Identification and application of synthesis gene cluster of cordycepin

A cordycepin and gene cluster technology, applied in the field of genes, can solve the problem of unclear synthesis mechanism of cordycepin

Active Publication Date: 2016-03-30
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, the synthesis mechanism of cordycepin in fungi has been unclear, which

Method used

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  • Identification and application of synthesis gene cluster of cordycepin
  • Identification and application of synthesis gene cluster of cordycepin
  • Identification and application of synthesis gene cluster of cordycepin

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Experimental program
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preparation example Construction

[0089] (1) Preparation of Cordyceps militaris conidia suspension: select fresh Cordyceps militaris cultured on a PDA (PotatoDextroseAgar, Difco) plate under light for a week, scrape an appropriate amount of colonies to 1ml sterile 0.05% Tween-20 aqueous solution, and vortex to isolate the bacteria Filament and conidia; remove mycelia by filtering with glass wool or three-layer lens-cleaning paper, and collect the filtrate; centrifuge the bacterial liquid at 12,000rpm for 1min to collect spores; discard the supernatant, and resuspend the spores with 1ml sterile water to remove residual nutrients ; 12,000rpm, 1min, centrifuge again to collect spores; repeat steps 4 to 5 once to completely remove residual nutrients on the surface of spores; Liquid concentration adjusted to 5×10 5 Spores / ml, used as a working solution for subsequent experimental operations. (2) Transformation of Cordyceps militaris: transfer the frozen AGL-1 bacteria that have been successfully transformed into t...

Embodiment 1

[0197] Example 1. Acquisition of potential gene clusters for cordycepin biosynthesis

[0198] Through the comparative genome analysis of Cordyceps militaris (CM01 strain) and the model fungus Aspergillus nidulans (Aspergillus nidulans, A4 strain), the inventors have found the potential gene cluster (gene cluster) of cordycepin biosynthesis, which are respectively Cm1 (GenBank accession number : CCM_04436), Cm2 (CCM_04437), Cm3 (CCM_04438) and Cm4 (CCM_04439) ( figure 1 On A), the corresponding homologous genes in Aspergillus nidulans are An1 (AN3333), An2 (AN3331), An3 (AN3330) and An4 (AN3329) ( figure 1 A below).

[0199] Using the dotplot method to compare the sequences of the cordycepin synthesis gene clusters of these two species, it was found that the coding regions of each gene showed good collinearity ( figure 1 B). Structural and functional domain analysis showed that three genes in this gene cluster encode related metabolic enzymes, namely, Cm1 encodes oxidordeuct...

Embodiment 2

[0203] Embodiment 2, Cordyceps militaris wild strain CM01 and the comparison of cpABC knockout mutant producing cordycepin

[0204] For the potential gene clusters obtained from the above analysis, the inventors firstly knocked out three enzyme genes cpA, cpB and cpC in the genome of the wild strain CMO1 of Cordyceps militaris.

[0205] 1. Construction of gene knockout vector

[0206] The construction of Cordyceps militaris gene deletion mutants relies on the gene replacement method based on homologous recombination and the fungal transformation technology mediated by Agrobacterium tumefaciens AGL-1. With the binary vector pDHt-Bar (DuanZ, ChenY, HuangW, ShangY, ChenP, WangC. (2013) Linkageofautophagytofungaldevelopment, lipidstorageandvirulenceinMetarhiziumrobertsii.Autophagy9 (4): 538-549) (containing glufosinate resistance gene (Bar), for The upstream homology arm-Bar-downstream homology arm structure is formed, and the corresponding segment gene in the genome is knocked o...

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Abstract

The invention relates to identification and application of a synthesis gene cluster of cordycepin that is a metabolite of cordyceps militaris (L.) link. The gene cluster of cordycepin biosynthesis is found for the first time. The gene cluster comprises Cm1 and Cm2 and/or Cm3, and corresponding homologous genes in aspergillus nidulans are An1, An2 and/or An3 respectively, and therefore a method of achieving efficient cordycepin biosynthesis is disclosed.

Description

technical field [0001] The invention relates to the field of gene technology, more specifically, the invention relates to the identification and application of a synthetic gene cluster of a metabolite of Cordyceps militaris - cordycepin. Background technique [0002] Cordycepin, also known as cordycepin, cordycepin, chemical name 3'-deoxyadenosine (3'-deoxyadenosine), is the first nucleoside antibiotic isolated from fungi. The fungi that can produce cordycepin include only a few Cordyceps genus such as Cordyceps militaris, Cordyceps Kyushu, and Aspergillus nidulans. Cordycepin has a good antibacterial effect, and it can improve the aging and immune function of the body caused by oxidative stress. It plays an important role in enhancing the regulation of adipocyte differentiation, preventing obesity, inhibiting inflammation, producing interleukin-10 in human peripheral blood mononuclear cells, and anti-leukemia. It is one of the important evaluation indicators for the quality...

Claims

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Application Information

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IPC IPC(8): C12P19/40C12N15/31C12N15/63
CPCC12N15/63C12P19/40
Inventor 王成树夏永亮商艳芳
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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