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Deep sea fungus FS140 oxidoreductase gene GliT promoter and application thereof

A promoter and gene technology, applied in the field of genetic engineering

Active Publication Date: 2021-04-30
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no research report on the GliT promoter of the deep-sea fungus Geosmithia pallida FS140 oxidoreductase

Method used

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  • Deep sea fungus FS140 oxidoreductase gene GliT promoter and application thereof
  • Deep sea fungus FS140 oxidoreductase gene GliT promoter and application thereof
  • Deep sea fungus FS140 oxidoreductase gene GliT promoter and application thereof

Examples

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Embodiment 1

[0025] Example 1 Obtaining the GliT promoter sequence of the deep-sea fungus Geosmithia pallida FS140 oxidoreductase gene

[0026] GliT promoter GliT 0Amplification: Inoculate the deep-sea fungus Geosmithia pallida FS140 on a YPD medium plate, culture at 28°C for 72 hours, pick fresh mycelia, and use the fungal DNA extraction kit to extract genome DNA. Genome walking kit was used to amplify the upstream sequence of the deep-sea fungal FS140 oxidoreductase gene GliT gene (GliT gene sequence is shown in SEQ ID NO.2) using TAIL-PCR technology, and the nucleotides of the upstream sequence were obtained by TA cloning and sequencing Acid sequence, using promoter analysis software to obtain the core region of the promoter GliT 0 and its key core areas. Design the specific reverse primers sp1, sp2 and sp3 in the GliT gene sequence (Table 1), and use the general forward primer AP3 of the Genome walking g kit for nested PCR amplification, and perform nested PCR reactions on FS140 thre...

Embodiment 2

[0029] Example 2 GliT promoter core region GliT 0 Functional verification of eukaryotic expression vectors

[0030] First, the hygromycin resistance gene hygromycin-B (GenBank Accession: XM_003071606) was inserted between the Xba I and Sal I restriction sites of the yeast vector YEp352-TEF1-CYC1 (YEp352-TEF1-CYC1 For the early construction of plasmids, carrying constitutive promoter TEF1 and terminator CYC1, which are 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 Saccharomycescerevisiae to produce(+)-valencene and its related sesquiterpenes, RSC Adv., 2019, 9, 30171, DOI: 10.1039 / c9ra05558d), constructing the positive control plasmid YEp352-TEF1-HYRB.

[0031] Secondly, the GliT promoter core region GliT 0 (The nucleotide sequence thereof is shown in SEQ ID NO.1), which is inserted into the yeast vector YEp352-TEF1-HYRB by replacing the element TEF1. First...

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Abstract

The invention discloses a deep sea fungus FS140 oxidordeuctase gene GliT promoter and an application thereof. The nucleotide sequence of the promoter is as shown in SEQ ID NO. 1. An upstream promoter sequence of a GliT gene is obtained through a chromosome walking technology, a promoter core region is predicted, a core region GliT0 of a deep sea fungus FS140 oxidordeuctase gene GliT promoter is obtained, and a key core region of the GliT promoter in a prokaryotic and eukaryotic expression system is successfully verified through point mutation. The promoter disclosed by the invention not only can efficiently promote the expression of hygromycin resistance gene hph, but also has the promotion efficiency similar to that of a constitutive promoter TEF1; the expression of the ampicillin Amp can be effectively promoted, so that a molecular biological foundation is laid for increasing the yield of the gliotoxin and obtaining more novel gliotoxin with high activity through transcription regulation and heterologous expression in the later period.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a deep-sea fungus Geosmithia pallida FS140 oxidoreductase gene GliT promoter and application thereof. Background technique [0002] Gliotoxin (GT) is an important hydrophobic fungal metabolite, which belongs to Epidithiodiketopiperazines (ETPs). ETPs are a class of active secondary metabolites mainly produced by fungi, whose structure is characterized by diketopiperazine (DKP) cores containing disulfide bonds, and have a wide range of biological activities, including antiproliferative, cytotoxic, immune Inhibitory, antiviral and antibacterial biological activities. The deep-sea fungus Geosmithia pallida FS140 is a fungus isolated from the sediments of the South China Sea. Twelve gliotoxins and their derivatives were isolated from its fermentation broth, including gliotoxin dimer derivatives with rare structures. Tests have shown that some compounds have strong antitumor activ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/81C12N15/70C12N15/80C12N1/21C12R1/865C12R1/19C12R1/645
CPCC12N9/0004C12N15/81C12N15/70C12N15/80Y02A50/30
Inventor 李赛妮章卫民
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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