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Myrothecium roridum A553 trichothecene synthase gene Tri5 promoter and application thereof

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

Active Publication Date: 2019-05-10
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 of plant endophytic fungi have not yet been discovered.

Method used

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  • Myrothecium roridum A553 trichothecene synthase gene Tri5 promoter and application thereof
  • Myrothecium roridum A553 trichothecene synthase gene Tri5 promoter and application thereof
  • Myrothecium roridum A553 trichothecene synthase gene Tri5 promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Obtaining the Trichothecene Synthase Gene Tri5 Promoter Sequence of Myrothecium roridum A553

[0026]Analysis of the expression level of trichothecene toxin biosynthetic genes: the endophytic fungus Myrothecium roridum A553 was inoculated on a YPD medium plate, cultured at 37°C for 72 hours, fresh mycelium was picked, and used Plant RNA Extraction Kit was used to extract RNA, and All-in-one RT Master Kit was used to reverse transcribe to obtain cDNA. Hiseq2000 was used for transcriptome sequencing, and primers for trichothecene biosynthesis-related genes Tri3, Tri4, Tri5, Tri6, Tri11, and Tri12 were designed based on the target gene sequence obtained by previous transcriptome sequencing. The primer sequence was: for the Tri3 gene (FP : 5′-ATGCTACTACAGGCAAATTTCAT-3′, RP: 5′-TCCGATATACGTCGCGAATCTAGC-3′), against the Tri4 gene (FP: 5′-ATGGATGCTCCGAAATCCAATG-3′, RP: 5′-GCCGATATATCTC GCATACCG-3′), against the Tri5 gene ( FP: 5′-ATGAGAGGACCAGACAGGAAGATCA-3′, RP: 5′...

Embodiment 2

[0031] Example 2 Functional verification of Tri5P in the core region of the Tri5 promoter

[0032] Insert Tri5P, the core region of the Tri5 promoter, into the yeast vector YEp352-TEF1-HYRB by homologous recombination (YEp352-TEF1-HYRB is an early constructed plasmid, carrying the hygromycin B resistance gene hph driven by the constitutive promoter TEF1 , is a known product in the prior art, and the vector map of YEp352-TEF1-HYRB is shown in image 3 A). Firstly, the upstream and downstream primers Tri5P-f0 (5'-AGCTCGGTACCCGGGGATCCCAATGTTCGCACTCTTAGTCAAG-3') and Tri5P-R (5'-GGTGAGTTCAGGCATCAGGGCTCAACTCAAGATCAAAGT-3') for the amplification of Tri5P (SEQ ID NO.1) in the core region of the Tri5 promoter were designed. Amplify the product. The vector YEp352-TEF1-HYRB was excised with BamH I and Xba I to excise TEF1 and recover the product, then the two products were recombined using ClonExpress II One Step Cloning Kit C112 (Vazyme) and transformed into DH5α to screen for positiv...

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Abstract

The invention discloses a myrothecium roridum A553 trichothecene synthase gene Tri5 promoter and application thereof. The nucleotide sequence of a promoter is shown in SEQ ID NO.1. According to the promoter, through a chromosome walking technology, the upstream promoter sequence of the Tri5 gene is obtained, the core region of the promoter is predicted, and the myrothecium roridum A553 trichothecene synthase gene Tri5 promoter Tri5P is obtained. By means of the promoter, expression of a hygromycin resistance gene can be efficiently started, the starting efficiency is close to that of a constitutive promoter TEF1, and a molecular biology basis is laid for increasing the yield of trichothecene toxin and obtaining more novel trichothecene toxin with high activities through transcriptional regulation and heterologous expression in the later period.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a Trichothecene synthase gene Tri5 promoter of Myrothecium dewlins A553 and application thereof. Background technique [0002] Myrothecium roridum (Myrothecium roridum) A553 is a fungus isolated from the plant Patchouli, from which a new compound trichothecene with strong anti-tumor activity was isolated. A total of 3 trichothecenes were obtained from Myrothecium dewlina A553, and one of them was a new type of trichothecene. Trichothecenes have anti-tumor, anti-fungal, anti-malarial and phytotoxic activities, so they have good research 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 factors and RNA polymerase to initiate transcription accurately. In recent years, due to the rapid developm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/81C12N1/19C12R1/865
Inventor 朱牧孜章卫民叶伟李赛妮岑由飞黄自磊李浩华刘洪新刘威振
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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