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A kind of glig promoter of ederia fs110 glutathione sulfur transferase gene and application thereof

A technology of FS110 and glutathione, applied in the field of genetic engineering, can solve the problem that the promoter has not yet been discovered

Inactive Publication Date: 2020-10-09
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the promoters of secondary metabolite biosynthesis genes in deep-sea fungi have not yet been discovered.

Method used

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  • A kind of glig promoter of ederia fs110 glutathione sulfur transferase gene and application thereof
  • A kind of glig promoter of ederia fs110 glutathione sulfur transferase gene and application thereof
  • A kind of glig promoter of ederia fs110 glutathione sulfur transferase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Acquisition of the promoter sequence of the gliotoxin biosynthetic gene GliG of Eydler's bacteria (Dichotomyces cejpii) FS110

[0026] Analysis of the expression level of gliotoxin biosynthetic genes: the deep-sea fungus Dichotomycescejpii (Dichotomycescejpii) FS110 was inoculated on a YPD medium plate, cultured at 37°C for 72 hours, fresh mycelium was picked, and RNA was extracted using a plant RNA extraction kit. -in-one RT Master Kit reverse transcribed to obtain cDNA, and used Hiseq2000 for transcriptome sequencing, and designed gliotoxin biosynthetic gene GliG, GliI, GliO primers based on the target gene sequence obtained by previous transcriptome sequencing. The primer sequence is: for GliG Gene (FP: 5′-atgaccgaacgaccttcttgatctcg-3′, RP: 5′-caatagtccatactccttctcgcc-3′), against GliI gene (FP: 5′-atgcctcacgcagaaacactcccc-3′, RP: 5′-ccacctcttatccaccccaatg-3′), against GliO Gene (FP: 5′-atggccaattctcgacccaacatcg-3′, RP: 5′-gttgaagttatccaggattgcg-3′), using...

Embodiment 2

[0030] Example 2: Functional verification of GliGP in the GliG promoter core region:

[0031] The forward and reverse primers of the 2μ replicon were designed for cloning, and the primers used were 2μori-U: 5’-GTTACCCCTGCAGGAACGAAGCATCTGTGCTTCATTT-3’, 2μori-D: 5’-GTTACCAAGCTTCATTGCGAATACCGCTTCCAC-3’. The 2μ replicon fragment amplified by PCR was double-digested with PstI and HindIII, and then inserted into the pAN7-1 vector, which was double-digested with PstI and HindIII. The positive clones were screened by bacterial liquid PCR and sequenced for verification, thus successfully cloning the yeast 2μ The replicon is inserted into the pAN7-1 vector (plasmid pAN7-1 is a broad host vector pAN7-1 with hygromycin B resistance gene hph and fungal promoter gpdA, which is a known product in the prior art), specifically The insertion site was located before the terminator tTRPC terminator, resulting in a 2μ-pAN7-1 vector ( image 3 A), and then use the method of enzyme-cut ligation to ...

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Abstract

The invention discloses an edyuillia FS110 glutathione S-transferase gene GliG promoter and application thereof. The nucleotide sequence of the promoter is as shown in SEQ ID NO.1. The upstream promoter sequence of the gene GliG is obtained by a chromosome walking technology, the core area of the promoter is predicted, and the edyuillia FS110 glutathione S-transferase gene GliG promoter is obtained. The promoter provided by the invention can efficiently start the expression of a hygromycin resistance gene hph, and the starting efficiency is obviously higher than that of a pgpdA promoter, so that a molecular biology base is laid for increasing the yield of gliotoxin through transcriptional control and heterogeneous expression in later period.

Description

Technical field: [0001] The invention belongs to the field of genetic engineering, and in particular relates to a GliG promoter of an ederia FS110 glutathione sulfur transferase gene and an application thereof. Background technique: [0002] Dichotomyces cejpii FS110 is a fungus isolated from deep sea. A new type of gliotoxin with strong anti-tumor and blood pressure-lowering activity was isolated from it. A total of 15 gliotoxins were obtained from E. dermatella FS110, 8 of which were new gliotoxins. Gliotoxin has anti-tumor, anti-fungal, anti-viral and immunomodulatory activities, so it has good development prospects in the development of anti-tumor, anti-hepatic fibrosis, anti-viral and immunosuppressant drugs. [0003] As an essential element for transcriptional regulation of structural genes and functional genes, promoters can recruit transcription factors and RNA polymerases to initiate transcription accurately. In recent years, due to the rapid development of filame...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/81C12N15/80C12N1/19C12N1/15C12R1/865
CPCC12N9/13C12N15/80C12N15/81C12Y208/01
Inventor 叶伟黄自磊章卫民李赛妮朱牧孜许建林李浩华刘桃妹
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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