生防菌密旋链霉菌Act12中SPA7074缺失突变株的构建及其次级代谢产物鉴定  

Construction of SPA7074-deficient mutant of biocontrol strain Streptomyces pactum Act12 and characterization of its secondary metabolites

作　　者：段雪梅[1] 赵飞扬 颜霞[1] 薛泉宏[2] 李晓霞[1] 文冰洁 贾良辉[1] 颜华[1] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]西北农林科技大学资源环境学院,陕西杨凌712100

出　　处：《微生物学报》2016年第12期1883-1891,共9页Acta Microbiologica Sinica

基　　金：陕西省农业科技创新与攻关(2015NY066);上海交通大学微生物代谢国家重点实验室开放基金(MMLKF14-09);中央高校基本科研业务费(Z109021426;Z109021432);高校博士点基金(20120204120034;20120204120042)~~

摘　　要：【目的】通过基因工程手段构建生防菌Act12转录调控因子SPA7074缺失突变株,并挖掘其中活性次级代谢产物资源和探讨其活性机理。【方法】利用同源重组方法敲除Act12基因组中可能的Tet R家族转录调控因子编码基因spa7074(accession number:KU955325),平板实验检测缺失突变株发酵液抑菌活性的变化,并通过HPLC比较代谢图谱,然后通过质谱及核磁共振对差异峰对应化合物进行结构鉴定。【结果】SPA7074缺失突变株对几种病原真菌的拮抗活性显著增强,比较代谢图谱表明出现数个差异峰,将最显著差异峰所对应化合物进行分离纯化鉴定,结果为寡霉素D。【结论】本研究通过基因工程手段敲除生防菌株Act12中的负转录调控转录因子,使得突变菌株抑菌活性显著增强,并获得了产量达野生型菌株7倍的寡霉素D高产菌株Δspa7074。[Objective] To disrupt spa7074, which encodes a member of the Tet R family transcriptional factors, in biocontrol strain Act12 and characterize the secondary metabolites in the mutant strain. [Methods] We disrupted the gene spa7074 by homologous recombination. The secondary metabolites of the mutant strain Δspa7074 and Act12 were detected by HPLC. The structure was analyzed by MS and NMR. [Results] Compared to the wild-type strain,the production of some unknown compounds in the mutant strain Δspa7074 increased obviously. We purified one of the compounds and identified as oligomycin D by MS and NMR analysis. [Conclusion] An oligomycin D-producing strain Δspa7074 was derived via genetic engineering.

关 键 词：生防菌 密旋链霉菌Act12 TetR家族 基因中断 寡霉素D 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治]