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Grifola frondosa glucan synthase, and encoding gene and application thereof

A technology of glucan synthase and coding gene, which is applied in the field of edible fungus genetics and genetic engineering, can solve the problems of no research results at the molecular level, and achieve obvious industrial production, slow growth and fast growth of bacteria

Inactive Publication Date: 2019-04-12
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no molecular level research results have been retrieved on the enzymes and synthesis mechanisms of the polysaccharide synthesis pathway of Grifola frondosa

Method used

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  • Grifola frondosa glucan synthase, and encoding gene and application thereof
  • Grifola frondosa glucan synthase, and encoding gene and application thereof
  • Grifola frondosa glucan synthase, and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the clone of Grifola frondosa glucan synthase coding gene

[0037] Grifola frondosa GF02 (purchased from American type culture collection, ATCC ® 60301™) was collected by centrifugation to cultivate mycelia, quickly added to liquid nitrogen and ground into a fine powder, and the genome was extracted by a plant or fungal genome extraction kit .

[0038] According to the coding gene of Grifola frondosa extracellular glucan synthase subunit fks-1-0 (GenBank ID: MK256943) 、 fks-1-1 (GenBank ID: MK256944) , fks-1-2 (GenBank ID: MK256945) and fks-1-3 (GenBankID: MK256946) designed primers, respectively:

[0039] fks 1-0-F (SEQ ID NO.13): 5'-acagaagggtctgcaccttaa-3',

[0040] fks 1-0-R (SEQ ID NO.14): 5'-gtatatgtcgtggagatctatagg-3';

[0041] fks 1-1-F (SEQ ID NO.15): 5'-ccgtaagagaaacgcacataga-3',

[0042] fks 1-1-R (SEQ ID NO.16): 5'-tgcagagcacaaataacacataac-3';

[0043] fks 1-2-F (SEQ ID NO .17): 5'-ccctcccacttagacccaac-3',

[0044] fks 1-2-R (SEQ ...

Embodiment 2

[0049] Example 2: Construction of the gene silencing vector pAN7- of the gene sequence of Grifola frondosa glucan synthase fks -1-0

[0050] According to the published full gene sequence of Grifola frondosa (GenBank assembly accession: GCA_001683735.1), two Dicer-Like amino acid sequences (GenBank: OBZ75102.1 and OBZ68881.1) were found in the Grifola frondosa genome, which were further conserved by NCBI Functional domain prediction analysis Dicer-Like protein 2, the results are as follows figure 2 , Grifola frondosa Dicer-Like protein contains three functional domains: HELICc, Dicer_dimer, and RIBOc, encoding 1438 amino acids, and has more than 95% homology with other species, indicating that there are typical RNAi key enzymes in Grifola frondosa. The possibility of gene silencing exists.

[0051] According to the gene sequence ( fks -1-0, fks -1-1, fks -1-2 and fks -1-3), design upstream and downstream primers according to the homologous region:

[0052] fks-F (S...

Embodiment 3

[0064] Example 3: Preparation of Grifola frondosa protoplasts by incomplete enzymolysis

[0065] Collect the mycelia obtained from the PDA liquid culture of Grifola frondosa, treat it with a tissue grinder under sterile conditions for 1 min, inoculate 100 mL of PDB medium with a 10% inoculum amount, and culture at 28 ° C for 4 days, 5000 g Centrifuge for 15 min to collect insoluble matter; wash twice with 0.6M mannitol solution, add 1 mL of 2% filamentous fungal wall-breaking enzyme solution, and enzymatically hydrolyze at 30°C for 4 h. Dilute the enzymatic solution at 5000 g Centrifuge for 15 minutes to collect insoluble matter, and sterile filter to obtain enzymatic protoplasts; add the prepared protoplasts to 50mL regeneration CYM medium (glucose 20g peptone 2g yeast extract 2g magnesium sulfate heptahydrate 0.5g dipotassium hydrogen phosphate 1.0g dihydrogen phosphate Potassium 0.46g agar 20g, hygromycin 100μg / mL), poured into the plate, cultured at 28°C for regeneration....

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Abstract

The invention belongs to the technical fields of genetic breeding and genetic engineering of edible fungi, and specifically relates to grifola frondosa glucan synthase as well as an encoding gene andapplication thereof. The encoding gene of glucan synthesis key enzyme-glucan synthase is obtained for the first time from clones of grifola frondosa mycelium genome by the invention; key roles of thegene in grifola frondosa mycelium growth and glucan synthesis are demonstrated by gene silencing; and the glucan synthase is over-expressed in grifola frondosa by adopting an genetic engineering technology so as to have thallus growth of recombinant strains and synthesis yield of glucan significantly increased. The grifola frondosa glucan synthase as well as the encoding gene and the application thereof are helpful for understanding synthesis mechanism of edible and medicinal fungal polysaccharides at molecular level, developing metabolic engineering study on edible and medicinal fungal polysaccharide synthesis, and providing important technical support and reference for producing glucan with stable quality by performing high-efficiency fermentation.

Description

technical field [0001] The invention belongs to the technical field of edible mushroom genetics and genetic engineering, and specifically relates to a grifola frondosa glucan synthase, its coding gene and application. Background technique [0002] Edible and medicinal fungal polysaccharides are one of the most active research fields at home and abroad. They play a key role in many important biological processes such as cell recognition, intercellular material transport, immune regulation and anti-tumor. Among them, dextran is a polysaccharide formed by the polymerization of D-glucose through β-1,3 and / or β-1,6 glycosidic linkages, which has significant immune regulation, anti-tumor, anti-aging, anti-virus and Physiological functions such as lowering blood fat, so they are called "biological response regulators". [0003] However, the synthesis process of edible and medicinal fungal polysaccharides is extremely complex, with many substrates and enzymes involved in the synthe...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/80C12N1/15C12P19/08C12R1/645
CPCC12N9/1051C12N15/80C12P19/08C12Y204/01021
Inventor 崔凤杰朱鸿安陶庭磊孙文敬昝新艺
Owner JIANGSU UNIV
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