CN108558992A

CN108558992A - The transcription factor PDD1 and its encoding gene of regulation and control needle mushroom fruit body development and application

Info

Publication number: CN108558992A
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: 2018-09-21
Anticipated expiration: 2038-01-12
Also published as: CN108558992B

Abstract

The invention discloses a kind of transcription factor PDD1 of regulation and control needle mushroom fruit body development and its encoding gene and applications.In the coding gene sequence such as sequence table of the transcription factor PDD1 of the present invention shown in sequence 1；In the amino acid sequence of the transcription factor such as sequence table shown in sequence 2.The present invention has found that pdd1 is required positive regulatory factor during Flammulina velutipes mycelium growth and fruit body development, affects needle mushroom former base phase and maturity period, fructification quantity, height and weight in wet base by the overexpression and RNAi experiments for carrying out pdd1 in needle mushroom.The invention also discloses one plant to have the needle mushroom mutant strain (pdd1 that the fruiting period is short, yield is high^OE#31), not only there is the characteristic to come to the ripening period in advance, additionally it is possible to which the yield for improving needle mushroom shortens the production cycle of needle mushroom, reduces energy consumption and cost of labor, has huge application prospect.

Description

The transcription factor PDD1 and its encoding gene of regulation and control needle mushroom fruit body development and application

Technical field

The present invention relates to gene engineering technology fields, and in particular to a positive regulation needle mushroom (Flammulinavelutipes) mycelia growth and the transcription factor PDD1 of fruit body development and one plant have the fruiting period The pdd1 short, yield is high is overexpressed mutant strain (pdd1^OE#31)。

Background technology

Needle mushroom (Flammulinavelutipes) also known as dried mushroom, plain mushroom, hair handle money mushroom etc., are typical load classes Edible mushroom, in the whole world, especially Asian countries cultivates extensively.Needle mushroom contains abundant protein, dietary fiber and carbohydrate, Additionally contain abundant vitamin B1, B2, B3 and vitamin C, D, E.Needle mushroom not only has as a kind of traditional edible mushroom There is abundant nutritive value, and there is important medical value.Recent studies indicate that needle mushroom have it is antitumor, prolong Long-life reduces cholesterol, antifatigue, immunological regulation and other effects.It is to realize that these nutritive and medicinal values, which are all with fructification, The yield and quality of carrier, fructification directly determines nutrition and medicinal value.

It is planted recently as needle mushroom large area, successively there are many research about needle mushroom yield-increasing technology and method, Main optimization and genetic breeding including nutritional condition.The optimization of nutritional condition is matched including improvement planting type, Optimum Cultivation material Side and addition promote volume increase substance.For example promote son using planting types such as narrow bag cultivating, feux rouges induction fruiting, horizontal both ends fruitings Entity is grown, or selects more suitably carbon source, nitrogen source, replaces corncob to promote mycelia growth with rice husk, or pass through external source Add the modes such as the material incentives fructifications such as triacontanol, special efficacy nutrient for plants (SPNE), soy noodle generation.But these Method stresses to be changed external environment to adapt to strain growth, and Increase production ability is limited, and needs to consider cost.And it is hereditary Breeding is to make a change the variation for deacclimatizing environment for bacterial strain itself, main applied to the mode of needle mushroom genetic breeding at present Including spontaneons screening, mutation breeding, crossbreeding and genetic engineering breeding, fruiting week is obtained such as through spontaneons screening and mutagenesis Merits bacterial strains such as " the spoke gold No.1s " of phase short " Sanming City No.1 ", Antimould power strong " western teacher 8001 " and high yield, and Precocious needle mushroom " agriculture gold six ", disease-resistant needle mushroom A45 for mating by different parents etc..Three of the above breeding method exists Development of Molecular Biology initial stage is more common, although the excellent bacterial strain of character can be selected, screening more blindly, and consumes Take the artificial and time.Another is genetic engineering breeding, with clearly defined objective, and the period is shorter, but the method needs to be based on exchanging Control the deep understanding of fruit body development gene.With application of the molecular biology in needle mushroom, gradually there are some about acupuncture needle The report of massee fruiting bodies development gene.If fvh1 is expressed in needle mushroom fruit body development period height, hydrophobin encoding gene Fv- Hyd1 shifts to an earlier date former base formation after being overexpressed, histidine kinase gene and the participation of GATA Zinc finger transcription factors are cold-induced Low adenosine deaminase coding is struck in former base forming process, the variant expression in former base and mycelia of Mads8, N1477, HK535 gene Gene Fv-ada can inhibit Flammulina velutipes mycelium to grow, expression and needle mushroom of phenylalanine ammoniacalyase encoding gene Fvpal Entity forms correlation, the analysis of needle mushroom mating type gene, and laccase encoding gene participates in the formation of acupuncture needle massee fruiting bodies, is overexpressed Fv-JRL1 promotes Flammulina velutipes mycelium growth and fruit body development, but these study limitations are in gene structure or gene work( The research of energy, need to be carried out to application of its gene in needle mushroom.

Invention content

The technical problem to be solved by the present invention is to how regulate and control Flammulina velutipes mycelium growth and fruit body development, yield is cultivated Needle mushroom high, the fruiting period is short.

In order to solve the above-mentioned technical problem, present invention firstly provides a kind of controllable Flammulina velutipes mycelium growth and fructifications The protein of development.Controllable Flammulina velutipes mycelium growth provided by the invention is entitled with the protein of fruit body development PDD1, the PDD1 protein are following protein a) or b) or c) or d)：

A) amino acid sequence is protein shown in sequence 2；

B) fused protein that the N-terminal of protein shown in sequence 2 and/or C-terminal connection label obtain；

C) by amino acid sequence shown in sequence 2 by one or several amino acid residues substitution and/or missing and/or Add obtained protein with the same function；

D) with amino acid sequence shown in sequence 2 with 75% or 75% or more homology and egg with the same function White matter.

In order to which the protein in making a) is convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 2 or The upper label as shown in Table 1 of carboxyl terminal connection.

The sequence of table 1, label

Label	Residue	Sequence
			Poly-Arg	5-6 (being usually 5)	RRRRR
Poly-His	2-10 (being usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

It is above-mentioned c) in protein, the substitution of one or several amino acid residues and/or lack and or add as not More than 10 amino acid residues substitution and/or lack and or add.

It is above-mentioned c) in protein can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression and obtain.

It is above-mentioned c) in the encoding gene of protein can be one or several by will be lacked in DNA sequence dna shown in sequence 1 The codon of amino acid residue, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end and/or 3 ' ends The coded sequence for connecting label shown in table 1 obtains.

It is above-mentioned d) in, " homology " include with the present invention sequence 2 shown in amino acid sequence have 75% or higher, or 80% or higher 85% or higher 90% or higher 95% or more high homology amino acid sequence.

In order to solve the above-mentioned technical problem, the present invention also provides with the relevant biomaterial of PDD1 protein.

Any one of provided by the invention with the relevant biomaterial of PDD1 protein is following A 1) to A8)：

A1 the nucleic acid molecules of PDD1 protein) are encoded；

A2) contain A1) expression cassettes of the nucleic acid molecules；

A3) contain A1) recombinant vectors of the nucleic acid molecules；

A4) contain A2) recombinant vector of the expression cassette；

A5) contain A1) recombinant microorganisms of the nucleic acid molecules；

A6) contain A2) recombinant microorganism of the expression cassette；

A7) contain A3) recombinant microorganism of the recombinant vector；

A8) contain A4) recombinant microorganism of the recombinant vector.

In above-mentioned biomaterial, A1) nucleic acid molecules be it is following 1) or 2) or 3) shown in gene：

1) its coded sequence is cDNA molecules or genomic DNA molecule shown in sequence 1；

2) there is 75% or 75% or more homogeneity with the nucleotide sequence 1) limited, and encodes the cDNA of PDD1 protein Molecule or genomic DNA molecule；

1) or 2) 3) and the cDNA molecules of PDD1 protein are encoded with the nucleotide sequence hybridization that limits under strict conditions Or genomic DNA molecule.

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules also may be used To be RNA, such as mRNA or hnRNA.

Those of ordinary skill in the art can easily adopt by known method, for example, orthogenesis and point mutation side Method is mutated the nucleotide sequence of the coding PDD1 protein of the present invention.Those are by manually modified, with coding The nucleotide sequence 75% of PDD1 protein or the nucleotide of higher homogeneity, as long as encoding PDD1 protein and with identical Function is the nucleotide sequence derived from the present invention and is equal to the sequence of the present invention.

Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair Shown in bright coded sequence 2 amino acid sequence form protein nucleotide sequence have 75% higher or 85% or Higher or 90% higher or 95% or higher homogeneity nucleotide sequence.Homogeneity can with the naked eye or computer software It is evaluated.Using computer software, homogeneity between two or more sequences can use percentage (%) to indicate, can be with For evaluating the homogeneity between correlated series.

Above-mentioned 75% or 75% or more homogeneity can be 80%, 85%, 90% or 95% or more homogeneity.

In above-mentioned biomaterial, A2) described in the expression cassettes of the nucleic acid molecules containing coding PDD1 protein be to refer to The DNA of pdd1 is expressed in host cell, which not only may include the promoter for starting pdd1 transcriptions, may also include termination The terminator of pdd1 transcriptions.Further, the expression cassette may also include enhancer sequence.In a specific embodiment of the present invention, The promoter is specially gpd promoter fragments, and the terminator is specially that trpC terminates sub-piece.It can be carried with existing expression Recombinant vector of the body structure containing the pdd1 expression casettes.

In above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.

In above-mentioned biomaterial, the microorganism can be yeast, bacterium, algae or fungi, such as Agrobacterium.

In order to solve the above-mentioned technical problem, the present invention also provides above-mentioned PDD1 protein or above-mentioned relevant biological materials New application.

It answers the present invention provides above-mentioned PDD1 protein or above-mentioned relevant biological material are any in following (b1)-(b6) With：

(b1) regulation and control Flammulina velutipes mycelium growth；

(b2) regulate and control needle mushroom fruit body development；

(b3) needle mushroom biomass and/or yield are improved；

(b4) shorten the fruiting period of needle mushroom；

(b5) the transgenosis needle mushroom that yield is high, the fruiting period is short is cultivated；

(b6) needle mushroom breeding.

It is described to be regulated to promote in above application；The raising needle mushroom biomass and/or yield are embodied in as follows (c1) any in-(c3)：

(c1) increase acupuncture needle massee fruiting bodies quantity；

(c2) increase acupuncture needle massee fruiting bodies height；

(c3) increase acupuncture needle massee fruiting bodies weight in wet base；

The fruiting period for shortening needle mushroom, which is embodied in, promotes the former base phase of needle mushroom and/or maturity period to shift to an earlier date.

The present invention constructs pdd1 in needle mushroom wild type and is overexpressed mutant strain (pdd1 respectively^OE) and pdd1 strike low table Up to mutant strain (pdd1^RNAi).To needle mushroom wild type, pdd1 be overexpressed mutant strain and pdd1 strike low expression mutant strain into Row mycelia growth experiment analyzes phenotype, it is found that pdd1 strikes low expression mutant strain mycelia on synthetic media and culture material and gives birth to Length is all slower than wild type.And pdd1 is overexpressed mutant strain mycelia growth in culture material and is faster than wild type, shortens mycelia life Long period.Illustrate that pdd1 has positive regulating and controlling effect to Flammulina velutipes mycelium growth.

The present invention is overexpressed mutant strain to needle mushroom wild type, pdd1 simultaneously and pdd1 strikes low expression mutant strain and carries out Fruiting is tested, and section is observed and taken pictures in different times.The fruiting phenotype of all bacterial strains is analyzed, is found wild Type bacterial strain generates former base (the initial stage fructification of needle mushroom) on the 7th day after stimulation fruiting is handled, and pdd1 is overexpressed mutant bacteria Strain shifts to an earlier date 1 day than wild type and generates greater area of former base.Continue mushroom producing culture, it is found that pdd1 is overexpressed mutant strain ratio Wild type shifts to an earlier date 4 days and enters ripe harvest time, and in pdd1 strikes low expression mutant strain, after pdd1 is struck low by a small margin, It is generated without former base in fruiting time identical as wild type, minute quantity former base can be generated by extending fruiting time；But work as pdd1 quilts Significantly strike it is low after, even if extend fruiting time can not generate former base.Illustrate that the expression quantity of pdd1 significantly affects needle mushroom The generation of former base and the development of fructification, pdd1 are required positive regulatory factors during needle mushroom fruit body development.

In order to solve the above-mentioned technical problem, the present invention finally provides that a kind of cultivation yield is high, the fruiting period is short turns base Because of the method for needle mushroom.

The method provided by the invention for cultivating yield height, fruiting period short transgenosis needle mushroom is including improving receptor acupuncture needle The expression quantity and/or activity of PDD1 protein in mushroom, the step of obtaining transgenosis needle mushroom；The yield of the transgenosis needle mushroom The fruiting period higher than the receptor needle mushroom and/or the transgenosis needle mushroom is shorter than the receptor needle mushroom.

In the above method, the yield of the transgenosis needle mushroom is embodied in following (d1)-higher than the receptor needle mushroom (d3) any in：(d1) the fructification quantity of transgenosis needle mushroom is more than the receptor needle mushroom；(d2) transgenosis needle mushroom Fructification height be higher than the receptor needle mushroom；(d3) the fructification weight in wet base of transgenosis needle mushroom is more than the receptor acupuncture needle Mushroom；The fruiting period of the transgenosis needle mushroom be shorter than the receptor needle mushroom be embodied in transgenosis needle mushroom former base phase and/ Or the maturity period is earlier than the receptor needle mushroom.

Specifically, the transgenosis needle mushroom that yield provided by the invention is high, the fruiting period is short is carried compared to receptor needle mushroom It generates within first 1 day former base and comes to the ripening period for 4 days in advance, fructification quantity increases by 19.85 ± 4.64%, and fructification height increases 40.36 ± 7.25%, total weight in wet base increases by 62.47 ± 8.88%, and not only former base phase and maturity period shift to an earlier date, and total biomass is notable Increase.

In the above method, it is described improve receptor needle mushroom in PDD1 protein expression quantity and/or active method be PDD1 protein is overexpressed in receptor needle mushroom.

In the above method, the method for the overexpression is that the encoding gene of PDD1 protein is imported receptor needle mushroom.

In the above method, the nucleotide sequence of the encoding gene of the PDD1 protein is DNA molecular shown in sequence 1.

In a specific embodiment of the present invention, the encoding gene of the PDD1 protein is by containing pdd1 expression casettes Recombinant expression carrier import in the recipient plant.The recombinant expression carrier be by gpd promoter fragments (Pgpd-OE), Pdd1OE segments and termination sub-piece (TtrpC-OE) are merged in XmnI restriction enzyme sites with plasmid pBHg-BCA1 with recombination kit The size obtained afterwards is the carrier of 11261bp.

In the above method, the receptor needle mushroom can be Strains of Flammulina velutipes FL19 (yellow cultivars).

The present invention has found transcription factor PDD1 and its encoding gene pdd1 in needle mushroom for the first time, and finds that pdd1 is acupuncture needle Required positive regulatory factor, affects former base phase and the maturity period of needle mushroom during the growth of mushroom silk and fruit body development, son Physical quantities, height and weight in wet base.The present invention also provides one plant to have the needle mushroom mutant strain that the fruiting period is short, yield is high (pdd1^OE#31), not only there is the characteristic for entering former base phase and maturity period in advance, additionally it is possible to improve the yield of needle mushroom, shorten gold The production cycle of needle mushroom reduces energy consumption and cost of labor, has huge application prospect in the selection and breeding of needle mushroom excellent species.

Description of the drawings

Fig. 1 is transcriptional level figure of the HMG-box structural protein coding genes in needle mushroom fruit body development different times.

Fig. 2 is the structure chart of transcription factor PDD1.

Fig. 3 is pdd1 transcriptional levels in wild type, pdd1 overexpression mutant strains and pdd1 strike low expression mutant strain Analysis result figure.

Fig. 4 is wild type, pdd1 is overexpressed mutant strain and pdd1 strikes mycelia of the low expression mutant strain on CYM tablets Growth result figure.

Fig. 5 is wild type, pdd1 is overexpressed mutant strain and pdd1 strikes mycelia of the low expression mutant strain in culture material Growth result figure.

Fig. 6 is wild type, pdd1 is overexpressed mutant strain and pdd1 strikes fruiting of the low expression mutant strain in culture material Result figure.

Fig. 7 is wild type, pdd1 is overexpressed mutant strain and pdd1 strikes the biomass statistics of low expression mutant strain fruiting Result figure.

Specific implementation mode

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.

The formula of culture medium and solution involved in following embodiments is as follows：

LB culture mediums：1% tryptone, 0.5% yeast extract, 1%NaCl add appropriate distillation water dissolution, pH7.0 fixed Hold, high pressure steam sterilization.

CYM culture mediums：1% maltose, 2% glucose, 0.2% tryptone, 0.2% yeast extract add appropriate steaming Distilled water dissolves, natural pH constant volumes, high pressure steam sterilization.

Fruiting culture material culture medium：30% sawdust, 43.5% cotton seed hulls, 25% wheat bran, 1% precipitated calcium carbonate, 0.5% stone Ash, 60% water after being mixed well after adding water and infiltrating 4-5 hour, are dispensed into the tissue culture bottle that volume is 350mL, per bottled 325g culture materials, 3 hours of autoclaving.

Agrobacterium-mediated Transformation inducing culture (IM)：2.05g K₂HPO₄, 0.15g NaCl, 0.5g MgSO₄·7H₂O, 0.067g CaCl₂·2H₂O, 0.0025g FeSO₄·7H₂O, 0.5g (NH₄)₂SO₄, 1.8g glucose, 5mL glycerine, 8.53g 2- (N-Morpholino) ethanesulfonic Acid (pH5.3, filtration sterilization), 200 μM of acetosyringones (filtration sterilization, It is added when paving plate).

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

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

Plasmid pBHg-BCA1 in following embodiments derives from University Of Agriculture and Forestry In Fujian's fungus research center, is recorded in document “Lu,Y.P.,et al.,A Jacalin-Related Lectin Regulated the Formation of Aerial Mycelium and Fruiting Body in Flammulina velutipes.Int J Mol Sci,2016.17 (12) in ".

Plasmid pCSN44 in following embodiments derives from genetic of fungi resource center of the U.S. (Fungal Genetics Stock Center), it is recorded in document " Chen, X., et al., De-repression of CSP-1activates adaptive responses to antifungal azoles.Sci Rep,2016.6:P.19447. in ".

Needle mushroom wild type in following embodiments is Strains of Flammulina velutipes FL19 (yellow), derives from University Of Agriculture and Forestry In Fujian Fungus research center.

The acquisition and its sequence analysis of embodiment 1, pdd1 genes and transcription factor PDD1

One, the amino acid sequence of the nucleotide sequence of pdd1 genes and transcription factor PDD1

8 are found in needle mushroom genome while there is the encoding gene of nuclear localization sequence and HMG-box structural proteins, With RNA-seq and quantitative PCR analysis this 8 genes in the transcriptional level of fruit body development different phase, one of base is found Because that, to elongation phase, maturity period gradually up-regulated expression (Fig. 1), may be played in needle mushroom fruit body development since former base period Critical function, and be pdd1 (primordium development defect1) by the unnamed gene, nucleotides sequence is classified as Sequence 1.The amino acid sequence of pdd1 gene coding transcripts factor PDD1, transcription factor PDD1 are sequence 2.

Two, sequence analysis

Pdd1 upstream region of gene and downstream sequence are respectively extended into 2000bp as sequence is referred to, with Zillions of Oligos Mapped (ZOOM) softwares navigate to the reads of transcript profile on reference sequences, and analysis obtains pdd1 genes from initiation codon It is 1204bp to terminator codon overall length, includes the introne of 1 49bp.

With SMART (http://smart.embl-heidelberg.de/) and software DNAMAN to transcription factor PDD1's Amino acid sequence is analyzed.As a result it shows：Transcription factor PDD1 is one with HMG-box structural domains and nuclear localization sequence Albumen (Fig. 2), molecular weight 42472.68Da, isoelectric point 5.64.

Embodiment 2, pdd1 are overexpressed mutant strain and pdd1 strikes the structure of low expression mutant strain

One, the structure of pdd1 over-express vectors

(1) PCR is carried out by template of the genomic DNA of needle mushroom wild-type strain with primer Pgpd-F and Pgpd-OE-R Amplification, obtains needle mushroom gpd promoter fragments (Pgpd-OE), and the gpd promoters are comprising gpd genes First Intron and outside Aobvious son, target sequence size are 920bp.Primer sequence is as follows：

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

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

PCR response procedures are：98℃ 30s；98 DEG C of 10s, 55 DEG C of 90s, 72 DEG C of 60s (25cycles)；72℃ 10min；4℃.

(2) PCR expansions are carried out by template of the genomic DNA of needle mushroom wild-type strain with primer pdd1-F and pdd1-R Increase, obtain pdd1OE segments, target sequence size is 1203bp, and primer sequence is as follows：

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

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

PCR response procedures are：98℃ 30s；98 DEG C of 10s, 59 DEG C of 90s, 72 DEG C of 60s (25cycles)；72℃ 10min；4℃.

(3) PCR amplification is carried out by template of plasmid pCSN44 with primer TtrpC-OE-F and TtrpC-R, obtain trpC ends Only sub-piece (TtrpC-OE), target sequence size are 720bp.Primer sequence is as follows：

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

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

PCR response procedures are：98℃ 30s；98 DEG C of 10s, 58 DEG C of 90s, 72 DEG C of 60s (25cycles)；72℃ 10min；4℃.

(4) it uses the small extraction reagent kit of ordinary plasmids to extract pBHg-BCA1 plasmids, restriction enzyme XmnI is used in combination to be digested The plasmid, while digestion products are recycled with Ago-Gel DNA QIAquick Gel Extraction Kits, obtain the pBHg-BCA1 after digestion recycling.

(5) segment recombination kit (Nanjing Vazyme Biotechnology Co., Ltd., article No. are used：C113-01) by Pgpd- OE, pdd1OE and TtrpC-OE segment are connected into the pBHg-BCA1 (being carried out according to kit specification operation) after digestion recycling, turn Change bacillus coli DH 5 alpha competence, then choose single bacterium colony, with verification primer Pgpd-detect-F and TtrpC-detect-R to list Bacterium colony carries out bacterium colony PCR verifications, and target sequence size is 1558bp, obtains pdd1 over-express vectors pBHg-BCA1-pdd1OE.Draw Object sequence is as follows：

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

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

PCR response procedures are：94℃ 5min s；94 DEG C of 30s, 58 DEG C of 90s, 72 DEG C of 90s (25cycles)；72℃ 10min；4℃.

Pdd1 over-express vectors pBHg-BCA1-pdd1OE be by gpd promoter fragments (Pgpd-OE), pdd1OE segments and Terminate what sub-piece (TtrpC-OE) was obtained with segment recombination kit after XmnI restriction enzyme sites are merged with plasmid pBHg-BCA1 Size is the carrier of 11936bp.

Two, pdd1 strikes the structure of low expression carrier

The present invention strikes low expression carrier using the RNAi technology structure pdd1 of hairpin structure.It is as follows：

(1) it is carried out with primer Pgpd-F and Pgpd-RNAi-R by template of the genomic DNA of needle mushroom wild-type strain PCR amplification, obtains needle mushroom gpd promoter fragments (Pgpd-RNAi), and target sequence size is 920bp.Primer sequence is as follows：

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

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

(2) it is with wild type needle mushroom genomic DNA with primer pdd1-antisense-F and pdd1-antisense-R Template carries out PCR amplification, obtains pdd1-antisense segments, and target sequence size is 489bp.Primer sequence is as follows：

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

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

PCR response procedures are：98℃ 30s；98 DEG C of 10s, 65 DEG C of 90s, 72 DEG C of 60s (25cycles)；72℃ 10min；4℃.

(3) PCR amplification is carried out by template of plasmid pCSN44 with primer TtrpC-RNAi-F and TtrpC-R, obtain trpC Sub-piece (TtrpC-RNAi) is terminated, target sequence size is 720bp.Primer sequence is as follows：

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

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

(5) segment recombination kit (Nanjing Vazyme Biotechnology Co., Ltd., article No. are used：C113-01) by Pgpd- RNAi, pdd1-antisense and TtrpC-RNAi segment are connected into the pBHg-BCA1 after digestion recycling (according to kit specification Operated), bacillus coli DH 5 alpha competence is converted, single bacterium colony is then chosen, with verification primer Pgpd-detect-F and TtrpC- Detect-R carries out bacterium colony PCR verifications to single bacterium colony, and target sequence size is 684bp, obtains plasmid pBHg-BCA1-pdd1- antisens.Primer sequence is as follows：

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

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

PCR response procedures are：94℃ 5min；94 DEG C of 30s, 58 DEG C of 90s, 72 DEG C of 60s (25cycles)；72℃ 10min；4℃.

(6) use primer pdd1-sense-F and pdd1-sense-R using the genomic DNA of needle mushroom wild-type strain as mould Plate carries out PCR amplification, obtains pdd1-sense segments, and target sequence size is 540bp.Primer sequence is as follows：

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

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

(7) use restriction enzyme BamHI simultaneously to plasmid pBHg-BCA1-pdd1-antisens and segment pdd1- After sense segments carry out digestion and recycle, the two segments are connected overnight with T4Ligase.

(8) connection product is converted into bacillus coli DH 5 alpha competence, then chooses single bacterium colony, with verification primer Pgpd- Detect-F and TtrpC-detect-R carries out bacterium colony PCR verifications to single bacterium colony, and target sequence size is 1224bp, obtains pdd1 and strikes Low expression carrier pBHg-BCA1-pdd1RNAi.Primer sequence is as follows：

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

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

PCR response procedures are：94℃ 5min；94 DEG C of 30s, 58 DEG C of 90s, 72 DEG C of 90s (25cycles)；72℃ 10min；4℃.

It is by gpd promoter fragments (Pgpd-RNAi), pdd1- that pdd1, which strikes low expression carrier pBHg-BCA1-pdd1RNAi, Antisense segments, pdd1-sense segments and trpC terminate sub-piece (TtrpC-RNAi) and are existed with segment recombination kit The size that XmnI restriction enzyme sites obtain after being merged with plasmid pBHg-BCA1 is the carrier of 11761bp.

Three, plasmid pBHg-BCA1-pdd1OE and pBHg-BCA1-pdd1RNAi converts Agrobacterium

Plasmid pBHg-BCA1-pdd1OE and pBHg-BCA1-pdd1RNAi prepared by step 1 and step 2 are turned respectively Change AGL-1 bacterial strains competent cell (Beijing Bo Maide gene technology Co., Ltd, article No.：BC302-01 in).Specific steps are such as Under：

(1) two pipes, 50 μ L AGL-1 competent cells are taken, 1 μ g plasmids pBHg-BCA1-pdd1OE and plasmid are separately added into PBHg-BCA1-pdd1RNAi places 10min on ice with the pressure-vaccum mixing of liquid-transfering gun gently.

(2) centrifuge tube is put in the quick-frozen 5min of liquid nitrogen kind.

(3) 37 DEG C are immediately placed on and stands 5min in water-bath, not shake the water surface.

(4) centrifuge tube is put back on ice, keeps 5min.

(5) 1mL LB liquid mediums, 28 DEG C of stationary culture 2-3h are added.

(6) it draws bacterium solution to be coated on the tablet of the LB containing 50 μ g/mL kanamycins and 50 μ g/mL rifampins, 28 DEG C First 1h is placed in front, then is inverted culture 48-72h.The picking single bacterium colony after single bacterium colony is grown, with verification primer Pgpd-detect- F and TtrpC-detect-R carries out bacterium colony PCR verifications to single bacterium colony, and target sequence size is respectively 1558bp (pBHg-BCA1- Pdd1OE) and 1224bp (pBHg-BCA1-pdd1RNAi), the agrobacterium strains AGL1- eventually with plasmid is respectively obtained Pdd1OE and AGL1-pdd1RNAi.Primer sequence is as follows：

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

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

Four, Agrobacterium-mediated Transformation needle mushroom wild-type strain

Agrobacterium strains AGL1-pdd1OE and AGL1-pdd1RNAi prepared by step 3 are converted into needle mushroom wild-type bacteria Strain (Strains of Flammulina velutipes FL19).It is as follows：

(1) prepare needle mushroom wild-type strain fungus block：It carries and a few days ago being cultivated the CYM for covering with Flammulina velutipes mycelium with card punch Base breaks into bullet (d=5mm), the stationary culture 48h in CYM fluid nutrient mediums.

(2) Agrobacterium (AGL1-pdd1OE and AGL1-pdd1RNAi) containing target fragment plasmid is transferred to respectively and is contained In the LB liquid medium of rifampin and kanamycins, 28 DEG C of shake culture 12-16h (150rpm).

(3) when the OD600 of Agrobacterium bacterium solution reaches 0.5-0.8, the 50mL centrifuge tubes of sterilizing are transferred to, after sealed membrane sealing, 4500rpm, 4 DEG C of centrifugation 12min, collects thalline.

(4) Agrobacterium is washed 2 times with IM culture mediums.Then the IM culture mediums (Agrobacterium thalline is resuspended) of addition 5mL, 28 DEG C, 150rpm, be protected from light culture 4-6 hour to OD600 be 0.3-0.5, to induce its conversion capability.

(5) ready fungus block in step (1) is added in the Agrobacterium after induction, after Liquid static culture 3-6h, Fungus block is gone on the IM culture mediums for being covered with glassine paper, 25 DEG C co-culture 3-6 days.

(6) it after the completion of co-culturing, with sterile water wash fungus block to remove Agrobacterium, goes to containing 12.5 μ g/mL hygromycin Bs In the CYM screening flat boards of 200 μM of Cefotaxime Sodiums, 3-4 weeks son to be transformed of 25 DEG C of stationary cultures is grown.

Five, the screening of needle mushroom transformant

1, preparation of samples

(1) single bacterium colony picked out from CYM screening flat boards is continued to be transferred to containing 12.5 μ g/mL hygromycin Bs and 200 The CYM screening flat boards of μM Cefotaxime Sodium screened for 5 generations.

(2) screening for 5 generations, still well-grown bacterial strain is transferred to the CYM for being covered with glassine paper without containing hygromycin B On tablet, mycelia is collected after covering with mycelia, a part is extracted for DNA, and another part is extracted for RNA.Simultaneously with needle mushroom Wild-type strain is as a contrast.

2, bacterial strain DNA extractions and verification

(1) needle mushroom wild type mycelia is collected, throws into Liquid nitrogen storage rapidly.

(2) 400 μ L Extraction buffers (SDEB) and 400 μ L 2 × CTAB buffer solutions are added in liquid nitrogen grinding mycelium, are vortexed Mixing.

(3) 800 μ L phenol/chloroform (1 is added:1) solution mixes well, and 12000rpm centrifuges 10min.

(4) it Aspirate supernatant and is transferred in new 1.5mL centrifuge tubes, 500 μ L chloroforms is added, mix well, 12000rpm, Centrifuge 10min.

(5) it draws appropriate supernatant and is transferred in new 1.5mL centrifuge tubes, the isopropanol of 0.6 times of volume of addition, mixing, 4 DEG C place 10~20min, 12000rpm centrifuge 10min, abandon supernatant.

Twice, 12000rpm centrifuges 10min to (6) 75% ethyl alcohol cleaning precipitation, abandons supernatant.

(7) extra alcohol is sucked out, dries by of short duration centrifugation.

(8) (the DNA lysates preparation of appropriate DNA lysates is added：10 μ L are added in 1mL 10mM Tris-HCl (pH8.0) RNaseA) dissolving DNA.

(9) 37 DEG C of water-bath 2h, -20 DEG C of preservations.

(10) mutant strain is overexpressed with needle mushroom wild type, pdd1 respectively and pdd1 strikes the DNA of low expression mutant strain For template, PCR amplification verification is carried out, bacterium is carried out to single bacterium colony with verification primer Pgpd-detect-F and TtrpC-detect-R Fall PCR verifications, target sequence be respectively without band (needle mushroom wild type), size be 1558bp (pdd1 be overexpressed mutant strain) and Size is 1224bp (pdd1 strikes low expression mutant strain), while respectively using corresponding plasmid as positive control.Primer sequence is such as Under：

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

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

3, bacterial strain RNA extractions and quantitative PCR verify transcriptional level

RNA is extracted to obtained Positive mutants body in above-mentioned steps 2, is carried out at the same time quantitative PCR experiment, concrete operations are such as Under：

(1) the bacterium sample of collection is placed in 1.5mL centrifuge tubes and put into liquid nitrogen rapidly and frozen.

(2) liquid nitrogen grinding mycelium, is added 0.7mL Trizol, and mixing is placed at room temperature for 5min.4 DEG C, 12000rpm centrifugations 10min draws supernatant in 1.5mL Axygen centrifuge tubes.

(3) 0.7mL chloroforms are added, slight oscillatory 15s on turbula shaker is placed at room temperature for 5min.4 DEG C, 12000rpm from Heart 15min.

(4) appropriate supernatant is drawn, the isopropanol of 0.6 times of volume precooling is added, mixing is placed at room temperature for 10min.4 DEG C, 12000rpm centrifuges 10min, abandons supernatant.

(5) 75% ice cold ethanol cleaning twice, dries alcohol.

(6) with 100 μ L RNA-free water dissolutions RNA.

(7) UV spectrophotometer measuring RNA records OD260, OD280 and Ratio value of RNA sample.Then according to public affairs Formula：The extension rate of the concentration of RNA sample=value × 40 OD260 × RNA sample, calculates the concentration of RNA.

(8) integrality of ordinary gel electrophoresis detection RNA：The Ago-Gel of 1% concentration, 2 μ g RNA sample applied sample amounts, Voltage is 180V, ultraviolet lower observation RNA bands after electrophoresis 15min, EB dyeing.

(9) all RNA samples are inverted to cDNA respectively with cDNA synthetic agent box, are used for quantitative PCR.

(10) using above-mentioned cDNA samples as template, pdd1 is determined with quantification PCR primer Q-pdd1-F and Q-pdd1-R Amount amplification, using 2^-ΔΔCtComputational methods experimental data is handled.It is simultaneously template as reference gene using actin, draws Object is Q-actin-F and Q-actin-R.Primer sequence is 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’。

Quantitative PCR response procedures are：95 DEG C of pre-degeneration 60s；95 DEG C of denaturation 15s, 58 DEG C of annealing 15s, 72 DEG C of extension 45s, 40 cycles；Solubility curve is analyzed：65 DEG C~95 DEG C, increases by 0.5 DEG C per 0.05s and detect.

(11) quantitative PCR data of gained is analyzed, chooses the wild type that compares, the bacterial strain of pdd1 up-regulated expressions is made It is overexpressed mutant strain for pdd1, the bacterial strain that pdd1 lowers expression strikes low expression mutant strain as pdd1.Pdd1 mistakes are obtained 3 plants of mutant strain is expressed, respectively：pdd1^OE#7、pdd1^OE#31And pdd1^OE#38, pdd1 strikes 3 plants of low expression mutant strain, respectively For：pdd1^RNAi#64、pdd1^RNAi#148And pdd1^RNAi#170, data result such as Fig. 3.The above bacterial strain is as experimental strain.

The application of embodiment 3, pdd1 in regulation and control Flammulina velutipes mycelium growth

One, the experiment of tablet mycelia observation on Growth is carried out to pdd1 mutant strains

(1) needle mushroom wild type, pdd1 are overexpressed mutant strain (pdd1^OE#7、pdd1^OE#31And pdd1^OE#38) and pdd1 Strike low expression mutant strain (pdd1^RNAi#64、pdd1^RNAi#148And pdd1^RNAi#170) respectively bacterium of the same size is made with card punch Block (d=5mm), and be inoculated into respectively on the CYM tablets of not drug containing, 25 DEG C of cultures are taken pictures for 7 days later.

(2) during culture, mycelia growth length was measured every 24 hours, and keep a record, it is flat calculates each bacterial strain Equal growth rate.

It is as follows to different strains growing state and growth rate analysis result：On CYM tablets, pdd1 is overexpressed mutant bacteria The growth of strain mycelia is similar to wild type, without significant difference, and pdd1 strike low expression mutant strain mycelia grow die down it is slack-off, Illustrate that pdd1 low expressions influence Flammulina velutipes mycelium growth (Fig. 4).

Two, the experiment of culture material mycelia observation on Growth is carried out to pdd1 mutant strains

Needle mushroom wild type, pdd1 are overexpressed mutant strain (pdd1^OE#7、pdd1^OE#31And pdd1^OE#38) and pdd1 strike it is low Express mutant strain (pdd1^RNAi#64、pdd1^RNAi#148And pdd1^RNAi#170) respectively fungus block (d of the same size is made with card punch =5mm), and it is inoculated into respectively in the tissue culture bottle equipped with 325g culture materials, 25 DEG C of cultures are taken pictures to there is mycelia to cover with bottom of bottle.

As seen from Figure 5, pdd1 is overexpressed mutant strain (12 days) and first covers with culture material than wild type (15 days), and pdd1 strikes Low expression mutant strain (22 days) covers with culture material the latest.In culture material, mycelial growth rate：Pdd1 is overexpressed mutant strain >Wild type>Pdd1 strikes low expression mutant strain.Illustrate that pdd1 high expression promotes mycelia growth, pdd1 low expressions to inhibit mycelia life Long, so that bacterial strain more adapts to the nutrient environment of culture material, pdd1 can be carried as molecule selection and breeding strain excellent for the high expression of pdd1 For reference.

The application of embodiment 4, pdd1 in regulating and controlling needle mushroom fruit body development

One, fruiting observation experiment is carried out to pdd1 mutant strains

(1) needle mushroom wild type, pdd1 are overexpressed mutant strain (pdd1^OE#7、pdd1^OE#31And pdd1^OE#38) and pdd1 Strike low expression mutant strain (pdd1^RNAi#64、pdd1^RNAi#148And pdd1^RNAi#170) respectively bacterium of the same size is made with card punch Block (d=5mm), and being inoculated into respectively in the tissue culture bottle equipped with 325g culture materials, at 25 DEG C, 70% humidity is trained under dark condition It supports 15-20 days, until all bacterial strain mycelia cover with culture bottle.

(2) mycelium stimulation is carried out to each culture bottle respectively, i.e., is gently scraped off at the top of culture bottle with the scalpel of sterilization treatment Thick mycelia continues at 25 DEG C, 70% humidity, covers with new mycelia at the top of culture to culture bottle under dark condition, obtains after covering bacterium Culture bottle.

(3) culture bottle after bacterium will be covered at 15 DEG C, 95% humidity cultivates under dark condition, carries out cold stimulation, cultivate 5-7 It has former base (fructification of needle mushroom most initial) and grows.Routine observation is simultaneously taken pictures.

It is as follows to fruiting interpretation of result：The 6th day after stimulating fruiting, pdd1, which is overexpressed mutant strain, former base generation, And wild-type strain generates former base on the 7th day after stimulation fruiting is handled, at this point, pdd1 be overexpressed mutant strain have it is more more dense Former base generate.Continue mushroom producing culture to harvest time, it is found that pdd1 is overexpressed mutant strain and shifts to an earlier date entrance in 4 days than wild type Ripe harvest time, and bacterial strain is taller and bigger (Fig. 6).At the same time, pdd1, which strikes in low expression mutant strain, still loses former base, when after After the long fruiting time that renews, pdd1 is struck low bacterial strain (pdd1 by a small margin^RNAi#148And pdd1^RNAi#170) in have extremely individual former bases Generation, and pdd1 is significantly struck low bacterial strain (pdd1^RNAi#64) in still lose former base (Fig. 6).It these results suggest that pdd1's Expression quantity significantly affects the formation of needle mushroom former base and the development of fructification, during pdd1 is needle mushroom fruit body development Required positive regulatory factor.

Two, mutant strain is overexpressed to needle mushroom wild type and pdd1 and carries out biomass estimation experiment

It does not generate or only generates only a few because pdd1 strikes low bacterial strain and grow short and small thin and weak fructification, do not have system Meter learns meaning, therefore the present invention is only limited to wild type to the measurement of biomass and pdd1 is overexpressed mutant strain.In order to more effective Carry out count, the present invention only to length>The fructification of 6cm carries out statistical analysis.

Analysis result shows：Pdd1 is overexpressed mutant strain and generally increased than wild-type biology amount, wherein bacterial strain pdd1^OE#31Biomass increase is the most notable, fructification quantity increase by 19.85 ± 4.64%, and fructification height increase by 40.36 ± 7.25%, total weight in wet base increases by 62.47 ± 8.88% (Fig. 7), is one plant of good volume increase bacterial strain, in conjunction with what is come to the ripening period in advance Characteristic can shorten the production cycle in production application, reduce energy consumption and cost of labor, be one plant of good low energy consumption high yield Production bacterial strain.

Sequence table

<110>Institute of Microorganism, Academia Sinica

<120>The transcription factor PDD1 and its encoding gene of regulation and control needle mushroom fruit body development and application

<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 55 60

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. protein is following protein a) or b) or c) or d)：

A) amino acid sequence is protein shown in sequence 2；

C) amino acid sequence shown in sequence 2 by the substitution of one or several amino acid residues and/or is lacked and ored add Obtained protein with the same function；

D) with amino acid sequence shown in sequence 2 with 75% or 75% or more homology and albumen with the same function Matter.

Any one of 2. it is following A 1 with the relevant biomaterial of protein described in claim 1) to A8)：

A1 the nucleic acid molecules of protein described in claim 1) are encoded；

A2) contain A1) expression cassettes of the nucleic acid molecules；

A3) contain A1) recombinant vectors of the nucleic acid molecules；

A4) contain A2) recombinant vector of the expression cassette；

A5) contain A1) recombinant microorganisms of the nucleic acid molecules；

A6) contain A2) recombinant microorganism of the expression cassette；

A7) contain A3) recombinant microorganism of the recombinant vector；

A8) contain A4) recombinant microorganism of the recombinant vector.

3. relevant biological material according to claim 2, it is characterised in that：A1) nucleic acid molecules be it is following 1) or 2) Or 3) shown in gene：

2) there is 75% or 75% or more homogeneity with the nucleotide sequence 1) limited, and encodes albumen described in claim 1 The cDNA molecules or genomic DNA molecule of matter；

1) or 2) 3) and protein described in claim 1 is encoded with the nucleotide sequence hybridization that limits under strict conditions CDNA molecules or genomic DNA molecule.

4. protein described in claim 1 or relevant biological material according to claim 2 or 3 are in following (b1)-(b6) Any application：

(b1) regulation and control Flammulina velutipes mycelium growth；

(b2) regulate and control needle mushroom fruit body development；

(b3) needle mushroom biomass and/or yield are improved；

(b4) shorten the fruiting period of needle mushroom；

(b6) needle mushroom breeding.

5. application according to claim 4, it is characterised in that：It is described to be regulated to promote；

Or, the raising needle mushroom biomass and/or yield be embodied in it is any in following (c1)-(c3)：

(c1) increase acupuncture needle massee fruiting bodies quantity；

(c2) increase acupuncture needle massee fruiting bodies height；

(c3) increase acupuncture needle massee fruiting bodies weight in wet base；

Or, the fruiting period for shortening needle mushroom is embodied in and the former base phase of needle mushroom and/or maturity period is promoted to shift to an earlier date.

6. a kind of method for cultivating yield height, fruiting period short transgenosis needle mushroom, including improve right in receptor needle mushroom It is required that the expression quantity and/or activity of the protein described in 1, the step of obtaining transgenosis needle mushroom；The transgenosis needle mushroom Yield is shorter than the receptor needle mushroom higher than the fruiting period of the receptor needle mushroom and/or the transgenosis needle mushroom.

7. according to the method described in claim 6, it is characterized in that：

The yield of the transgenosis needle mushroom is embodied in any in following (d1)-(d3) higher than the receptor needle mushroom：

(d1) the fructification quantity of transgenosis needle mushroom is more than the receptor needle mushroom；

(d2) the fructification height of transgenosis needle mushroom is higher than the receptor needle mushroom；

(d3) the fructification weight in wet base of transgenosis needle mushroom is more than the receptor needle mushroom；

Or, the fruiting period of the transgenosis needle mushroom is shorter than the former base phase that the receptor needle mushroom is embodied in transgenosis needle mushroom And/or the maturity period is earlier than the receptor needle mushroom.

8. the method described according to claim 6 or 7, it is characterised in that：In the raising receptor needle mushroom described in claim 1 Protein expression quantity and/or active method be protein described in claim 1 is overexpressed in receptor needle mushroom.

9. according to any method in claim 6-8, it is characterised in that：The method of the overexpression is by claim The encoding gene of protein described in 1 imports receptor needle mushroom.

10. according to any method in claim 6-9, it is characterised in that：The nucleosides of the encoding gene of the protein Acid sequence is DNA molecular shown in sequence 1.