Cordyceps militaris glyceraldehyde-3-phosphate dehydrogenase gene promoter

A technology of Cordyceps sinensis glycerin and phosphate dehydrogenase, applied in the direction of microorganism-based methods, DNA/RNA fragments, microorganisms, etc., to achieve high activity and change the effect of resource scarcity

Inactive Publication Date: 2012-06-13
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report related to the promoter of the Cordyceps militaris gene, so it is necessary to select the endogenous promoter of the Cordyceps militaris gene in order to achieve high-efficiency expression in the gene-specific vector

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Step 1, cloning of the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of Cordyceps militaris

[0016] 1. The cultivation of Cordyceps militaris

[0017] Put the Cordyceps militaris strain preserved on the slant of the test tube (Cordyceps militaris number: 51534, derived from the Culture Collection Center of the Chinese Academy of Agricultural Sciences) to 28°C for activation for 24 hours, pick the mycelium of the strain on the slant of the test tube and connect it to the comprehensive medium containing potato ( Potato Dextrose Agar, PDA) plate, 28 ° C constant temperature culture for 7 days. 1cm 2 The left and right strains grown on the PDA plate were inoculated into the PDA liquid medium, and cultured at 28° C. and 120 rpm for 5 days.

[0018] 2. Isolation of Cordyceps militaris DNA

[0019] Genomic DNA was extracted by high-salt and low-pH method, including the following steps:

[0020] (1) Put about 1 g of the sample ground by liquid nitrogen into t...

Embodiment 2

[0101] Step 1, PCR amplification of the missing promoter fragment

[0102] (1) Synthesis of primers

[0103] According to the full-length sequence of the Cordyceps militaris glyceraldehyde-3-phosphate dehydrogenase gene, a sequence (501-2515 nucleotides in SEQ ID NO.1) capable of amplifying a 2000 bp promoter region is designed. The primer sequence is: upstream primer: 5'-AGACGGCTTTGGGCGGCTGCTTGC-3'; downstream primer: 5'-TGTTCTTGATTAGAAAAGTGAGGTG-3';

[0104] (2) PCR amplification

[0105] Using the Cordyceps militaris genomic DNA as a template, using the primers described in step (1), after PCR amplification, the partial sequence (about 2000bp) of the 5' flank of the Cordyceps militaris glyceraldehyde-3-phosphate dehydrogenase gene was cloned into Intermediate vector (such as pMD 18-T vector), the sequencing result is compared with the 5' flanking sequence of the obtained 2515bp CmGDP1P, and the identity with the 501-2515th nucleotide in SEQ ID NO.1 is 100%, indicating tha...

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Abstract

The invention discloses a cordyceps militaris glyceraldehyde-3-phosphate dehydrogenase gene promoter, belonging to the technical field of gene engineering. The promoter is provided with a nucleotide sequence shown in the first bit to 2515th bit in SEQ ID NO.1 or a nucleotide sequence hybridized by the nucleotide sequence shown in the first bit to 2515th bit in SEQ ID NO.1 under a highly strict condition. The promoter of the invention can start a reporter gene, namely the gusA gene to have efficient expression in mycelium and sporocarp of fungi, and has higher activity especially in the mycelium and the sporocarp of the cordyceps militaris.

Description

technical field [0001] The invention relates to a gene promoter in the technical field of genetic engineering, in particular to a gene promoter of glyceraldehyde-3-phosphate dehydrogenase from Cordyceps militaris. Background technique [0002] Cordyceps is a large class of medicinal fungi, belonging to Eumycota, Ascomycotina, Pyenomycetes, Sphaerides, Clavicipiracese, Cordyceps The genus (Cordyceps). More than 350 species of this genus have been reported, and more than 30 species have been found in China. The types of Cordyceps fungi with medicinal value in China include: Cordyceps sineusis, referred to as Cordyceps; C.militaris, also known as Cordyceps militaris; C.hauntessi (C.hauntessi), also known as Hawks Cordyceps; Liangshan Cordyceps (C.liangshanensis Zhang, Liuet Hu); C. barnesii (Berk)); Cocoon grass; Gansu Cordyceps (C.gansueasis Zhang.Wang et Yan sp.nov); Xinyi Cordyceps (C.gracilis (Gray) Dur.Et Mont); Brazil Cordyceps (C.brasihexisis Henn); ); Cordyceps chine...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12R1/645
Inventor 周选围龚振华唐克轩
Owner SHANGHAI JIAOTONG UNIV
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