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Deep sea fungus FS140 anti-gliotoxin self-protection gene mfs-get and application thereof

A gliotoxin and gene protection technology, applied in the field of genetic engineering, can solve problems such as reducing immune activity, inducing apoptosis, and redox reaction imbalance

Active Publication Date: 2021-04-23
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

[0004] The toxicity of gliotoxin to the host mainly includes: inducing apoptosis; causing redox reaction imbalance; inhibiting proteasome activity, leading to the inhibition of NF-κB activity, reducing immune activity, etc.

Method used

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  • Deep sea fungus FS140 anti-gliotoxin self-protection gene mfs-get and application thereof
  • Deep sea fungus FS140 anti-gliotoxin self-protection gene mfs-get and application thereof
  • Deep sea fungus FS140 anti-gliotoxin self-protection gene mfs-get and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 Obtaining the self-protection gene sequence of the deep-sea fungus Geosmithia pallida FS140 against gliotoxin

[0026] Amplification of the gene mfs-get: Inoculate the deep-sea fungus Geosmithia pallida FS140 on a YPD medium plate, culture at 37°C for 72 hours, pick fresh mycelium, use the fungal RNA extraction kit to extract RNA, and then use All-in- cDNA was obtained by reverse transcription with one RT Master Kit. According to the results of transcriptome sequencing, the sequence encoding the anti-trichothecene self-protection gene mfs-get was predicted, and the upstream and downstream primers YEp352-mfs-get-F and YEp352-mfs-get-R were designed and amplified using the cDNA library as a template to obtain PCR product ( figure 1 ). The product was recovered and cloned by TA with the pEASY-T1 kit, transformed into Escherichia coli competent cells, spread on the ampicillin-resistant plate to screen out positive clones, and used universal primers M13-F (5′-GT...

Embodiment 2

[0027] Example 2 Functional verification of the anti-gliotoxin self-protection gene mfs-get

[0028] The gene mfs-get was inserted into the yeast vector YEp352-TEF1-CYC1 by homologous recombination (YEp352-TEF1-CYC1 is an early constructed plasmid, carrying a constitutive promoter TEF1 and a terminator CYC1, see the vector map image 3 A, known products in the prior art: Xiaodan Ouyang, Yaping Cha, Wen Li, Chaoyi Zhu, Muzi Zhu, Shuang Li, Min Zhuo, Shaobin Huang and Jianjun Li. Stepwise engineering of Saccharomyces cerevisiae to produce(+)-valencene and its related sequiterpenes, RSC Adv., 2019, 9, 30171, DOI: 10.1039 / c9ra05558d). First design the upstream and downstream primers YEp352-mfs-get-F and YEp352-mfs-get-R for gene mfs-get (SEQ ID NO.1) amplification, and its primer sequence is YEp352-mfs-get-F: 5' - AATCTAAGTCTAGA ATGAATAGGCTGATCAGAGGCCAG-3'; YEp352-mfs-get-R: 5'- ATGCGGCCCGTCGAC CTATGCCTGCCCATTGTCGG-3 (the underlined sequence is the fragment of the homology arm...

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PUM

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Abstract

The invention discloses a deep sea fungus FS140 anti-gliotoxin self-protection gene mfs-get and application thereof. The nucleotide sequence of the anti-gliotoxin self-protection gene mfs-get is as shown in SEQ ID NO.1. Therefore, an anti-gliotoxin self-protection gene mfs-get sequence is obtained from a cDNA library of deep sea fungi FS140, and is successfully introduced into S.cerevisiae BJ5464 for anti-toxic function verification, so that the molecular biology foundation is laid for improving the anti-gliotoxin capability of the saccharomyces cerevisiae, improving the heterologous expression level of the gliotoxin and obtaining novel gliotoxin in a later period.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a deep-sea fungus Geosmithia pallida FS140 anti-gliotoxin self-protection gene mfs-get and an application thereof. Background technique [0002] In the early stage, our research group isolated more than 20 gliotoxin compounds from the deep-sea fungus Geosmithia pallida FS140, including gliotoxin dimer compounds with rare structures, most of which have obvious antitumor activity . [0003] Gliotoxin (GT) is a kind of diketopiperazine (epipolythiodioxopiperazine, ETP), ETP as an important virulence factor, it produces specific toxicity to various cells through different pathways, in invasive aspergillosis play an important role in synergy. The toxic and side effects of ETP are mainly through the cross-linking of disulfide bonds and sulfhydryl groups with the target protein to inactivate the protein activity, and can generate toxic reactive oxygen species (reactive o...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/81C12N15/80C12N1/19C12N1/15C12R1/865C12R1/645
CPCC07K14/37C12N15/81C12N15/80
Inventor 李赛妮章卫民朱牧孜
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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