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A kind of trichothecene synthase gene tri5 promoter of Myromyces dewlina a553 and its application

A technology of trichothecenes and wet paint spot, which is applied in the field of genetic engineering and can solve the problem that the promoter has not yet been discovered.

Active Publication Date: 2022-06-24
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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  • A kind of trichothecene synthase gene tri5 promoter of Myromyces dewlina a553 and its application
  • A kind of trichothecene synthase gene tri5 promoter of Myromyces dewlina a553 and its application
  • A kind of trichothecene synthase gene tri5 promoter of Myromyces dewlina a553 and its application

Examples

Experimental program
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Embodiment 1

[0025] Example 1 Acquisition of Tri5 promoter sequence of trichothecene synthase gene of Myrothecium roridum A553

[0026]Analysis of the expression level of trichothecenes biosynthesis genes: The endophytic fungus Myrothecium roridum A553 was inoculated on a YPD medium plate, cultured at 37°C for 72h, and the fresh mycelia were picked and used RNA was extracted with a plant RNA extraction kit, and cDNA was obtained by reverse transcription using the All-in-one RT Master Kit. Hiseq2000 was used for transcriptome sequencing, and the target gene sequences obtained by previous transcriptome sequencing were used to design trichothecenes biosynthesis-related genes Tri3, Tri4, Tri5, Tri6, Tri11, Tri12 primers. The primer sequences are: for the Tri3 gene (FP : 5′-ATGCTACTACAGGCAAATTTCAT-3′, RP: 5′-TCCGATATACGTCGCGAATCTAGC-3′), for Tri4 gene (FP: 5′-ATGGATGCTCCGAAATCCAATG-3′, RP: 5′-GCCGATATATCTC GCATACCG-3′), for Tri5 gene ( FP: 5′-ATGAGAGGACCAGACAGGAAGATCA-3′, RP: 5′-CGAGAACCTCCTGA...

Embodiment 2

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

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

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Abstract

The invention discloses a Trichothecene synthase gene Tri5 promoter of Myrothecium roridum A553 and application thereof. The nucleotide sequence of the promoter is shown in SEQ ID NO.1. The invention obtains the upstream promoter sequence of the Tri5 gene through the chromosome walking technology and predicts the core region of the promoter, and obtains the Tri5P promoter Tri5P of the Trichothecene synthase gene of Myrotheca dewlins A553. The promoter of the present invention can efficiently promote the expression of the hygromycin resistance gene hph, and the promoter efficiency is similar to that of the constitutive promoter TEF1, so as to increase the production of trichothecenes and obtain More highly active new trichothecenes lay the foundation of molecular biology.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a Tri5 promoter of the trichothecenes synthase gene of M. var. A553 and its application. Background technique [0002] Myrothecium roridum A553 is a fungus isolated from the broad medicinal plant Patchouli, from which a new compound trichothecenes with strong antitumor activity was isolated. A total of 3 trichothecenes were obtained from the fungus A553, one of which was a novel trichothecenes. Trichothecenes have antitumor, antifungal, antimalarial and phytotoxic activities, so they have good research prospects in the development of antitumor, antifungal infection and antimalarial drugs. [0003] As an essential element in the transcriptional regulation of structural and functional genes, promoters can recruit transcription factors and RNA polymerases to precisely initiate transcription. In recent years, due to the rapid development of filamentous fungi in the di...

Claims

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Application Information

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Patent Type & Authority Patents(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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