Myrothecium roridum A553 trichothecene-resistant self-protection gene mfs1, and application thereof

A technology of trichothecenes and lacquer spot fungus, applied in the field of genetic engineering, can solve problems such as not

Active Publication Date: 2020-03-24
东岱(济南)智能技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The trichothecene biosynthetic gene cluster in Fusarium graminearum has been reported, and Tri12 and Tri101 genes in the gene cluster have been confirmed to be involved in host resistance to trichothecenes, but they are not the only ones mechanism

Method used

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  • Myrothecium roridum A553 trichothecene-resistant self-protection gene mfs1, and application thereof
  • Myrothecium roridum A553 trichothecene-resistant self-protection gene mfs1, and application thereof
  • Myrothecium roridum A553 trichothecene-resistant self-protection gene mfs1, and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1 Obtaining the mfs1 sequence of anti-trichothecene self-protection gene mfs1 of Myrothecium roridum A553

[0023] Amplification of gene mfs1: inoculate endophytic fungus Myrothecium roridum A553 on YPD medium plate, culture at 37°C for 72 hours, pick fresh mycelium, and use fungal RNA extraction kit to extract RNA , and then use the All-in-one RT Master Kit to reverse transcribe to obtain cDNA. According to the transcriptome sequencing results, the anti-trichothecene self-protection gene mfs1 sequence was predicted, and the upstream and downstream primers msf1-F: 5'-ATGTTTATCGCCGCCCGTGT-3' and mfs1-R: 5'-TTAATCAAGAGGCCCACCCGT-3' were designed to generate cDNA library For template amplification, the PCR product ( figure 1). The recovered product was purified and cloned by TA using the pEASY-T1 kit, transformed into Escherichia coli DH5α competent cells, spread on the ampicillin resistance plate to select positive clones, and used the universal primer M13-F (5...

Embodiment 2

[0024] Example 2 Functional verification of anti-trichothecene self-protection gene mfs1

[0025] The gene mfs1 was inserted into the yeast vector YEp352-TEF1-CYC1 by homologous recombination (YEp352-TEF1-CYC1 is an early constructed plasmid, which carries a constitutive promoter TEF1 and a terminator CYC1. For the vector map of YEp352-TEF1-CYC1, see figure 2 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 msf1-U:5'- for the amplification of gene mfs1 (SEQ ID NO.1) CAATCTAATCTAAGTCTAGA ATGTTTATCGCCGCCCGTGT-3';msf1-D:5'- TGCGGCCCGTCGAC TTAATCAAGAGGCCCACCCGT-3' (the underlined sequence is the fragment of the homology arm), using the cDNA library in Example 1 as a template, the ...

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Abstract

The invention discloses a myrothecium roridum A553 trichothecene-resistant self-protection gene mfs1 and an application thereof. The nucleotide sequence of the myrothecium roridum A553 trichothecene-resistant self-protection gene mfs1 is as shown in SEQ ID NO. 1. According to the invention, a transcriptome sequencing result is used to predict a gene sequence, a cDNA library is used as a template,and amplification is carried out to obtain the myrothecium roridum A553 trichothecene-resistant self-protection gene mfs1. The myrothecium roridum A553 trichothecene-resistant self-protection gene mfs1 can efficiently assist saccharomyces cerevisiae in resisting trichothecene toxins, and lay a molecular biological foundation for improving the trichothecene-resistant capacity of saccharomyces cerevisiae in the later period, improving the heterologous expression level of trichothecene toxins and obtaining novel trichothecene toxins.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an anti-trichothecene self-protection gene mfs1 of Myrothecium dewwet bacteria A553 and its application. Background technique [0002] Myrothecium roridum (Myrothecium roridum) A553 is a fungus isolated from the Cantonese medicinal plant Patchouli, and a new compound trichothecene was isolated from its fermentation broth, which has strong antitumor activity . 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] The toxicity of trichothecenes is mainly due to targeting Rpl3, which is generally conserved in eukaryotic cells, and inhibits protein synthesis, so it is not only toxic to animal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/37C12N15/81C12N15/80
CPCC07K14/37C12N15/80C12N15/81
Inventor 朱牧孜李赛妮章卫民岑由飞叶伟刘洪新李浩华
Owner 东岱(济南)智能技术有限公司
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