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Grifola frondosa UDP-glucose pyrophosphorylase and application thereof

A technology of glucose pyrolysis and phosphorylase, applied in the field of edible fungus genetics and genetic engineering

Active Publication Date: 2019-08-13
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the research on the synthesis pathway of Grifola frondosa polysaccharides (mycelial polysaccharides and exopolysaccharides), the roles and functions of key enzymes is still blank, and comprehensive and systematic research is still needed

Method used

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  • Grifola frondosa UDP-glucose pyrophosphorylase and application thereof
  • Grifola frondosa UDP-glucose pyrophosphorylase and application thereof
  • Grifola frondosa UDP-glucose pyrophosphorylase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the cloning of Grifola frondosa UDP-glucose pyrophosphorylase coding gene

[0028] Grifola frondosa GF02 (purchased from American type culture collection, ATCC ® 60301™) was collected by centrifugation to cultivate mycelium, quickly added to liquid nitrogen and ground into a fine powder, and the genome was extracted with a plant or fungal genome extraction kit.

[0029]Primers were designed according to the UGP sequence of UDP-glucose pyrophosphorylase gene (GenBank ID: A0H81_11820) and its upstream and downstream 500bp, respectively:

[0030] UGP-F (SEQ ID NO .7): 5'-ATCTCCCTTTCCTCTACCCATC-3',

[0031] UGP-R (SEQ ID NO.8): 5'-GATCAGAAGACAGGGTCCAACA-3'.

[0032] Using the published Grifola frondosa genome (GenBank assembly accession: GCA_001683735.1) as a template, the above primers were used to amplify the full length of the gene. The PCR reaction program: 94°C pre-denaturation for 3 min; 94°C denaturation for 20 s, annealing for 30 s , annealing temper...

Embodiment 2

[0034] Example 2: Construction of Grifola frondosa UDP-glucose pyrophosphorylase gene silencing vector pAN7-ugp-dual

[0035] According to the Grifola frondosa UDP-glucose pyrophosphorylase gene sequence obtained by cloning in Example 1, the upstream and downstream primers designed according to the homologous region are respectively:

[0036] EUGP-F (SEQ ID NO.13):

[0037] 5'-CGGGGTACCTCAACACTCACGAGGATACGCT-3',

[0038] EUGP-R (SEQ ID NO.14):

[0039] 5'-GCTCTAGAGCGCCCAAGTTGTCAGAGTT-3'.

[0040] The conserved sequence of the Grifola frondosa UDP-glucose pyrophosphorylase gene was obtained by PCR amplification using the Grifola frondosa cDNA obtained in Example 1 as a template.

[0041] Grifola frondosa gpd Gene sequence (GenBank: A0H81_05461), the designed GPD promoter primers are:

[0042] gpd-F (SEQ ID NO. 15): 5'-cgggatcccgttcgcattacacacattg-3';

[0043] gpd-R (SEQ ID NO. 16): 5'-ggggtaccactggtgggtacaaatgacg-3'.

[0044] The target gene was amplified by PCR using...

Embodiment 3

[0050] Example 3: Construction of UDP-glucose pyrophosphorylase gene overexpression vector pAN7-o-ugp

[0051] Similar to Example 2, according to the Grifola frondosa UDP-glucose pyrophosphorylase gene sequence cloned in Example 1, the upstream and downstream primers were designed according to the homologous region as follows:

[0052] UGP-F (SEQ ID NO.19):

[0053] 5'–CGGGATCCATCTCCCCTTTCCTCTACCCATC-3',

[0054] UGP-R (SEQ ID NO.20):

[0055] 5'-GCTCTAGAGATCAGAAGACAGGGTCCAACA-3'.

[0056] Using Grifola frondosa cDNA as a template to amplify the gene encoding UDP-glucose pyrophosphorylase from Grifola frondosa ugp .

[0057] According to the Aspergillus nidulans 35s promoter sequence SEQ ID NO.6 in the plasmid pAN7-1 (purchased from Hunan Fenghui Biotechnology Co., Ltd.), the primers for the 35s promoter were designed as follows:

[0058] 35s-F (SEQ ID NO.17): 5'-CCCAAGCTTGATTTCGGCACGGCTACG-3';

[0059] 35s-R (SEQ ID NO.18): 5'-GCTCTAGAAAAGCTGCCTACCAGGGACT-3.

[0060...

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Abstract

The invention belongs to the technical field of edible fungi hereditary and genetic engineering, and particularly relates to grifola frondosa UDP-glucose pyrophosphorylase and application thereof. Through the genetic engineering technology, the UDP-glucose pyrophosphorylase gene is overexpressed or silenced in grifola frondosa, the quantity of mycelia in grifola frondosa recombinant bacteria and the yield of mycelia polysaccharides and exopolysaccharides can be significantly increased or reduced separately, and the composition of monosaccharide in the mycelia polysaccharides and exopolysaccharides of the grifola frondosa is significantly affected. The grifola frondosa UDP-glucose pyrophosphorylase provides a research basis for mastering a controlling mechanism for synthesis / metabolism of grifola frondosa polysaccharide and efficiently fermenting and producing a stable-quality polysaccharide product.

Description

technical field [0001] The invention belongs to the technical field of edible mushroom genetics and genetic engineering, in particular to a grifola frondosa UDP-glucose pyrophosphorylase and application thereof. Background technique [0002] Microbial polysaccharides are a class of biologically active macromolecular compounds, mainly including bacterial polysaccharides and fungal polysaccharides, which play an important role in the growth of bacteria and can be used in health care products, cosmetics and other fields. At present, there have been many studies involving bacterial exopolysaccharide biosynthesis pathway and gene control. [0003] Edible and medicinal fungal polysaccharides are one of the active research fields at home and abroad. However, the synthesis process of edible and medicinal fungal polysaccharides is extremely complicated, and there are many substrates and enzymes involved in the synthesis. Therefore, in order to achieve efficient and stable synthesis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/80C12N1/15C12P19/08C12R1/645
CPCC12N9/1051C12N15/80C12P19/08C12Y204/01021
Inventor 崔凤杰陶庭磊昝新艺孙文敬吴喜红
Owner JIANGSU UNIV
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