTT-3’；

TtrpC-detect-R:5’-AACACCATTTGTCTCAACTCCG-3’。

the PCR reaction program is: 94 ℃ for 5min s; 94 ℃ for 30s, 58 ℃ for 90s, 72 ℃ for 90s (25 cycles); 10min at 72 ℃; 4 ℃ is prepared.

pdd1 overexpression vector pBHg-BCA1-pdd1OE is 11936bp vector obtained by fusing gpd promoter fragment (Pgpd-OE), pdd1OE fragment and terminator fragment (TtrpC-OE) with a fragment recombination kit at XmnI cleavage site and plasmid pBHg-BCA 1.

Second, construction of pdd1 knockdown expression vector

The pdd1 knockdown expression vector is constructed by utilizing the RNAi technology of the hairpin structure. The method comprises the following specific steps:

(1) PCR amplification is carried out by using primers Pgpd-F and Pgpd-RNAi-R and using the genome DNA of the wild strain of the flammulina velutipes as a template to obtain a flammulina velutipes gpd promoter fragment (Pgpd-RNAi), wherein the size of a target sequence is 920 bp. The primer sequences are as follows:

Pgpd-F:5’-CAGATCCCCCGAATTATTCGAGCTCGGTACAGTCGTG-3’；

Pgpd-RNAi-R:5’-CCCGACTTGGAACATGACCTGTAAAATGGTGAGCAAGAC-3’。

(2) Carrying out PCR amplification by using primers pdd1-antisense-F and pdd1-antisense-R and using wild-type flammulina velutipes genome DNA as a template to obtain a pdd1-antisense fragment, wherein the size of a target sequence is 489 bp. The primer sequences are as follows:

pdd1-antisense-F:5’-CATTTTACAGGTCATGGGCCCATGTTCCAAGTCGGGTTTAAAGGC-3’；

pdd1-antisense-R:5’-GAGGACTTACCGTCAAGAAAGAACCCACCGTTG-3’。

the PCR reaction program is: 30s at 98 ℃; 98 ℃ for 10s, 65 ℃ for 90s, 72 ℃ for 60s (25 cycles); 10min at 72 ℃; 4 ℃ is prepared.

(3) Primers TtrpC-RNAi-F and TtrpC-R were used to perform PCR amplification using plasmid pCSN44 as template to obtain trpC terminator fragment (TtrpC-RNAi) with a target sequence size of 720 bp. The primer sequences are as follows:

TtrpC-RNAi-F:5’-CCCGACTTGGAACATGGATCCACTTAACGTTACTGAAATCA-3’；

TtrpC-R:5’-AATTAACGCCGAATTCATGCCTGCAGGTCGAGAAAG-3’。

(5) Pgpd-RNAi, pdd1-antisense and TtrpC-RNAi fragments are ligated into enzyme-cleaved and recovered pBHg-BCA1 (according to the kit instructions) by using a fragment recombination kit (Nanjing Nozao Tenza Biotech Co., Ltd., product No.: C113-01), Escherichia coli DH5 α is transformed into competence, then single colonies are picked up, colony PCR verification is carried out on the single colonies by using verification primers Pgpd-detect-F and TtrpC-detect-R, the size of a target sequence is 684bp, and plasmid pBHg-BCA1-pdd 1-antisense is obtained, wherein the primer sequences are as follows:

Pgpd-detect-F:5’-AACCGCCATCTTCCACACTT-3’；

TtrpC-detect-R:5’-AACACCATTTGTCTCAACTCCG-3’。

the PCR reaction program is: 5min at 94 ℃; 94 ℃ for 30s, 58 ℃ for 90s, 72 ℃ for 60s (25 cycles); 10min at 72 ℃; 4 ℃ is prepared.

(6) PCR amplification is carried out by using primers pdd1-sense-F and pdd1-sense-R and genome DNA of the wild strain of the flammulina velutipes as a template to obtain a pdd1-sense fragment, wherein the size of a target sequence is 540 bp. The primer sequences are as follows:

pdd1-sense-F:5’-CATTTTACAGGTCATGGGCCCATGTTCCAAGTCGGGTTTAAAGGC-3’；

pdd1-sense-R:5’-GAGGACTTACCGTCAAGAAAGAACCCACCGTTG-3’。

(7) After the plasmid pBHg-BCA1-pdd1-antisens and the fragment pdd1-sense fragment were digested simultaneously with the restriction enzyme BamHI and recovered, the two fragments were ligated overnight with T4 Ligase.

(8) Transforming the ligation product into escherichia coli DH5 α competence, then picking single colonies, and carrying out colony PCR verification on the single colonies by using verification primers Pgpd-detect-F and TtrpC-detect-R, wherein the size of a target sequence is 1224bp, and the pdd1 knock-down expression vector pBHg-BCA1-pdd1RNAi primer sequence is obtained as follows:

Pgpd-detect-F:5’-AACCGCCATCTTCCACACTT-3’；

TtrpC-detect-R:5’-AACACCATTTGTCTCAACTCCG-3’。

the PCR reaction program is: 5min at 94 ℃; 94 ℃ for 30s, 58 ℃ for 90s, 72 ℃ for 90s (25 cycles); 10min at 72 ℃; 4 ℃ is prepared.

pdd1 knockdown expression vector pBHg-BCA1-pdd1RNAi is a 11761bp vector obtained by fusing a gpd promoter fragment (Pgpd-RNAi), a pdd1-antisense fragment, a pdd1-sense fragment and a trpC terminator fragment (TtrpC-RNAi) with a fragment recombination kit at an XmnI cleavage site and plasmid pBHg-BCA 1.

Third, plasmid pBHg-BCA1-pdd1OE and pBHg-BCA1-pdd1RNAi transformation Agrobacterium

In the RNAi transformation of the AGL-1 strain competent cells (Beijing Bomaide Gene technology Co., Ltd., Cat.: BC302-01) from the plasmids pBHg-BCA1-pdd1OE and pBHg-BCA1-pdd1 prepared in step and step two, respectively, the specific steps are as follows:

(1) two tubes of 50. mu.L AGL-1 competent cells were added with 1. mu.g of plasmid pBHg-BCA1-pdd1OE and plasmid pBHg-BCA1-pdd1RNAi, respectively, gently mixed by pipetting with a pipette, and left on ice for 10 min.

(2) And (5) placing the centrifugal tube in liquid nitrogen for quick freezing for 5 min.

(3) Immediately placed in a standing water bath at 37 ℃ for 5min without shaking the water surface.

(4) The centrifuge tubes were placed back on ice and held for 5 min.

(5) Adding 1mL LB liquid culture medium, standing and culturing at 28 deg.C for 2-3 h.

(6) The bacterial liquid is absorbed and spread on a flat plate containing LB with 50 mug/mL kanamycin and 50 mug/mL rifampicin, and the bacterial liquid is firstly placed on the front for 1h at the temperature of 28 ℃ and then is cultured for 48-72h in an inverted way. After the single colony grows out, picking the single colony, and carrying out colony PCR verification on the single colony by using verification primers Pgpd-detect-F and TtrpC-detect-R, wherein the sizes of target sequences are 1558bp (pBHg-BCA1-pdd1OE) and 1224bp (pBHg-BCA1-pdd1RNAi) respectively to obtain the final plasmid-carrying agrobacterium strains AGL1-pdd1OE and AGL1-pdd1RNAi respectively. The primer sequences are as follows:

Pgpd-detect-F:5’-AACCGCCATCTTCCACACTT-3’；

TtrpC-detect-R:5’-AACACCATTTGTCTCAACTCCG-3’。

Fourth, agrobacterium transformation of wild strain of needle mushroom

And (3) converting the agrobacterium strains AGL1-pdd1OE and AGL1-pdd1RNAi prepared in the step three into a wild-type flammulina velutipes strain (flammulina velutipes strain FL 19). The method comprises the following specific steps:

(1) preparing a golden mushroom wild type strain block: the CYM culture medium full of needle mushroom mycelia is beaten into small round pieces (d is 5mm) by a puncher two days in advance, and is subjected to static culture in a CYM liquid culture medium for 48 hours.

(2) Agrobacterium containing the desired fragment plasmid (AGL1-pdd1OE and AGL1-pdd1RNAi) was transferred to LB liquid medium containing rifampicin and kanamycin, respectively, and shake-cultured at 28 ℃ for 12-16h (150 rpm).

(3) When the OD600 of the agrobacterium liquid reaches 0.5-0.8, transferring the agrobacterium liquid into a sterilized 50mL centrifuge tube, sealing the centrifuge tube by a sealing film, centrifuging at 4500rpm at 4 ℃ for 12min, and collecting thalli.

(4) Agrobacterium was washed 2 times with IM medium. Then 5mL of IM medium (resuspended Agrobacterium cells) was added, incubated at 28 ℃ and 150rpm for 4-6 hours in the dark to OD600 of 0.3-0.5 to induce transformation.

(5) Adding the bacterium blocks prepared in the step (1) into induced agrobacterium, standing and culturing for 3-6h, transferring the bacterium blocks to an IM (instant Messaging) culture medium coated with cellophane, and culturing for 3-6 days at 25 ℃.

(6) After the co-culture was completed, the pellet was washed with sterile water to remove Agrobacterium, transferred to a CYM screening plate containing 12.5. mu.g/mL hygromycin B and 200. mu.M cefotaxime sodium, and subjected to static culture at 25 ℃ for 3-4 weeks until transformants grew out.

Fifthly, screening of flammulina velutipes transformants

1. Sample preparation

(1) Single colonies picked from CYM screening plates were transferred to CYM screening plates containing 12.5. mu.g/mL hygromycin B and 200. mu.M cefotaxime sodium, and screened for 5 generations.

(2) Transferring the strains which still grow well after 5 generations of selection to a CYM plate which does not contain hygromycin B and is covered with cellophane, collecting the mycelia after the mycelia grow, using parts for DNA extraction and parts for RNA extraction, and using the wild flammulina velutipes strains as a control.

2. Strain DNA extraction and validation

(1) Collecting wild mycelium of Flammulina velutipes, and rapidly throwing into liquid nitrogen for storage.

(2) The mycelium was triturated with liquid nitrogen, 400. mu.L of extraction buffer (SDEB) and 400. mu.L of 2 × CTAB buffer were added, vortexed and mixed.

(3) Add 800. mu.L phenol/chloroform (1:1) solution, mix well, centrifuge at 12000rpm for 10 min.

(4) The supernatant was aspirated and transferred to a new 1.5mL centrifuge tube, 500. mu.L chloroform was added, mixed well, 12000rpm, and centrifuged for 10 min.

(5) And (3) sucking a proper amount of supernatant, transferring the supernatant into a new 1.5mL centrifuge tube, adding isopropanol with the volume of 0.6 time, uniformly mixing, standing at 4 ℃ for 10-20 min, centrifuging at 12000rpm for 10min, and discarding the supernatant.

(6) The precipitate was washed twice with 75% ethanol, centrifuged at 12000rpm for 10min, and the supernatant was discarded.

(7) Centrifuging for a short time, sucking out the excessive alcohol, and air drying.

(8) An appropriate amount of a DNA dissolving solution (DNA dissolving solution preparation: 10. mu.L of 10mM Tris-HCl (pH 8.0)) was added to dissolve the DNA.

(9) Water bath at 37 deg.c for 2 hr and storing at-20 deg.c.

(10) Respectively taking DNA of a needle mushroom wild type strain, pdd1 overexpression mutant strain and pdd1 knock-down expression mutant strain as templates, carrying out PCR amplification verification, carrying out colony PCR verification on a single colony by using verification primers Pgpd-detect-F and TtrPC-detect-R, respectively taking target sequences of no band (needle mushroom wild type), 1558bp (pdd1 overexpression mutant strain) and 1224bp (pdd1 knock-down expression mutant strain) as target sequences, and respectively taking corresponding plasmids as positive controls. The primer sequences are as follows:

Pgpd-detect-F:5’-AACCGCCATCTTCCACACTT-3’；

TtrpC-detect-R:5’-AACACCATTTGTCTCAACTCCG-3’。

3. Strain RNA extraction and quantitative PCR verification of transcription level

Extracting RNA from the positive mutant obtained in the step 2, and simultaneously performing a quantitative PCR experiment, wherein the specific operations are as follows:

(1) the collected bacterial samples were placed in 1.5mL centrifuge tubes and quickly placed into liquid nitrogen for cryopreservation.

(2) The mycelia were ground with liquid nitrogen, added to 0.7mL of Trizol, mixed well and left at room temperature for 5 min. Centrifuge at 12000rpm for 10min at 4 ℃ and aspirate the supernatant into a 1.5mL Axygen centrifuge tube.

(3) 0.7mL of chloroform was added, and the mixture was vortexed and gently shaken on a vortex shaker for 15 seconds, and allowed to stand at room temperature for 5 min. Centrifuge at 12000rpm for 15min at 4 ℃.

(4) Sucking appropriate amount of supernatant, adding 0.6 times volume of precooled isopropanol, mixing, and standing at room temperature for 10 min. Centrifuge at 12000rpm for 10min at 4 ℃ and discard the supernatant.

(5) Washing with 75% ice-cold ethanol twice, and air drying.

(6) The RNA was dissolved in 100. mu.L of RNA-free water.

(7) Detecting RNA by an ultraviolet spectrophotometer, and recording OD260, OD280 and Ratio values of the RNA sample. Then according to the formula: the concentration of RNA sample was calculated as OD260 value × 40 × dilution multiple of RNA sample.

(8) General gel electrophoresis for RNA integrity detection: agarose gel with concentration of 1%, loading 2 μ g of RNA sample, voltage of 180V, electrophoresis for 15min, and observing RNA band under ultraviolet after EB staining.

(9) All RNA samples were separately inverted into cDNA using a cDNA synthesis kit for quantitative PCR.

(10) Using the cDNA sample as template, quantitative PCR primer Q-pdd1-F and Q-pdd1-R were used to quantitatively amplify pdd1, 2 was used^-ΔΔCtThe experimental data are processed by the calculation method (2). Meanwhile, actin is taken as a template as an internal reference gene, and primers are Q-actin-F and Q-actin-R. The primer sequences are as follows:

Q-pdd1-F:5’-TCAGCAATGCGTCTGACACG-3’；

Q-pdd1-R:5’-CGTTCATGTTCCAAGTCGGGT-3’；

Q-actin-F:5’-CACCATGTTCCCTGGTATTG-3’；

Q-actin-R:5’-CACCAATCCAGACAGAGTATTT-3’。

the quantitative PCR reaction procedure was: pre-denaturation at 95 ℃ for 60 s; denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 15s, and extension at 72 ℃ for 45s for 40 cycles; dissolution curve analysis: and the temperature is increased by 0.5 ℃ every 0.05s at 65-95 ℃ and detected.

(11) The obtained quantitative PCR data are analyzed, and a strain with pdd1 up-regulated expression compared with the wild type is selected as a pdd1 overexpression mutant strain, and a strain with pdd1 down-regulated expression is selected as a pdd1 knockdown expression mutant strain. Pdd1 overexpression mutant strains 3 are obtained in total, and are respectively as follows: pdd1^OE#7、pdd1^OE#31And pdd1^OE#38Pdd1 knock-down expression mutant strain 3, which are: pdd1^RNAi#64、pdd1^RNAi#148And pdd1^RNAi#170The data results are shown in FIG. 3. The above strains were used as experimental strains.

Example 3, pdd1 application in regulating and controlling the growth of Flammulina velutipes hypha

Flat hypha growth observation experiment of pdd1 mutant strain

(1) Wild type needle mushroom pdd1 overexpression mutant strain (pdd 1)^OE#7、pdd1^OE#31And pdd1^OE#38) And pdd1 mutant strains with reduced expression (pdd 1)^RNAi#64、pdd1^RNAi#148And pdd1^RNAi#170) The pellets (d: 5mm) were prepared into -sized pellets by using a punch, and the pellets were inoculated onto CYM plates containing no drug, and photographed after culturing at 25 ℃ for 7 days.

(2) During the culture process, the growth length of hyphae is measured every 24 hours, and the average growth rate of each strain is calculated.

The results of the growth and growth rate analysis for different strains were as follows: on CYM plates, the growth of pdd1 overexpression mutant strain hyphae is similar to that of wild type, and has no obvious difference, while the growth of pdd1 knock-down expression mutant strain hyphae is weakened and slowed down, which shows that pdd1 underexpression affects the growth of flammulina velutipes hyphae (figure 4).

Second, pdd1 mutant strain is subjected to cultivation material hypha growth observation experiment

Wild type needle mushroom pdd1 overexpression mutant strain (pdd 1)^OE#7、pdd1^OE#31And pdd1^OE#38) And pdd1 mutant strains with reduced expression (pdd 1)^RNAi#64、pdd1^RNAi#148And pdd1^RNAi#170) The resulting culture medium was punched into -sized clumps (d ═ 5mm) and inoculated into tissue culture bottles containing 325g of culture medium, respectively, and cultured at 25 ℃ until the bottom of the bottle was filled with the mycelia and photographed.

As can be seen in fig. 5, pdd1 overexpressing mutant strain (12 days) grew up in the compost earlier than wild type (15 days), while pdd1 knock-down expressing mutant strain (22 days) grew up in the compost at the latest. On the compost, hyphal growth rate: pdd1 overexpression mutant Strain > wild type > pdd1 knock-down expression mutant strain. The result shows that pdd1 high expression promotes hypha growth, pdd1 low expression inhibits hypha growth, the high expression of pdd1 makes the strain more suitable for the nutrient environment of the cultivation material, and pdd1 can be used as a reference for molecular breeding of excellent strains.

Example 4, pdd1 application in regulating and controlling the development of needle mushroom fruiting body

fruiting observation experiment of pdd1 mutant strain

(1) Wild type needle mushroom pdd1 overexpression mutant strain (pdd 1)^OE#7、pdd1^OE#31And pdd1^OE#38) And pdd1 mutant strains with reduced expression (pdd 1)^RNAi#64、pdd1^RNAi#148And pdd1^RNAi#170) The culture medium was prepared into -sized clumps (d ═ 5mm) by punch, and the clumps were inoculated into 325g culture medium in tissue culture bottles, and cultured at 25 ℃ and 70% humidity in the dark for 15-20 days until all the strains grew over the culture bottles.

(2) And (3) scratching each cultivation bottle, namely lightly scraping thick hyphae on the top of the cultivation bottle by using a sterilized scalpel, and continuously culturing at 25 ℃ and 70% humidity in the dark until the top of the cultivation bottle is full of new hyphae to obtain the cultivation bottle covered with the hyphae.

(3) Culturing the culture bottle coated with the strain at 15 deg.C under 95% humidity in dark condition, cold stimulating, and culturing for 5-7 days to allow primordium (the most initial fruiting body of Flammulina velutipes) to grow out. Periodically observed and photographed.

The fruiting results were analyzed as follows: on day 6 after the fruiting stimulation, pdd1 overexpressing the mutant strain had primordia produced, while the wild type strain produced primordia on day 7 after the fruiting stimulation treatment, at which time pdd1 overexpressing the mutant strain had more and denser primordia produced. The fruiting culture was continued to harvest and pdd1 overexpressing mutant strains were found to enter mature harvest 4 days earlier than wild type and were larger (FIG. 6). Meanwhile, no primordia were found in the pdd 1-knockdown mutant strain, and pdd1 was slightly knocked down when the fruiting time was continued to be prolonged (pdd 1)^RNAi#148And pdd1^RNAi#170) Among them, very individual primordia were produced, and pdd1 was greatly knocked down the strain (pdd 1)^RNAi#64) The primordia were not found (FIG. 6). The results show that the expression level of pdd1 significantly influences the formation of primordia of flammulina velutipes and the development of sporocarp, and pdd1 is a positive regulatory factor required in the process of the sporocarp development of the flammulina velutipes.

Secondly, biomass measurement experiment is carried out on the wild type flammulina velutipes and the pdd1 overexpression mutant strain

Since pdd1 knockdown strains do not produce or produce very few dwarf and slimy fruiting bodies and are not statistically significant, the biomass determination of the invention is limited to wild-type and pdd1 overexpressing mutant strains. For more efficient statistics, the present invention only performs statistical analysis on fruiting bodies >6cm in length.

The analysis result shows that: pdd1 overexpression mutant strains generally have increased biomass compared to wild type strains, wherein strain pdd1^OE#31The biomass is increased most obviously, the number of sporocarps is increased by 19.85 +/-4.64 percent, the height of the sporocarps is increased by 40.36 +/-7.25 percent, the total wet weight is increased by 62.47 +/-8.88 percent (figure 7), the strain is a good yield-increasing strain of strains, and the strain is combined with the characteristic of entering a maturation stage in advance, so that the production period can be shortened in production application, the energy consumption and the labor cost are reduced, and the strain is a good production strain of strains with low energy consumption and high yield.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> transcription factor PDD1 for regulating and controlling fruiting body development of needle mushroom, and coding gene and application thereof

<160>2

<170>PatentIn version 3.5

<210>1

<211>1204

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

atgcctccct cccgaactct gtcgtcctct cctacggaaa tcgtctggac tcttccgaat 60

tcctcttttg ggcaacttga cactgccccc ggcagcacta ccacaaaaca cgccaagaag 120

aagcctgcta ctcacatccc ccgtccacca aatgccttca ttctttttcg ctcgtccttc 180

atcaagtctc aacacgtatc caccgatgtc gagacgaacc atagcacgct cagcaaaatc 240

attggtatga cttggcaggg catgaaggac gaggagagga aggtctggca cgacaaggcc 300

cgggtcgcgt tggaggaaca caggaaaaga tttcccgcgt acgctttcag gccgtccggt 360

ggtggtgcca aaggaggtac cccagacggc ggtggtggag gagtcaagag acggaaagta 420

cgggaggtcg aaccgaaaga taccaaacgc tgccagaaaa tcgcggagct tctggtagag 480

ggcctcaagg gcgctacgct ggacgatgcg atccgcgagt tcgacaagac gcacgtcaag 540

gagatcgtaa cgcgtttcga ggtgcctatc actgagagcg cgtacaggaa gaaggaagaa 600

aagaaggcgg cacaggatgt taaggtcatc gttgtaagtc ctcacttcat cgtctcgatg 660

gcgcgttctt aatcgttctt aggacgtcaa gaaagaaccc accgttgaag aaatggtcga 720

ccaacaattg tactatcccg acccggcaaa cgactattcg tattgccata cgccaacttc 780

accatatccc aacacgccga tttcttcata tccaacatcc cctatcgatc acaattcgcc 840

agttgatatg acctcgttcc cctgttcccc ctctgctcac agttcgtatc catgctctcc 900

ggcggcagac aacttcttca atcctgaatt cgttgggtct tgtcagcttc catacccccc 960

ggcgccctct tacgatacgc tttcaccgcc gtcttacgaa ctcgacgctc ccgttccggg 1020

cttcttgcct ccaccagaat ctgttcttga ctcattcttc agcaatgcgt ctgacacgtt 1080

cacattcgaa tttccgggtg cccccgacgg cgagttcctt acagacttca tggattttga 1140

aatgcccatg cctttaaacc cgacttggaa catgaacgga gaaggtagtg gccttgcatt 1200

ttga 1204

<210>2

<211>384

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Met Pro Pro Ser Arg Thr Leu Ser Ser Ser Pro Thr Glu Ile Val Trp

1 5 10 15

Thr Leu Pro Asn Ser Ser Phe Gly Gln Leu Asp Thr Ala Pro Gly Ser

20 25 30

Thr Thr Thr Lys His Ala Lys Lys Lys Pro Ala Thr His Ile Pro Arg

35 40 45

Pro Pro Asn Ala Phe Ile Leu Phe Arg Ser Ser Phe Ile Lys Ser Gln

50 5560

His Val Ser Thr Asp Val Glu Thr Asn His Ser Thr Leu Ser Lys Ile

65 70 75 80

Ile Gly Met Thr Trp Gln Gly Met Lys Asp Glu Glu Arg Lys Val Trp

85 90 95

His Asp Lys Ala Arg Val Ala Leu Glu Glu His Arg Lys Arg Phe Pro

100 105 110

Ala Tyr Ala Phe Arg Pro Ser Gly Gly Gly Ala Lys Gly Gly Thr Pro

115 120 125

Asp Gly Gly Gly Gly Gly Val Lys Arg Arg Lys Val Arg Glu Val Glu

130 135 140

Pro Lys Asp Thr Lys Arg Cys Gln Lys Ile Ala Glu Leu Leu Val Glu

145 150 155 160

Gly Leu Lys Gly Ala Thr Leu Asp Asp Ala Ile Arg Glu Phe Asp Lys

165 170 175

Thr His Val Lys Glu Ile Val Thr Arg Phe Glu Val Pro Ile Thr Glu

180 185 190

Ser Ala Tyr Arg Lys Lys Glu Glu Lys Lys Ala Ala Gln Asp Val Lys

195 200 205

Val Ile Val Asp Val Lys Lys Glu Pro Thr Val Glu Glu Met Val Asp

210 215 220

Gln Gln Leu Tyr Tyr Pro Asp Pro Ala Asn Asp Tyr Ser Tyr Cys His

225 230 235 240

Thr Pro Thr Ser Pro Tyr Pro Asn Thr Pro Ile Ser Ser Tyr Pro Thr

245 250 255

Ser Pro Ile Asp His Asn Ser Pro Val Asp Met Thr Ser Phe Pro Cys

260 265 270

Ser Pro Ser Ala His Ser Ser Tyr Pro Cys Ser Pro Ala Ala Asp Asn

275 280 285

Phe Phe Asn Pro Glu Phe Val Gly Ser Cys Gln Leu Pro Tyr Pro Pro

290 295 300

Ala Pro Ser Tyr Asp Thr Leu Ser Pro Pro Ser Tyr Glu Leu Asp Ala

305 310 315 320

Pro Val Pro Gly Phe Leu Pro Pro Pro Glu Ser Val Leu Asp Ser Phe

325 330 335

Phe Ser Asn Ala Ser Asp Thr Phe Thr Phe Glu Phe Pro Gly Ala Pro

340 345 350

Asp Gly Glu Phe Leu Thr Asp Phe Met Asp Phe Glu Met Pro Met Pro

355 360 365

Leu Asn Pro Thr Trp Asn Met Asn Gly Glu Gly Ser Gly Leu Ala Phe

370 375 380

Claims

1. A protein, which is a) or b) as follows:

a) the amino acid sequence is a protein shown in a sequence 2;

b) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the sequence 2.

2. The protein-related biomaterial of claim 1, which is any of the following A1) to A8):

A1) a nucleic acid molecule encoding the protein of claim 1;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising the recombinant vector of a 4).

3. The related biological material according to claim 2, wherein: A1) the nucleic acid molecule is a gene shown in the following 1) or 2):

2) a cDNA molecule or a genomic DNA molecule having identity of of 75% or more than 75% with the nucleotide sequence defined in 1) and encoding the protein of claim 1.

4. The protein of claim 1 or the related biomaterial of claim 2 or 3 for use as of any one of (b1) - (b6) as follows:

(b1) regulating and controlling the growth of the flammulina velutipes hyphae;

(b2) regulating the development of needle mushroom fruiting bodies;

(b3) the biomass and/or the yield of the flammulina velutipes are/is improved;

(b4) shortening the fruiting period of the needle mushrooms;

(b6) and (5) breeding needle mushrooms.

5. Use according to claim 4, characterized in that: the regulation is promotion;

or, the biomass and/or yield of the flammulina velutipes is increased by in any one of (c1) - (c3) as follows:

(c1) increasing the number of needle mushroom fruiting bodies;

(c2) increasing the height of needle mushroom fruiting bodies;

(c3) increasing the wet weight of needle mushroom fruiting bodies;

or, the shortening of the fruiting period of the flammulina velutipes is realized by promoting the primordial stage and/or the mature stage of the flammulina velutipes to be advanced.

6, method for cultivating transgenic flammulina velutipes with high yield and short fruiting period, which comprises the step of increasing the expression quantity and/or activity of the protein in claim 1 in a receptor flammulina velutipes to obtain the transgenic flammulina velutipes, wherein the yield of the transgenic flammulina velutipes is higher than that of the receptor flammulina velutipes and/or the fruiting period of the transgenic flammulina velutipes is shorter than that of the receptor flammulina velutipes.

7. The method of claim 6, wherein:

the yield of the transgenic needle mushroom is higher than that of the acceptor needle mushroom which is embodied in any of (d1) - (d3) as follows:

(d1) the number of fruiting bodies of the transgenic flammulina velutipes is more than that of the recipient flammulina velutipes;

(d2) the fruiting body of the transgenic needle mushroom is higher than that of the receptor needle mushroom;

(d3) the wet weight of the fruiting body of the transgenic flammulina velutipes is larger than that of the receptor flammulina velutipes;

or the fruiting period of the transgenic needle mushroom is shorter than the primordial period and/or the mature period of the receptor needle mushroom expressed in the transgenic needle mushroom and earlier than the receptor needle mushroom.

8. The method according to claim 6 or 7, characterized in that: the method for improving the expression amount and/or activity of the protein in the receptor flammulina velutipes, namely, the protein in the receptor flammulina velutipes is over-expressed.

9. The method of claim 8, wherein: the overexpression method is to introduce the gene encoding the protein of claim 1 into recipient flammulina velutipes.

10. The method of claim 9, wherein: the nucleotide sequence of the coding gene of the protein is a DNA molecule shown in sequence 1.