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Promoter of trichothecene synthase gene Tri12 of Leucosphaeria dothidea A553 and application of promoter

A technology of exposed lacquer spot bacteria and promoter, applied in the field of genetic engineering, can solve problems such as few reports on research

Active Publication Date: 2020-04-17
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, there are few reports on the promoters of endophytic fungal secondary metabolite biosynthesis genes.

Method used

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  • Promoter of trichothecene synthase gene Tri12 of Leucosphaeria dothidea A553 and application of promoter
  • Promoter of trichothecene synthase gene Tri12 of Leucosphaeria dothidea A553 and application of promoter
  • Promoter of trichothecene synthase gene Tri12 of Leucosphaeria dothidea A553 and application of promoter

Examples

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Embodiment 1

[0024] Example 1 Obtaining the Trichothecene Synthase Gene Tri12 Promoter Sequence of Myrothecium roridum A553

[0025] Amplification of the Tri12 promoter Tri12P: Inoculate the plant endophytic fungus Myrothecium roridum A553 on a YPD medium plate, culture at 37°C for 72 hours, pick fresh mycelium, and use the fungal DNA extraction kit Extract genome DNA. Genome walking kit was used to amplify the upstream sequence of Trichothecene synthase gene Tri12 gene (Tri12 gene sequence is shown in SEQ ID NO.2) of Myrotheca dewyere A553 by TAIL-PCR technology, and cloned by TA The nucleotide sequence of the upstream sequence was obtained by sequencing, and the core region Tri12P of the promoter was obtained by using the promoter analysis software. Design the specific reverse primers sp1, sp2 and sp3 in the Tri12 gene sequence (Table 1), and use the general forward primer AP3 of the Genomewalking kit to carry out nested PCR amplification. Amplify, obtain corresponding amplified produc...

Embodiment 2

[0028] Example 2 Functional verification of the Tri12 promoter core region Tri12P

[0029] 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 Saccharomyces cerevisiaeto produce(+)-valencene and its related sesquiterpenes, RSC Adv., 2019, 9, 30171, DOI: 10.1039 / c9ra05558d), construction of positive control plasmid YEp352-TEF1-HYRB (YEp352-TEF -HYRB vector map see figure 2 A).

[0030] Secondly, the Tri12 promoter core region Tri12P (its nucleotide sequence is shown in SEQ ID NO.1) was inserted into the yeast vector YEp352-TEF1-HYRB by replacing the element TEF1 ...

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Abstract

The invention discloses a promoter of a trichothecene synthase gene Tri12 of Leucosphaeria dothidea A553 and the application of the promoter. The nucleotide sequence of the promoter is as shown in SEQID NO. 1. According to the invention, an upstream promoter sequence of the Tri12 gene is obtained through a chromosome walking technology, and a promoter core region is predicted so as to obtain thepromoter Tri12P of the trichothecene synthase gene Tri12 of the Leuconostoc dunalianum A553. The promoter can efficiently start expression of the hygromycin resistance gene hph, and the starting efficiency is similar to the starting efficiency of a constitutive promoter TEF1, so a molecular biological foundation is laid for increasing the yield of trichothecene toxin and obtaining more high-activity novel trichothecene toxin through transcription regulation and heterologous expression in a later period.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a Tri12 promoter of the Trichothecene synthase gene of Myrotheca dewlins A553 and an application thereof. Background technique [0002] Macrocyclic trichothecenes are a class of macrolide antibiotics with biological activities such as antitumor, antifungal, antimalarial and phytotoxicity. Myrothecium roridum A553 is an endophytic fungus isolated from Patchouli. Three macrocyclic trichothecenes were isolated from its fermentation broth, one of which was a new macrocyclic trichothecene Cyclotrichothecenes, activity tests show that they all have strong anti-tumor activity. Therefore, macrocyclic trichothecenes have good research and development prospects in the development of anti-tumor, anti-fungal infection and anti-malarial drugs. [0003] As an essential element for transcriptional regulation of structural and functional genes, promoters can recruit transcription...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/80C12N15/81C12N1/15C12N1/19C12R1/645C12R1/865
CPCC12N9/00C12N15/81C12N15/80
Inventor 朱牧孜岑由飞章卫民李赛妮叶伟刘洪新李浩华
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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