CN113637691A - Grifola frondosa glucosyltransferase gfgel4 and coding gene and application thereof - Google Patents

Abstract

The invention discloses a grifolan glucosyltransferase gfgel4, and a coding gene and application thereof, belongs to the technical field of edible fungus heredity and genetic engineering, and particularly relates to grifolan glucosyltransferase gfgel4, and a coding gene and application thereof; the invention clones the coding gene of the key enzyme beta-1, 3 glucosyltransferase for forming the branch structure in the glucosan synthesis process from the grifola frondosa mycelium genome for the first time, and proves the key role of the gene in the growth of the grifola frondosa mycelium, the glucosan synthesis and the formation of the glucosan branch structure through gene silencing; finally, beta-1, 3 glucosyltransferase is overexpressed in the grifola frondosa by a genetic engineering technology, so that the glucan synthesis yield is improved, and the branching degree of glucan is improved; the invention is helpful to understand the synthesis mechanism of edible and medicinal fungi polysaccharide from molecular level, and provides technical scheme and guidance for improving glucan synthesis path of edible and medicinal fungi by using genetic engineering technology.

Description

Grifola frondosa glucosyltransferase gfgel4 and coding gene and application thereof

Technical Field

The invention belongs to the technical field of edible fungus heredity and genetic engineering, and particularly relates to glucosyltransferase gfgel4 and an encoding gene and application thereof.

Background

Edible and medicinal fungal polysaccharide is one of the fields which are actively researched at home and abroad at present, is widely involved in various vital metabolic activities in cells, has various biological activities such as anti-tumor, anti-virus, anti-oxidation, immunoregulation and the like, wherein glucan is polysaccharide formed by linking and polymerizing D-glucose through beta-1, 3 and/or beta-1, 6 glycosidic bonds, has various biological activities, and polysaccharide with moderate branching degree usually has higher biological activity, for example, grifola frondosa glucan with the branching degree of 0.31-0.36 has stronger immune activity.

However, the synthesis of edible and medicinal fungal polysaccharides is very complicated, the substrates and enzyme systems involved in the synthesis are numerous, and the technical methods available for reference are seriously insufficient, so that the understanding and the recognition thereof are very limited. Researches show that when glucose is used as a carbon source for ganoderma lucidum fermentation, besides the growth requirement of the strain, the glucose can be converted into corresponding nucleotide sugars such as UDP-glucose, UDP-galactose, GDP-mannose, dTDP-glucose pyrophosphorylase and the like by a plurality of nucleotide sugar synthetases (such as UDP-glucose pyrophosphorylase, UDP-glucose-4-epimerase, GDP-mannose pyrophosphorylase, dTDDP-glucose pyrophosphorylase and the like), and it is presumed that glycosyltransferase can have close relation with the formation of polysaccharide branched chains, so that the functions of enzyme systems related to glucan synthesis need to be determined to realize the efficient and stable synthesis of glucan for food and medicine, and targeted metabolic regulation means are adopted to effectively strengthen the synthesis.

Grifola frondosa (Grifola frondosa) is an edible fungus rich in nutrients such as polysaccharides and proteins. The existing research results show that glucan (D-fraction) purified from the fruit body of Grifola frondosa has remarkable activities of resisting tumors, regulating immunity and the like. It has been proved by research that the monosaccharide composition and content of polysaccharide of Grifola frondosa mycelia or exopolysaccharide are directly related to the activity of nucleotide sugar synthetase, and the branching degree of glucan of Grifola frondosa extracellular glucan is also closely related to glycosyltransferase. However, most edible fungal glucans contain different proportions of branching structures, and thus how these branching structures are formed and which enzymes are involved in glucan branching reconstruction have not been systematically studied.

Disclosure of Invention

The invention aims to provide a technical means for simply modifying the primary structure of the extracellular glucan of the grifola frondosa and effectively changing the synthesis amount of grifola frondosa polysaccharide. Specifically, the invention aims to provide a grifola frondosa polysaccharide synthesis related enzyme, namely beta-1, 3 glucosyltransferase, and simultaneously provides a grifola frondosa beta-1, 3 glucosyltransferase encoding gene sequence, a gene silencing and gene overexpression plasmid, a gene engineering bacterium and a production method.

In order to realize the aims of efficiently and stably synthesizing the polysaccharide of the grifola frondosa mycelium and further improving the quality of a grifola frondosa fermentation product, the invention clones a coding gene of a polysaccharide synthesis key enzyme-beta-1, 3-glucosyltransferase from a grifola frondosa mycelium genome for the first time, and proves the key role of the gene in the growth of the grifola frondosa mycelium and the synthesis process of the polysaccharide through gene silencing;

finally, beta-1, 3-glucosyltransferase is over-expressed in grifola frondosa by a genetic engineering technology, so that the polysaccharide synthesis yield of the recombinant strain is remarkably improved, and the branching degree of extracellular polysaccharide is improved. The invention is helpful to understand the synthesis mechanism of edible and medicinal fungal polysaccharide from molecular level, and provides important technical support and reference for developing metabolic engineering research of edible and medicinal fungal polysaccharide synthesis and efficient fermentation production of polysaccharide with stable quality.

The invention carries out the gene silencing of the beta-1, 3-glucosyltransferase of the grifola frondosa by an RNA interference (RNAi) technology and carries out the gene overexpression of the beta-1, 3-glucosyltransferase of the grifola frondosa by a homologous recombination technology, thereby providing an effective solution strategy and a method for understanding the synthesis mechanism of fungal polysaccharide for food and medicine and efficiently producing immunopotentiator polysaccharide at the molecular level, and the constructed recombinant strain can synthesize the polysaccharide by substrates such as glucose, starch hydrolysate, lignocellulose and the like.

The invention provides a grifola frondosa beta-1, 3-glucanotransferase (1,3-beta-glucanosyltransferase-gfgel4), wherein the full length of a gene sequence for coding the gfgel4 is 1945bp, and is shown as SEQ ID NO. 1; the enzyme comprises 1 complete open reading frame and codes 554 amino acids in total, as shown in SEQ ID NO. 2.

The invention also provides the use of said beta-1, 3-glucanotransferase of Grifola frondosa for altering the amount of polysaccharide synthesis, said alteration comprising up-regulating or down-regulating the amount of polysaccharide synthesis of Grifola frondosa; further, the up-regulation of the polysaccharide synthesis amount is realized by expressing a gene encoding beta-1, 3-glucanotransferase of grifola frondosa; the down-regulation of the synthesis amount of polysaccharide is realized by gene silencing and coding beta-1, 3-glucosyltransferase gene of grifola frondosa; further, the regulation of the synthesis amount of the grifola frondosa polysaccharide is realized by expressing the complete sequence (SEQ ID NO.1) of the grifola frondosa beta-1, 3-glucanotransferase gene, and the regulation of the synthesis amount of the grifola frondosa polysaccharide is realized by gene silencing the conserved sequence (SEQ ID NO.3) of the grifola frondosa beta-1, 3-glucanotransferase gene.

The invention also provides the use of said grifola frondosa beta-1, 3-glucanotransferase in altering the degree of extracellular glucan branching, the alteration comprising increasing or decreasing the degree of branching of grifola frondosa extracellular glucan; further, the increasing or decreasing the degree of extracellular glucan branching is achieved by expressing or gene silencing a gene encoding grifola frondosa beta-1, 3-glucanotransferase.

The invention also provides two recombinant vectors, namely an overexpression vector, wherein the vector comprises the grifola frondosa beta-1, 3-glucanotransferase gene gDNA sequence (SEQ ID NO.1) for increasing the branching degree of grifola frondosa extracellular glucan and up-regulating the grifola frondosa polysaccharide synthesis amount. The recombinant vector is obtained by amplifying a gDNA sequence (SEQ ID NO.1) of a beta-1, 3-glucosyltransferase gene and connecting the gDNA sequence with plasmids pAN7-gpd and pAN7-35s respectively through homologous recombination ligase; according to one embodiment of the invention, the gene overexpression vectors are pAN7-gpd-gfgel4 and pAN7-35s-gfgel 4.

The recombinant vector also comprises a grifola frondosa gpd promoter (SEQ ID NO.4) and an Aspergillus nidulans 35s promoter (SEQ ID NO.5), namely a gene silencing vector. Plasmid pAN7-1 (purchased from lake Nanfenghui Biotech Co., Ltd.) is subjected to Hind III single enzyme digestion, and the enzyme digestion product is connected with the obtained target fragment of the 35s promoter (SEQ ID NO.5) through homologous recombination ligase to obtain pAN7-35s plasmid; the pAN7-35s plasmid was digested with Sbf I and BamH I, and the digested product was ligated with the gpd promoter (SEQ ID NO.4) obtained above using homologous recombination ligase to obtain pAN7-35s-gpd plasmid. The pAN7-35s-gpd plasmid is subjected to single Sbf I restriction enzyme digestion, and the restriction enzyme digestion product is connected with the obtained conserved sequence (SEQ ID NO.3) of the grifola frondosa beta-1, 3-glucosyltransferase gene through homologous recombination ligase to obtain a vector pAN7-gfgel4-dual of the conserved region of the bidirectional promoter silencing beta-1, 3-glucosyltransferase gene.

The invention also provides a recombinant engineering bacterium, which comprises the gene silencing vector or the overexpression vector. The engineering bacteria are obtained by preparing an incomplete enzymolysis grifola frondosa protoplast and transforming the protoplast with the recombinant vector by adopting a PEG mediated transformation method.

Wherein the preparation of the incompletely enzymolyzed Grifola frondosa protoplast is prepared by washing cultured Grifola frondosa mycelia with 0.3-0.6M mannitol solution, adding 0.5-4.0% muramidase (purchased from Guangdong institute of microorganisms) for enzymolysis at 25-45 deg.C for 1-5h, centrifuging to collect the insoluble substance after enzymolysis, and filtering.

The PEG mediated transformation method is used for transferring the gene silencing vector or the over-expression vector into the protoplast, and the gene silencing vector pAN7-gfgel4-dual or the gene over-expression vectors pAN7-gpd-gfgel4 and pAN7-35s-gfgel4 and PEG/CaCl₂Mixing the solution, adding into Maitake Mushroom protoplast, ice-bathing, and adding PEG/CaCl₂The solution is subjected to constant volume centrifugation by using STC buffer, then is resuspended and coated on a resistance plate of a selective regeneration CYM medium (hygromycin 100 mu g/mL), and is cultured and screened at the temperature of 28 ℃.

The invention also provides the application of the recombinant engineering bacteria in changing the branching degree of glucan; the alteration includes increasing or decreasing the degree of branching of the extracellular glucan of Grifola frondosa.

The invention also provides a production method of the high-yield grifola frondosa polysaccharide, which adopts the grifola frondosa recombinant engineering bacteria to produce by substrates such as glucose, starch hydrolysate or lignocellulose and the like, and the method comprises the following steps:

(1) preparing a fermentation medium and a seed culture medium, wherein a carbon source of the fermentation medium and the seed culture medium is glucose, or starch hydrolysate or lignocellulose or two of the glucose, the starch hydrolysate and the lignocellulose, and the concentration of the carbon source is 5.0-100.0 g/L;

(2) culturing the engineering bacteria, activating engineering bacteria seed liquid in a culture medium, and preparing seed culture liquid in a fermentation tank with a corresponding scale in a step-by-step amplification manner;

(3) inoculating the engineering bacteria seed liquid into a shake flask or a fermentation tank containing a fermentation culture medium in an inoculation amount of 5.0% -15.0%.

The culture conditions of the fermentation tank are as follows: culturing at 20-30 deg.C, ventilation amount of 0.5-2.0vvm, stirring rotation speed of 50-500rpm for 3-10 d; the shake flask conditions were: culturing at 20-30 deg.C and rotation speed of 80-200rpm for 3-10 days.

The total yield of polysaccharide obtained by fermenting the recombinant grifola frondosa by using the method is more than 2.0-50.0 g/L.

(1) The invention regulates and controls the synthesis of the grifola frondosa polysaccharide by adopting an overexpression technology or a gene silencing technology. The gene-silenced grifola frondosa recombinant strain has relatively slow growth speed of the strain, the polysaccharide synthesis amount and synthesis speed are reduced to different degrees compared with the original strain, and the branching degree of extracellular glucan is correspondingly reduced, which indicates that beta-1, 3-glucanotransferase has irreplaceable effect in the polysaccharide synthesis process. Compared with the original strain, the polysaccharide synthesis amount and synthesis speed of the gene over-expressed grifola frondosa recombinant strain are obviously improved, and the branching degree of extracellular glucan is relatively increased. The yield of polysaccharide fermented by the gene over-expression grifola frondosa recombinant bacteria in a 25L fermentation tank can be improved by more than 30%, the solubility of extracellular glucan is better, and the biological activity is higher; the method also has the advantages of fast growth, cheap culture medium, stable heredity, high yield and the like, and has obvious potential of industrial production. The invention provides a technical basis and a method guide for improving the polysaccharide synthesis way of edible and medicinal fungi by using a genetic engineering technology.

Drawings

FIG. 1 shows the cloning results of the beta-1, 3-glucanotransferase (gfgel4) gene from Grifola frondosa; in the figure, A is the gDNA full length of the gfgel4 gene; the full length of cDNA of the gfgel4 gene; m1: DNA 5000 marker, 1 is gfgel4-gDNA amplification product; m2: DNA 2000Maker, 2 is the gfgel4-cDNA amplification product. .

FIG. 2 is a schematic diagram of the construction of the silencing vector pAN7-gfgel4-dual for the beta-1, 3-glucanotransferase gene of Grifola frondosa.

FIG. 3 is a schematic diagram of the construction of an overexpression vector pAN7-35s-gfgel4 for the beta-1, 3-glucanotransferase gene of Grifola frondosa.

FIG. 4 is a schematic diagram of the construction of an overexpression vector pAN7-gpd-gfgel4 for the beta-1, 3-glucanotransferase gene of Grifola frondosa.

FIG. 5 shows the results of growth of grifola frondosa beta-1, 3-glucanotransferase PEG-mediated transformation and regeneration silencing recombinant bacteria in a medium containing 100. mu.g/mL hygromycin, wherein WT indicates that protoplasts were grown in a medium not containing hygromycin.

FIG. 6 shows the result of the growth of Grifola frondosa beta-1, 3-glucanotransferase PEG-mediated transformation and regeneration over-expression recombinant bacteria in a medium containing 100. mu.g/mL hygromycin.

FIG. 7 is a graph comparing the growth of Grifola frondosa recombinant bacteria with beta-1, 3-glucanotransferase gene silencing and gene overexpression on PDA plates for 15 d.

FIGS. 8 and 9 show the results of polysaccharide synthesis by fermentation of Grifola frondosa recombinant bacteria with beta-1, 3-glucanotransferase gene silencing and gene overexpression in 25-L fermenter. In the figure, WT: a control strain; GFGEL 4-i: silencing recombinant bacteria; GFGEL4-35s, GFGEL 4-gpd: and (3) over-expressing the recombinant bacteria.

FIG. 10 is a monosaccharide composition of exopolysaccharides of Grifola frondosa recombinant bacteria with beta-1, 3-glucanotransferase gene silencing and gene overexpression in Grifola frondosa, wherein WT: a control strain; GFGEL 4-i: silencing recombinant bacteria; GFGEL4-35s, GFGEL 4-gpd: and (3) over-expressing the recombinant bacteria.

Description of the attached tables

Table 1 shows methylation results of Grifola frondosa recombinant extracellular polysaccharide of Grifola frondosa subjected to gene silencing and gene overexpression by beta-1, 3-glucanotransferase.

TABLE 1

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-10, the present invention provides the following technical solutions: the specific implementation of the invention is further explained by combining the following examples and the accompanying drawings, and the plasmid, PCR reagent and the like used in the following examples are commercial products, and the specific operation is performed according to the instructions. However, embodiments of the invention are not so limited and other non-specified experimental manipulations and process parameters are carried out in accordance with conventional techniques.

The dextran assay described in the examples of the invention is as follows: collecting the fermented grifola frondosa mash, centrifuging by 10000g, collecting supernatant, concentrating, and precipitating by using 95% ethanol according to a volume ratio of 1: 3; 10000g of alcohol precipitate is collected by centrifugation, and the product is dried in vacuum at 50 ℃ to constant weight, namely the weight of the produced glucan is recorded.

The method for determining the monosaccharide composition in glucan disclosed by the embodiment of the invention comprises the following steps: a40 mg sample of dextran was weighed, added to 3mL of a 2mol/L TFA solution, hydrolyzed at 110 ℃ for 3h, cooled to room temperature, transferred to a rotary evaporator flask, spun dry at 40 ℃, then added to 3mL of methanol, spun dry at 40 ℃ (repeated 4 times to completely remove TFA), dissolved in 3mL of water, and filtered through a 0.22. mu.L filter for use. The standard substances of glucose, mannose, galactose, xylose, arabinose and N-acetylglucosamine are respectively 5mg and dissolved in a 5mL volumetric flask, and are filtered through a 0.22 mu L filter membrane for standby. The chromatographic conditions were as follows: CarboPac PA20(3 mm. times.150 mm) anion exchange chromatography column, CarboPac PA20(3 mm. times.50 mm) guard column, eluted with sodium hydroxide solution (7mmol/L) and detected by pulsed amperometric detector.

The method for analyzing the methylation in the glucan disclosed by the embodiment of the invention comprises the following steps: weighing 10mg dried Grifola frondosa extracellular crude polysaccharide in 1.5ml of colloidal gold phosphate solution (MSO), and performing ultrasonic water bath for 20 min; fully dissolving the sample, adding excessive fresh ground NaOH powder, and continuing to perform ultrasonic water bath for 20 min; cooling in ice bath until the sample liquid is solidified, and dropwise adding excessive CH under the protection of nitrogen₃Performing ultrasonic treatment in a water bath at the temperature of 18-20 ℃ for 30 min; 1mL of Na 4mmol/L was added₂S₂O₃The methylation reaction is terminated. After methylation, products are hydrolyzed, reduced and acetylated to obtain partially methylated sugar alcohol acetyl ester derivatives, and then GC-MS analysis is carried out to analyze mass spectrograms of GC peaks.

Example 1: cloning of beta-1, 3-glucanotransferase encoding gene of grifola frondosa

Grifola frondosa GF02 (purchased from American type culture Collection,

60301^TM) Culturing mycelium, rapidly adding liquid nitrogen, grinding into fine powder, and extracting genome with plant or fungal genome extraction kit.

Primers were designed based on the published beta-1, 3-glucanotransferase gene of Grifola frondosa (GenBank ID: A0H 81-00473) as follows:

gfgel4-F(SEQ ID NO.7)：5'CCACCTCCGCACACGCATC 3'，

gfgel4-R(SEQ ID NO.8)：5'CGTGTCCACCACTCTACCCTC 3'。

the full length of the gene was amplified using the above primers using the published genome of Grifola frondosa (GenBank association access: GCA-001683735.1) as a template, and PCR reaction was performed: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 20s, annealing at 30s, annealing at 52 ℃ (temperature determined by upstream and downstream primers), extension at 72 ℃ for 2min for 30s (time determined by target gene length), and reaction for 35 cycles; the PCR amplification products were separated by electrophoresis on 1% agarose gel, and the target gene fragment (shown in FIG. 1) was recovered by agarose gel DNA recovery kit and sequenced, then the sequence was sequenced to obtain the gfgel4 gene sequence, which was confirmed to be the gene sequence encoding beta-1, 3-glucanotransferase of Grifola frondosa by BLAST alignment in NCBI.

Cultured Grifola frondosa GF02 mycelia were collected and rapidly ground in liquid nitrogen to powder to extract total RNA. According to RACE technique, TAKARA 3' -FULL RACE Core Set V2.0 kit (Takara Code: D314) was used and a specific primer 3RACE1(SEQ ID NO.9) was designed: 5'-ttctgggtatccatcctcgc-3', 3RACE2(SEQ ID NO. 10): 5'-gatacccagaagtaggtca-3', using cDNA reverse transcription of total RNA of Grifola frondosa as a template, PCR amplifying to obtain a 3 'cDNA specific fragment of gfgel4 gene, using TAKARA 5' -FULL RACE Kit (Takara Code: D315) Kit and designing a specific primer 5RACE1(SEQ ID NO. 11): 5'-cccatcagttctcttttgcg-3', 5RACE2(SEQ ID NO. 12): 5'-aagaagaagaggctcgggca-3', using cDNA after dephosphorylation reaction, decapping reaction, 5'RACE Adaptor connection and reverse transcription reaction as template, PCR amplifying to obtain 5' cDNA specific fragment of gfgel4 gene, splicing to obtain complete cDNA sequence of gfgel4 gene shown in SEQ ID NO. 6.

Example 2: construction of Gene silencing vector pAN7-gfgel4-dual and Gene overexpression vectors pAN7-gpd-gfgel4, pAN7-35s-gfgel4 of beta-1, 3-glucosyltransferase Gene sequence of Grifola frondosa

Based on the gene sequence of the beta-1, 3-glucanotransferase of Grifola frondosa cloned in example 1 (gfgel4), PCR primers containing homology arms at the upstream and downstream were designed according to the homology region, respectively:

gfgel4-i-TF(SEQ ID NO.13)：5'-TTGTACCCACCAGTACCTGCAGGAAAAATGGCCTCGCCTTCAGCT-3'，

gfgel4-i-TR(SEQ ID NO.14)：5'-GCAGCTTTAAGCTTGCATGCCTGCAGGTTTTTTACCGCTGAAGTCGCAC-3'，

gfgel4-TF35s(SEQ ID NO.15)：5'-CACTCCACATCTCCACTCGACCTGCAGGCCACCTCCGCACACGCATC-3'，

gfgel4-TR35s(SEQ ID NO.16)：5'–GGCAGCTTTAAGCTTGCATGCCTGCAGGCGTGTCCACCACTCTACCCTC-3'，

gfgel4-TFgbd(SEQ ID NO.17)：5'–ACCAGTACCTGCAGGCATGCAAGCTT CGTGTCCACCACTCTACCCTC-3'，

gfgel4-TRgbd(SEQ ID NO.18)：5'–TGTAAAACGACGGCCAGTGCCAAGCTTCCACCTCCGCACACGCATC-3'。

the fragment gfgel4-i (SEQ ID NO.3) of the target gene obtained by PCR amplification using the Grifola frondosa cDNA obtained in example 1 as a template and the fragment GFUGT-o (SEQ ID NO.1) of the target gene obtained by PCR amplification using the Grifola frondosa gDNA as a template.

Based on the GPD gene sequence of Grifola frondosa (GenBank: A0H 81-05461), GPD promoter primers are designed as follows:

GPD-F(SEQ ID NO.19)：5'-GAGTAGATGCCGACCGCGGGATCC AATCGTAATTAGGTGGCT-3'；

GPD-R(SEQ ID NO.20)：5'-CAGCTTTAAGCTTGCATGCCTGCAGG TACTGGTGGGTACAAATGA-3'。

the desired gene (SEQ ID NO.4) was amplified by PCR using the total DNA of Grifola frondosa obtained in example 1 as a template.

According to the sequence of the Aspergillus nidulans 35s promoter in the plasmid pAN7-1 (purchased from lake Nanfenghui Biotech Co., Ltd.), a 35s promoter primer is designed as follows:

35S-F(SEQ ID NO.21)：5'-CGACCTGCAGGCATGCAAGCTT AAAGCTGCCTACCAGGGACT-3'；

35S-R(SEQ ID NO.22)：5'-AAAACGACGGCCAGTGCCAAGCTT GATTTCGGCACGGCTACGG-3'。

the target gene (SEQ ID NO.5) was amplified by PCR using plasmid pAN7-1 as a template.

Carrying out double enzyme digestion on plasmid pAN7-1 (purchased from lake Nanfenghui Biotechnology Co., Ltd.) on Sbf I and BamH I, and connecting the enzyme digestion product with the obtained gpd promoter target fragment through homologous recombination ligase to obtain pAN7-gpd plasmid; carrying out Hind III single enzyme digestion on the plasmid pAN7-1, and connecting the enzyme digestion product with the obtained 35s promoter target fragment through homologous recombination ligase to obtain a pAN7-35s plasmid; the pAN7-35s plasmid is subjected to double enzyme digestion by Sbf I and BamH I, and the enzyme digestion product is connected with the GPD target fragment homologous recombination ligase to obtain the pAN7-35s-GPD plasmid.

The pAN7-35s-gpd plasmid is subjected to single Sbf I restriction enzyme digestion, and the restriction enzyme digestion product is connected with the obtained gfgel4-i fragment through homologous recombination ligase to obtain a vector pAN7-gfgel4-dual with a bidirectional promoter silencing beta-1, 3-glucosyltransferase gene conserved region. (schematic vector construction is shown in FIG. 2).

The pAN7-35s plasmid is subjected to single Sbf I enzyme digestion, and the enzyme digestion product is connected with the GFUGT-o fragment obtained by the single Sbf I enzyme digestion to obtain an overexpression vector pAN7-35s-gfgel4 of the beta-1, 3-glucanotransferase gene. (schematic vector construction is shown in FIG. 3).

The pAN7-gpd plasmid is subjected to single enzyme digestion by Hind III, and the digestion product is connected with the GFUGT-o fragment obtained above through homologous recombination ligase to obtain an overexpression vector pAN7-gpd-gfgel4 of the beta-1, 3-glucanotransferase gene. (schematic vector construction is shown in FIG. 4).

Example 3: preparation of Grifola frondosa protoplast by incomplete enzymolysis

Collecting mycelium obtained by PDA liquid culture of Grifola frondosa, treating for 1min under aseptic condition with tissue pulverizer, inoculating in 100mL PDB culture medium with 10% inoculum size, standing and culturing at 28 deg.C for 4d, centrifuging for 15min at 5000g, and collecting insoluble substance; washing with 0.6M mannitol solution for 2 times, adding 1mL of 2% filamentous fungus wall-breaking enzyme solution, and performing enzymolysis at 30 deg.C for 4 hr. Centrifuging the enzymolysis liquid for 15min at 5000g, collecting insoluble substances, and performing sterile filtration to obtain an enzymolysis protoplast; the prepared protoplast was added to 50mL of regenerated CYM medium (glucose 20g peptone 2g yeast extract 2g magnesium sulfate heptahydrate 0.5g dipotassium hydrogen phosphate 1.0g potassium dihydrogen phosphate 0.46g agar 20g, hygromycin 100. mu.g/mL), mixed well, poured onto a plate, and cultured at 28 ℃ for regeneration.

Example 4: construction of beta-1, 3-glucanotransferase gene silencing and overexpression recombinant bacteria GFGEL4-i, GFGEL4-35s and GFGEL4-gpd of grifola frondosa

Take 10. mu.g of plasmid pAN7-gfgel4-dual and 50. mu.L of PEG/CaCl₂Solution (25% PEG4000 and 50mM CaCl₂) Adding into 100 μ L Grifola frondosa protoplast solution, and incubating on ice for 30 min; 0.5mL of PEG/CaCl was again added₂Standing the solution at room temperature for 20 min; fixing the volume to 1mL by using STC buffer, reversing and uniformly mixing, and standing for 5min at room temperature; centrifuging at 4000r/min for 10min, and removing the supernatant; selection plated with 100. mu.g/mL hygromycin B by STC bufferCarrying out culture and screening on the sexual regeneration CYM culture medium at 28 ℃ to obtain the grifola frondosa beta-1, 3-glucanotransferase gene silencing recombinant strain GFGEL4-i, as shown in figure 5. Over-expression recombinant bacteria GFGEL4-35s and GFGEL4-gpd of the beta-1, 3-glucanotransferase gene of Grifola frondosa were obtained by screening in the same manner, as shown in FIG. 6.

As can be seen from FIGS. 5 and 6, the regeneration of protoplasts prepared from the original strain of Grifola frondosa was inhibited in the 100. mu.g/mL hygromycin-resistant plates; the empty vector carrying the hygromycin resistance gene and the silent transformant can be regenerated after being transferred into the protoplast and cultured for 10 days at 28 ℃; the grifola frondosa starting strain WT, a Control strain Control, a GFGEL4-i silent strain and GFGEL4-gpd and GFGEL4-35s overexpression strains are inoculated on a PDA plate by using a hole puncher with a unified specification and cultured for 15d, the growth conditions of the strains are shown in a figure 7, and therefore, the growth conditions of silent transformants and overexpression transformants are greatly different, the silent transformants grow relatively slowly compared with the growth conditions of the Control strains, and the overexpression strains grow relatively quickly; over-expressed transformants constructed from different plasmids also showed different growth conditions, and the growth rate of the GFGEL4-gpd over-expressed transformant was faster than that of the GFGEL4-35s over-expressed transformant. The gene silencing of beta-1, 3-glucosyltransferase is shown to result in the growth of the grifola frondosa mycelium being slowed down, and the over-expression of the gene results in the growth of the grifola frondosa mycelium being accelerated.

Example 5: production of glucan by fermentation of Grifola frondosa recombinant bacteria GFGEL4-i, GFGEL4-35s and GFGEL4-gpd 25-L with glucose as carbon source

(1) Slant medium (g/L): potato 200, glucose 20, peptone 5, KH₂PO₄1.5，MgSO₄·7H₂O0.75, pH is natural. The culture medium is used for strain preservation and activation.

The primary seed culture medium is (g/L): glucose 20, peptone 5, KH₂PO₄1.5，MgSO₄·7H₂O0.75, pH is natural.

The secondary seed culture medium is (g/L): glucose 30, peptone 6, KH₂PO₄3，MgSO₄·7H₂O1.5, pH is natural.

(2) Hair-like deviceThe fermentation culture medium is (g/L): glucose 60, peptone 6, KH₂PO₄6，MgSO₄·7H₂O1, soybean oil 50mL, pH natural.

(3) Fresh recombinant mycelium blocks of 5mm × 5mm are cut and placed on a slant culture medium, and cultured in a constant temperature incubator at 28 ℃ for 5 d.

First and second seed culture media were prepared separately. Inoculating 10 recombinant bacteria blocks of 5mm × 5mm, loading liquid in 75/250mL, shake culturing at 28 deg.C and 150rpm for 3d, and making into primary seed liquid. Inoculating the primary seed solution into a secondary seed culture medium, wherein the inoculation amount is 10% (v/v), the liquid loading amount is 150/500mL, and culturing for 3d by a shaking table (28 ℃, 150rpm) to obtain a secondary seed solution.

(4) Inoculating the cultured secondary seed liquid into a 25L fermentation tank with the inoculation amount of 10%, controlling the liquid loading amount to be 60%, controlling the temperature to be 28 ℃, culturing for 7d, and controlling the stirring speed and the ventilation amount to be 90rpm and 0.8vvm respectively. Taking fermented mash every 24h, centrifuging for 10min at 10000g to obtain Grifola frondosa mycelium precipitate, repeatedly washing thallus with distilled water, freeze-drying, and weighing to calculate the dry weight of the mycelium; and (3) concentrating the supernatant, precipitating with ethanol, drying, weighing and calculating the glucan amount, and detecting the monosaccharide composition in the glucan sample. After fermentation for 6 days, the original strain of Grifola frondosa has a bacterial weight of 16.77g/L (shown in FIG. 8), and the extracellular glucan yield is 5.65g/L (shown in FIG. 9); the bacterial body quantity of the grifola frondosa beta-1, 3-glucosyltransferase gene silencing recombinant bacterium GFGEL4-i is only 12.7g/L, the glucan yield is 3.38g/L, and the glucose composition is reduced to 78% compared with 83.5% of that of the WT strain; the bacterial body quantities of the beta-1, 3-glucanotransferase gene overexpression strains GFGEL4-35s and GFGEL4-gpd are improved by more than 5%, wherein the bacterial body quantity of the GFGEL4-gpd is improved by 32% to the maximum to reach 22.1g/L, the glucan yield reaches 8.23g/L, and the glucose composition reaches more than 88.07% (as shown in figure 10). This also indicates that overexpression of the β -1, 3-glucanotransferase gene or silencing of a conserved region thereof has varying degrees of influence on both grifola frondosa mycelium growth and glucan synthesis.

Example 6: structural change of branching degree of extracellular glucan produced by Grifola frondosa recombinant bacteria GFGEL4-i and GFGEL4-gpd

The product of the methylation treatment of the extracellular glucan of the grifola frondosa is subjected to hydrolysis, reduction and acetylation, and then is detected and analyzed by GC-MS, so that the type and the proportional relation of glycosidic bonds are determined. Of these, 2,3,4, 6-tetra-O-methyl-glucitol, indicates the presence of Glcp-1 →; 2,4, 6-tri-O-methyl-glucitol, corresponding → 3-Glcp-1 → bond type; 2, 4-di-O-methyl-glucitol, corresponding → 3,6-Glcp-1 → bond type; wherein the proportion of three derivatization products of the extracellular glucan of the original strain of the grifola frondosa is Glcp-1 → 3-Glcp-1 → 3,6-Glcp-1 → 14.17%: 61.8%: 13.15% (as shown in Table 1); the proportion of three derivatization products of the grifola frondosa beta-1, 3-glucosyltransferase gene silencing recombinant bacterium GFGEL4-i is Glcp-1 → 3-Glcp-1 → 3,6-Glcp-1 → 12.19%: 62.87%: 11.23%; the beta-1, 3-glucanotransferase gene is overexpressed to obtain the ratio of three derivatization products of the grifola frondosa beta-1, 3-glucanotransferase gene overexpression recombinant strain GFGEL4-gpd, wherein the ratio of the three derivatization products is Glcp-1 → 3-Glcp-1 → 3,6-Glcp-1 → 17.15%: 58.1%: 15.42%. The proportion of beta-1, 3-linked glucose residues in a beta-1, 3-glucosyltransferase silencing strain is increased, the proportion of beta-1, 3-linked glucose residues in a beta-1, 3-glucosyltransferase overexpression strain is correspondingly reduced, the proportion of terminal glucose and residues at a branch point → 3,6-Glcp-1 → is slightly increased, which shows that the branching degree of the gene-silenced strain is obviously reduced, and the branching degree of the overexpression strain is higher than that of the original strain, which shows that the grifola frondosa beta-1, 3-glucosyltransferase can obviously regulate and control the formation of a glucan branch structure.

SEQ ID NO. 1: grifola frondosa beta-1, 3-glucanotransferase gfgel4 gene sequence

atgtacaaccttccgcggttagcgaccgtgattgctggcgtcgccgccttcgcaagtggtgtccatgccatccagaatgttacaagaacgggcaggtacctctatactgctgatggaaacaggttcttcatcaagggtattgcataccaagaacaaggtgtgctggtcattcgtctcgtatgccaccttataacgagcggtttcaggcgccatctccacggacccaaacaatccattcttggagccctcaaatttcaccgaccccctcattgacggagcagcgtgttcgcgcgatcttcctttcctccagcagctcggggtcaacgccatccgtgtctacagtgtgaattcttcgcttaaccacgatgcctgcatgagcaccttcagcaatgcgggcatctataccatgtgcgaaattgtctccgtaatgtgttccacagaattctaacaaaactgcgattttcgcagtattgaccttgcactccccgtgaatgggtcgatcgaccgtgcttccccggcttggacgaccaatctcttggacctttacctcaatacgatcaacgtcttcaacaagtacgacaatgttttggcgtacaatgtaggtaacgaggttgtcatcgaccccactggcactggcgctgcagccttcatcaaggctgccgctcgcgacgtcaaatcatacctgtatggctcatgattaattacgttccttggcacgcgctgacgccgtctcggcactagcaaatccatttcatcgtctgcacttgttggctatgctgccattgatggagactccacctgggtggaccctctcgccaattacctcgactgtgatccctcgggctccaattccggagatacctccattgacctcttcggcctcaataactagtacgttgactgcgcattgttctattctcgtgcttacccacattcttttcccagcgagtggtgcgggaatgccaccgcaagcgtctatgccaccaagaacggtgacttcgcaggctacaacgttgcggcgtacttcagcgagttcggttgcattacctcccccccacgcctctggaccgaggtccaggcgctgttcagcagccccatgaccgacatctggtcgggtggcctcgccttcagctatttccctgcgaccagcagccagggccagttcggcattgtcaacatttcctctgacggatccacggtgacgacgggcgatgacttcagccgtctcaagacgcagtacagcgaggtctcgccgccgaacagccctaccctgagcagcgcaggtccgaatacgttctcggcgtgcccccaggagaactcgacattcctcgcgtcctcgacgcttcccccaacgcccaacgacgcggcatgtagctgcctggagagccagctgagctgccagttcacgcccaagaccacgaacaccagtggtatcgtcggcacgctcctcgataccgcgtgctcgttgcttggcggcactggcggctcctgcgccgccatctccgccaacgggaccaccggaacgtacgggagcgttgcgttctgcgaccctggtgcgcatctcggcttcttgttcttgccttctattcagtgctgacctcccgatattgcagacgtgaagctctcgttcgtgatgagcgagtattatgaggctacgggccgcctggcgacgtcgtgcgacttcagcggtaacgcgacggtcaacagcggggcgccctcgtccggcgcgaccgctgctgtcacgtcgtgtctggcgaacccttctgcgacgttcacccccagtgccccgactgtcaccccaggcggctcatcaacaggaacgtccagcgctggatcgtctgggtccagcaataacaagaacggcgctgtcgggctgctgggcaacccgcaggcgttggtggggctggcgtcgcgtgcatcttcagcgtcttgggcggtgtgttga

SEQ ID NO. 2: grifola frondosa beta-1, 3-glucanotransferase gfgel4 gene encoding amino acid sequence

MYNLPRLATVIAGVAAFASGVHAIQNVTRTGRYLYTADGNRFFIKGIAYQEQGAISTDPNNPFLEPSNFTDPLIDGAACSRDLPFLQQLGVNAIRVYSVNSSLNHDACMSTFSNAGIYTIIDLALPVNGSIDRASPAWTTNLLDLYLNTINVFNKYDNVLAYNVGNEVVIDPTGTGAAAFIKAAARDVKSYLKSISSSALVGYAAIDGDSTWVDPLANYLDCDPSGSNSGDTSIDLFGLNNYEWCGNATASVYATKNGDFAGYNVAAYFSEFGCITSPPRLWTEVQALFSSPMTDIWSGGLAFSYFPATSSQGQFGIVNISSDGSTVTTGDDFSRLKTQYSEVSPPNSPTLSSAGPNTFSACPQENSTFLASSTLPPTPNDAACSCLESQLSCQFTPKTTNTSGIVGTLLDTACSLLGGTGGSCAAISANGTTGTYGSVAFCDPDVKLSFVMSEYYEATGRLATSCDFSGNATVNSGAPSSGATAAVTSCLANPSATFTPSAPTVTPGGSSTGTSSAGSSGSSNNKNGAVGLLGNPQALVGLASRASSASWAVC*

Seq.id No. 3: grifola frondosa beta-1, 3-glucanotransferase gene conserved sequence

tggcctcgccttcagctatttccctgcgaccagcagccagggccagttcggcattgtcaacatttcctctgacggatccacggtgacgacgggcgatgacttcagccgtctcaagacgcagtacagcgaggtctcgccgccgaacagccctaccctgagcagcgcaggtccgaatacgttctcggcgtgcccccaggagaactcgacattcctcgcgtcctcgacgcttcccccaacgcccaacgacgcggcatgtagctgcctggagagccagctgagctgccagttcacgcccaagaccacgaacaccagtggtatcgtcggcacgctcctcgataccgcgtgctcgttgcttggcggcaccggcggctcctgcgccgccatctccgccaacgggaccaccggaacgtacgggagcgttgcgttctgcgaccctgacgtgaagctctcgttcgtgatgagcgagtattatgaggctacgggccgcctggcgacgtcgtgcgacttcagcggt

Seq.id No. 4: grifola frondosa GPD promoter

tcgggatccccgtcgcattacacacattgttcgaacatgtacagagagtttgacagacaaaacagtaacacttgtttcgagagagacgctcgtagctaaacccgatgtccgaagaggaccctccccacgcgagtccgcaagatgaatgcgacagttggttgccacgagcacagagagcggcaaataccctcgagtcatcgtacggagcttgtcaatccagcaaatgtacacgagcatggcaggatccaatcgtaattaggtggctgagatctgaccaccgagaatgtgcgccctaggggatgagtaaacgcacgtttgcgcgtgaatcagcgatgatgctgtacggtggtgcttagagatacgaaaagttgcaagtgaacgtaaatggaggaaagggactggttgggaatattcatgacaagctggctagaacaagtcggaaatctagtctgaggcaaagccaccagcggagagccgttcgcggccttgcggtgacagtcgggcaacggccggaagctgcccggtgtaatcatccatcttagataacgatcaccaccccaccctataagacccctctccatctctgctcttctccccatccttcgtctccaaaaccattatcctcagcaatgccagtgagtcctgcagacaatctgcatcgtcttcgagcatccgtctcacccgtggttttcacaggtcaaggtcggaatcaacgggtgcgtcggctgtgggtgtgtgaacgttcagactgattaataccgtttctcgtgtcgccctactccagcttcggtaagaacttgcatatttgctggcttcgccgtgctcacggcagtgtgtaggtcgcattggccgtattgtgctccgtaatgctctcctcaaccccgaaatcgaggtcgtcgctgtgaacgagtgcgtattagttgatccccacccaatctccactgacgtcatttgtacccaccagta

Seq.id No. 5: aspergillus nidulans 35s promoter

gatttcggcacggctacggaagacggagaagccaccttcagtggactcgagtaccatttaattctatttgtgtttgatcgagacctaatacagcccctacaacgaccatcaaagtcgtatagctaccagtgaggaagtggactcaaatcgacttcagcaacatctcctggataaactttaagcctaaactatacagaataagataggtggagagcttataccgagctcccaaatctgtccagatcatggttgaccggtgcctggatcttcctatagaatcatccttattcgttgacctagctgattctggagtgacccagagggtcatgacttgagcctaaaatccgccgcctccaccatttgtagaaaaatgtgacgaactcgtgagctctgtacagtgaccggtgactctttctggcatgcggagagacggacggacgcagagagaagggctgagtaataagccactggccagacagctctggcggctctgaggtgcagtggatgattattaatccgggaccggccgcccctccgccccgaagtggaaaggctggtgtgcccctcgttgaccaagaatctattgcatcatcggagaatatggagcttcatcgaatcaccggcagtaagcgaaggagaatgtgaagccaggggtgtatagccgtcggcgaaatagcatgccattaacctaggtacagaagtccaattgcttccgatctggtaaaagattcacgagatagtaccttctccgaagtaggtagagcgagtacccggcgcgtaagctccctaattggcccatccggcatctgtagggcgtccaaatatcgtgcctctcctgctttgcccggtgtatgaaaccggaaaggccgctcaggagctggccagcggcgcagaccgggaacacaagctggcagtcgacccatccggtgctctgcactcgacctgctgaggtccctcagtccctggtaggcagcttt

SEQ ID NO. 6: grifola frondosa beta-1, 3-glucanotransferase gene cDNA sequence

atgtacaaccttccgcggttagcgaccgtgattgctggcgtcgccgccttcgcaagtggtgtccatgccatccagaatgttacaagaacgggcaggtacctctatactgctgatggaaacaggttcttcatcaagggtattgcataccaagaacaaggcgccatctccacggacccaaacaatccattcttggagccctcaaatttcaccgaccccctcattgacggagcagcgtgttcgcgcgatcttcctttcctccagcagctcggggtcaacgccatccgtgtctacagtgtgaattcttcgcttaaccacgatgcctgcatgagcaccttcagcaatgcgggcatctataccattattgaccttgcactccccgtgaatgggtcgatcgaccgtgcttccccggcttggacgaccaatctcttggacctttacctcaatacgatcaacgtcttcaacaagtacgacaatgttttggcgtacaatgtaggtaacgaggttgtcatcgaccccactggcactggcgctgcagccttcatcaaggctgccgctcgcgacgtcaaatcatacctcaaatccatttcatcgtctgcacttgttggctatgctgccattgatggagactccacctgggtggaccctctcgccaattacctcgactgtgatccctcgggctccaattccggagatacctccattgacctcttcggcctcaataactacgagtggtgcgggaatgccaccgcaagcgtctatgccaccaagaacggtgacttcgcaggctacaacgttgcggcgtacttcagcgagttcggttgcattacctcccccccacgcctctggaccgaggtccaggcgctgttcagcagccccatgaccgacatctggtcgggtggcctcgccttcagctatttccctgcgaccagcagccagggccagttcggcattgtcaacatttcctctgacggatccacggtgacgacgggcgatgacttcagccgtctcaagacgcagtacagcgaggtctcgccgccgaacagccctaccctgagcagcgcaggtccgaatacgttctcggcgtgcccccaggagaactcgacattcctcgcgtcctcgacgcttcccccaacgcccaacgacgcggcatgtagctgcctggagagccagctgagctgccagttcacgcccaagaccacgaacaccagtggtatcgtcggcacgctcctcgataccgcgtgctcgttgcttggcggcaccggcggctcctgcgccgccatctccgccaacgggaccaccggaacgtacgggagcgttgcgttctgcgaccctgacgtgaagctctcgttcgtgatgagcgagtattatgaggctacgggccgcctggcgacgtcgtgcgacttcagcggtaacgcgacggtcaacagcggggcgccctcgtccggcgcgaccgctgctgtcacgtcgtgtctggcgaacccttctgcgacgttcacccccagtgccccgactgtcaccccaggcggctcatcaacaggaacgtccagcgctggatcgtctgggtccagcaataacaagaacggcgctgtcgggctgctgggcaacccgcaggcgttggtggggctggcgtcgcgtgcatcttcagcgtcttgggcggtgtgttga。

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.

Sequence listing

<120> Grifola frondosa glucosyltransferase gfgel4 and encoding gene and application thereof

<140> 202110521577.7

<141> 2021-07-08

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1945

<212> DNA

<213> Grifola frondosa (Grifola frondosa)

<400> 1

atgtacaacc ttccgcggtt agcgaccgtg attgctggcg tcgccgcctt cgcaagtggt 60

gtccatgcca tccagaatgt tacaagaacg ggcaggtacc tctatactgc tgatggaaac 120

aggttcttca tcaagggtat tgcataccaa gaacaaggtg tgctggtcat tcgtctcgta 180

tgccacctta taacgagcgg tttcaggcgc catctccacg gacccaaaca atccattctt 240

ggagccctca aatttcaccg accccctcat tgacggagca gcgtgttcgc gcgatcttcc 300

tttcctccag cagctcgggg tcaacgccat ccgtgtctac agtgtgaatt cttcgcttaa 360

ccacgatgcc tgcatgagca ccttcagcaa tgcgggcatc tataccatgt gcgaaattgt 420

ctccgtaatg tgttccacag aattctaaca aaactgcgat tttcgcagta ttgaccttgc 480

actccccgtg aatgggtcga tcgaccgtgc ttccccggct tggacgacca atctcttgga 540

cctttacctc aatacgatca acgtcttcaa caagtacgac aatgttttgg cgtacaatgt 600

aggtaacgag gttgtcatcg accccactgg cactggcgct gcagccttca tcaaggctgc 660

cgctcgcgac gtcaaatcat acctgtatgg ctcatgatta attacgttcc ttggcacgcg 720

ctgacgccgt ctcggcacta gcaaatccat ttcatcgtct gcacttgttg gctatgctgc 780

cattgatgga gactccacct gggtggaccc tctcgccaat tacctcgact gtgatccctc 840

gggctccaat tccggagata cctccattga cctcttcggc ctcaataact agtacgttga 900

ctgcgcattg ttctattctc gtgcttaccc acattctttt cccagcgagt ggtgcgggaa 960

tgccaccgca agcgtctatg ccaccaagaa cggtgacttc gcaggctaca acgttgcggc 1020

gtacttcagc gagttcggtt gcattacctc ccccccacgc ctctggaccg aggtccaggc 1080

gctgttcagc agccccatga ccgacatctg gtcgggtggc ctcgccttca gctatttccc 1140

tgcgaccagc agccagggcc agttcggcat tgtcaacatt tcctctgacg gatccacggt 1200

gacgacgggc gatgacttca gccgtctcaa gacgcagtac agcgaggtct cgccgccgaa 1260

cagccctacc ctgagcagcg caggtccgaa tacgttctcg gcgtgccccc aggagaactc 1320

gacattcctc gcgtcctcga cgcttccccc aacgcccaac gacgcggcat gtagctgcct 1380

ggagagccag ctgagctgcc agttcacgcc caagaccacg aacaccagtg gtatcgtcgg 1440

cacgctcctc gataccgcgt gctcgttgct tggcggcact ggcggctcct gcgccgccat 1500

ctccgccaac gggaccaccg gaacgtacgg gagcgttgcg ttctgcgacc ctggtgcgca 1560

tctcggcttc ttgttcttgc cttctattca gtgctgacct cccgatattg cagacgtgaa 1620

gctctcgttc gtgatgagcg agtattatga ggctacgggc cgcctggcga cgtcgtgcga 1680

cttcagcggt aacgcgacgg tcaacagcgg ggcgccctcg tccggcgcga ccgctgctgt 1740

cacgtcgtgt ctggcgaacc cttctgcgac gttcaccccc agtgccccga ctgtcacccc 1800

aggcggctca tcaacaggaa cgtccagcgc tggatcgtct gggtccagca ataacaagaa 1860

cggcgctgtc gggctgctgg gcaacccgca ggcgttggtg gggctggcgt cgcgtgcatc 1920

ttcagcgtct tgggcggtgt gttga 1945

<210> 2

<211> 554

<212> PRT

<213> Grifola frondosa (Grifola frondosa)

<400> 2

Met Tyr Asn Leu Pro Arg Leu Ala Thr Val Ile Ala Gly Val Ala Ala

1 5 10 15

Phe Ala Ser Gly Val His Ala Ile Gln Asn Val Thr Arg Thr Gly Arg

20 25 30

Tyr Leu Tyr Thr Ala Asp Gly Asn Arg Phe Phe Ile Lys Gly Ile Ala

35 40 45

Tyr Gln Glu Gln Gly Ala Ile Ser Thr Asp Pro Asn Asn Pro Phe Leu

50 55 60

Glu Pro Ser Asn Phe Thr Asp Pro Leu Ile Asp Gly Ala Ala Cys Ser

65 70 75 80

Arg Asp Leu Pro Phe Leu Gln Gln Leu Gly Val Asn Ala Ile Arg Val

85 90 95

Tyr Ser Val Asn Ser Ser Leu Asn His Asp Ala Cys Met Ser Thr Phe

100 105 110

Ser Asn Ala Gly Ile Tyr Thr Ile Ile Asp Leu Ala Leu Pro Val Asn

115 120 125

Gly Ser Ile Asp Arg Ala Ser Pro Ala Trp Thr Thr Asn Leu Leu Asp

130 135 140

Leu Tyr Leu Asn Thr Ile Asn Val Phe Asn Lys Tyr Asp Asn Val Leu

145 150 155 160

Ala Tyr Asn Val Gly Asn Glu Val Val Ile Asp Pro Thr Gly Thr Gly

165 170 175

Ala Ala Ala Phe Ile Lys Ala Ala Ala Arg Asp Val Lys Ser Tyr Leu

180 185 190

Lys Ser Ile Ser Ser Ser Ala Leu Val Gly Tyr Ala Ala Ile Asp Gly

195 200 205

Asp Ser Thr Trp Val Asp Pro Leu Ala Asn Tyr Leu Asp Cys Asp Pro

210 215 220

Ser Gly Ser Asn Ser Gly Asp Thr Ser Ile Asp Leu Phe Gly Leu Asn

225 230 235 240

Asn Tyr Glu Trp Cys Gly Asn Ala Thr Ala Ser Val Tyr Ala Thr Lys

245 250 255

Asn Gly Asp Phe Ala Gly Tyr Asn Val Ala Ala Tyr Phe Ser Glu Phe

260 265 270

Gly Cys Ile Thr Ser Pro Pro Arg Leu Trp Thr Glu Val Gln Ala Leu

275 280 285

Phe Ser Ser Pro Met Thr Asp Ile Trp Ser Gly Gly Leu Ala Phe Ser

290 295 300

Tyr Phe Pro Ala Thr Ser Ser Gln Gly Gln Phe Gly Ile Val Asn Ile

305 310 315 320

Ser Ser Asp Gly Ser Thr Val Thr Thr Gly Asp Asp Phe Ser Arg Leu

325 330 335

Lys Thr Gln Tyr Ser Glu Val Ser Pro Pro Asn Ser Pro Thr Leu Ser

340 345 350

Ser Ala Gly Pro Asn Thr Phe Ser Ala Cys Pro Gln Glu Asn Ser Thr

355 360 365

Phe Leu Ala Ser Ser Thr Leu Pro Pro Thr Pro Asn Asp Ala Ala Cys

370 375 380

Ser Cys Leu Glu Ser Gln Leu Ser Cys Gln Phe Thr Pro Lys Thr Thr

385 390 395 400

Asn Thr Ser Gly Ile Val Gly Thr Leu Leu Asp Thr Ala Cys Ser Leu

405 410 415

Leu Gly Gly Thr Gly Gly Ser Cys Ala Ala Ile Ser Ala Asn Gly Thr

420 425 430

Thr Gly Thr Tyr Gly Ser Val Ala Phe Cys Asp Pro Asp Val Lys Leu

435 440 445

Ser Phe Val Met Ser Glu Tyr Tyr Glu Ala Thr Gly Arg Leu Ala Thr

450 455 460

Ser Cys Asp Phe Ser Gly Asn Ala Thr Val Asn Ser Gly Ala Pro Ser

465 470 475 480

Ser Gly Ala Thr Ala Ala Val Thr Ser Cys Leu Ala Asn Pro Ser Ala

485 490 495

Thr Phe Thr Pro Ser Ala Pro Thr Val Thr Pro Gly Gly Ser Ser Thr

500 505 510

Gly Thr Ser Ser Ala Gly Ser Ser Gly Ser Ser Asn Asn Lys Asn Gly

515 520 525

Ala Val Gly Leu Leu Gly Asn Pro Gln Ala Leu Val Gly Leu Ala Ser

530 535 540

Arg Ala Ser Ser Ala Ser Trp Ala Val Cys

545 550

<210> 3

<211> 514

<212> DNA

<213> Grifola frondosa (Grifola frondosa)

<400> 3

tggcctcgcc ttcagctatt tccctgcgac cagcagccag ggccagttcg gcattgtcaa 60

catttcctct gacggatcca cggtgacgac gggcgatgac ttcagccgtc tcaagacgca 120

gtacagcgag gtctcgccgc cgaacagccc taccctgagc agcgcaggtc cgaatacgtt 180

ctcggcgtgc ccccaggaga actcgacatt cctcgcgtcc tcgacgcttc ccccaacgcc 240

caacgacgcg gcatgtagct gcctggagag ccagctgagc tgccagttca cgcccaagac 300

cacgaacacc agtggtatcg tcggcacgct cctcgatacc gcgtgctcgt tgcttggcgg 360

caccggcggc tcctgcgccg ccatctccgc caacgggacc accggaacgt acgggagcgt 420

tgcgttctgc gaccctgacg tgaagctctc gttcgtgatg agcgagtatt atgaggctac 480

gggccgcctg gcgacgtcgt gcgacttcag cggt 514

<210> 4

<211> 966

<212> DNA

<213> Grifola frondosa (Grifola frondosa)

<400> 4

tcgggatccc cgtcgcatta cacacattgt tcgaacatgt acagagagtt tgacagacaa 60

aacagtaaca cttgtttcga gagagacgct cgtagctaaa cccgatgtcc gaagaggacc 120

ctccccacgc gagtccgcaa gatgaatgcg acagttggtt gccacgagca cagagagcgg 180

caaataccct cgagtcatcg tacggagctt gtcaatccag caaatgtaca cgagcatggc 240

aggatccaat cgtaattagg tggctgagat ctgaccaccg agaatgtgcg ccctagggga 300

tgagtaaacg cacgtttgcg cgtgaatcag cgatgatgct gtacggtggt gcttagagat 360

acgaaaagtt gcaagtgaac gtaaatggag gaaagggact ggttgggaat attcatgaca 420

agctggctag aacaagtcgg aaatctagtc tgaggcaaag ccaccagcgg agagccgttc 480

gcggccttgc ggtgacagtc gggcaacggc cggaagctgc ccggtgtaat catccatctt 540

agataacgat caccacccca ccctataaga cccctctcca tctctgctct tctccccatc 600

cttcgtctcc aaaaccatta tcctcagcaa tgccagtgag tcctgcagac aatctgcatc 660

gtcttcgagc atccgtctca cccgtggttt tcacaggtca aggtcggaat caacgggtgc 720

gtcggctgtg ggtgtgtgaa cgttcagact gattaatacc gtttctcgtg tcgccctact 780

ccagcttcgg taagaacttg catatttgct ggcttcgccg tgctcacggc agtgtgtagg 840

tcgcattggc cgtattgtgc tccgtaatgc tctcctcaac cccgaaatcg aggtcgtcgc 900

tgtgaacgag tgcgtattag ttgatcccca cccaatctcc actgacgtca tttgtaccca 960

ccagta 966

<210> 5

<211> 977

<212> DNA

<213> Aspergillus nidulans (Aspergillus nidulans)

<400> 5

gatttcggca cggctacgga agacggagaa gccaccttca gtggactcga gtaccattta 60

attctatttg tgtttgatcg agacctaata cagcccctac aacgaccatc aaagtcgtat 120

agctaccagt gaggaagtgg actcaaatcg acttcagcaa catctcctgg ataaacttta 180

agcctaaact atacagaata agataggtgg agagcttata ccgagctccc aaatctgtcc 240

agatcatggt tgaccggtgc ctggatcttc ctatagaatc atccttattc gttgacctag 300

ctgattctgg agtgacccag agggtcatga cttgagccta aaatccgccg cctccaccat 360

ttgtagaaaa atgtgacgaa ctcgtgagct ctgtacagtg accggtgact ctttctggca 420

tgcggagaga cggacggacg cagagagaag ggctgagtaa taagccactg gccagacagc 480

tctggcggct ctgaggtgca gtggatgatt attaatccgg gaccggccgc ccctccgccc 540

cgaagtggaa aggctggtgt gcccctcgtt gaccaagaat ctattgcatc atcggagaat 600

atggagcttc atcgaatcac cggcagtaag cgaaggagaa tgtgaagcca ggggtgtata 660

gccgtcggcg aaatagcatg ccattaacct aggtacagaa gtccaattgc ttccgatctg 720

gtaaaagatt cacgagatag taccttctcc gaagtaggta gagcgagtac ccggcgcgta 780

agctccctaa ttggcccatc cggcatctgt agggcgtcca aatatcgtgc ctctcctgct 840

ttgcccggtg tatgaaaccg gaaaggccgc tcaggagctg gccagcggcg cagaccggga 900

acacaagctg gcagtcgacc catccggtgc tctgcactcg acctgctgag gtccctcagt 960

ccctggtagg cagcttt 977

<210> 6

<211> 1665

<212> DNA

<213> Grifola frondosa (Grifola frondosa)

<400> 6

atgtacaacc ttccgcggtt agcgaccgtg attgctggcg tcgccgcctt cgcaagtggt 60

gtccatgcca tccagaatgt tacaagaacg ggcaggtacc tctatactgc tgatggaaac 120

aggttcttca tcaagggtat tgcataccaa gaacaaggcg ccatctccac ggacccaaac 180

aatccattct tggagccctc aaatttcacc gaccccctca ttgacggagc agcgtgttcg 240

cgcgatcttc ctttcctcca gcagctcggg gtcaacgcca tccgtgtcta cagtgtgaat 300

tcttcgctta accacgatgc ctgcatgagc accttcagca atgcgggcat ctataccatt 360

attgaccttg cactccccgt gaatgggtcg atcgaccgtg cttccccggc ttggacgacc 420

aatctcttgg acctttacct caatacgatc aacgtcttca acaagtacga caatgttttg 480

gcgtacaatg taggtaacga ggttgtcatc gaccccactg gcactggcgc tgcagccttc 540

atcaaggctg ccgctcgcga cgtcaaatca tacctcaaat ccatttcatc gtctgcactt 600

gttggctatg ctgccattga tggagactcc acctgggtgg accctctcgc caattacctc 660

gactgtgatc cctcgggctc caattccgga gatacctcca ttgacctctt cggcctcaat 720

aactacgagt ggtgcgggaa tgccaccgca agcgtctatg ccaccaagaa cggtgacttc 780

gcaggctaca acgttgcggc gtacttcagc gagttcggtt gcattacctc ccccccacgc 840

ctctggaccg aggtccaggc gctgttcagc agccccatga ccgacatctg gtcgggtggc 900

ctcgccttca gctatttccc tgcgaccagc agccagggcc agttcggcat tgtcaacatt 960

tcctctgacg gatccacggt gacgacgggc gatgacttca gccgtctcaa gacgcagtac 1020

agcgaggtct cgccgccgaa cagccctacc ctgagcagcg caggtccgaa tacgttctcg 1080

gcgtgccccc aggagaactc gacattcctc gcgtcctcga cgcttccccc aacgcccaac 1140

gacgcggcat gtagctgcct ggagagccag ctgagctgcc agttcacgcc caagaccacg 1200

aacaccagtg gtatcgtcgg cacgctcctc gataccgcgt gctcgttgct tggcggcacc 1260

ggcggctcct gcgccgccat ctccgccaac gggaccaccg gaacgtacgg gagcgttgcg 1320

ttctgcgacc ctgacgtgaa gctctcgttc gtgatgagcg agtattatga ggctacgggc 1380

cgcctggcga cgtcgtgcga cttcagcggt aacgcgacgg tcaacagcgg ggcgccctcg 1440

tccggcgcga ccgctgctgt cacgtcgtgt ctggcgaacc cttctgcgac gttcaccccc 1500

agtgccccga ctgtcacccc aggcggctca tcaacaggaa cgtccagcgc tggatcgtct 1560

gggtccagca ataacaagaa cggcgctgtc gggctgctgg gcaacccgca ggcgttggtg 1620

gggctggcgt cgcgtgcatc ttcagcgtct tgggcggtgt gttga 1665

Claims (10)

1. The coding gene of the beta-1, 3-glucanotransferase of the grifola frondosa, which comprises a nucleotide sequence shown in SEQ ID NO. 1.

2. The coding gene of the beta-1, 3-glucosyltransferase of the grifola frondosa comprises a grifola frondosa glucosyltransferase gene conserved sequence shown in SEQ ID NO. 3.

3. A protein encoded by the encoding gene of any one of claims 1-2.

4. The recombinant expression vector of the nucleotide sequence shown in SEQ ID NO.1 according to claim 1.

5. The recombinant expression vector of the nucleotide sequence shown in SEQ ID NO.3 according to claim 2.

6. The recombinant expression vector according to claim 5, further comprising a Grifola frondosa gpd promoter sequence and an Aspergillus nidulans 35s promoter sequence, said Grifola frondosa gpd promoter sequence being represented by SEQ ID No.4, said Aspergillus nidulans 35s promoter sequence being represented by SEQ ID No. 5.

7. A recombinant engineered bacterium of the recombinant expression vector of claim 4 or 6.

8. A method for regulating the synthesis amount and structure of grifola frondosa glucan, which is realized by using the recombinant engineering bacterium of claim 7.

9. The method for modifying the amount of glucan synthesized by Grifola frondosa according to claim 8, wherein said method is used for producing glucose, starch hydrolysate or lignocellulose, and comprises the following steps:

(1) preparing a fermentation medium and a seed culture medium, wherein a carbon source of the fermentation medium and the seed culture medium is glucose, or starch hydrolysate or lignocellulose or two of the glucose, the starch hydrolysate and the lignocellulose, and the concentration of the carbon source is 5.0-100.0 g/L;

(2) culturing the recombinant engineering bacteria of claim 8, activating the seed solution of the engineering bacteria in the culture medium, and preparing the seed culture solution in a fermentation tank with corresponding scale by stage amplification;

(3) inoculating the engineering bacteria seed liquid into a shake flask or a fermentation tank containing a fermentation culture medium in an inoculation amount of 5.0% -15.0%.

10. The method of claim 8, wherein the method is methylation and assay analysis is performed by GC-MS.

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