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 cheap culture medium, fast growth, and increased glucan synthesis Effect

Active Publication Date: 2019-08-13
JIANGSU UNIV
View PDF10 Cites 1 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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] Since the coding gene sequence of Grifola frondosa extracellular glucan synthase is relatively long and it is difficult to amplify the complete sequence fragment, this example is based on the published coding gene of Grifola frondosa extracellular glucan synthase (Genbank ID: MK808019 ) middle subunit sequence, divide the gene sequence into two segments (named GFGLS1 and GFGLS2) and design primers for amplification and splicing, respectively:

[0039] GFGLS1-F (SEQ ID NO .7): 5'-acagaagggtctgcaccttaa-3',

[0040] GFGLS1-R (SEQ ID NO.8): 5'-gtatatgtcgtggagatctatagg-3'; and

[0041] GFGLS2-F (SEQ ID NO .9):...

Embodiment 2

[0046] Example 2: Construction of the gene silencing vector pAN7- of the gene sequence of Grifola frondosa glucan synthase gfgls-dual

[0047] 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.

[0048] According to the gene sequence ( GFGLS ), design the upstream and downstream primers of overlap-PCR according to the homologous region, respectively:

[0049] GFGLS-F (SEQ I...

Embodiment 3

[0061] Example 3: Preparation of Grifola frondosa protoplasts by incomplete enzymolysis

[0062] Collect the mycelium obtained from the PDA liquid culture of Grifola frondosa, treat it with a tissue grinder under sterile conditions for 1 min, and inoculate 100 mL of PDB medium with a 10% inoculum amount, 28 o C after static culture 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, 30 o C enzymatic hydrolysis for 4h. 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), mix well, pour into the plate, 28 o C culture regene...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention belongs to the technical field of edible fungi hereditary and genetic engineering, and particularly relates to grifola frondosa glucan synthase and an encoding gene and application thereof. The glucan key synthetase-glucan synthetase encoding gene is cloned in grifola frondosa mycelium genome for the first time, and through gene silencing, it is proved that the gene plays a key rolein the grifola frondosa mycelium growth process and the glucan synthesis process; finally, through the genetic engineering technology, glucan synthetase is overexpressed in grifola frondosa, and the recombination strain cell growth and the glucan synthesis yield are significantly improved. The encoding gene is beneficial for understanding the edible and medical fungal polysaccharide synthesis mechanism at the molecular level, and by using the genetic engineering technology, a technical scheme and guidance are provided for modifying an edible and medical fungal polysaccharide synthesis route.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap