ECF-σ^5因子参与阿维链霉菌中阿维菌素合成和环境胁迫的研究  

ECF-σ^5 in Sreptomyces avermitilis is involved in regulation of avermectin biosynthesis and stress response

作　　者：罗帅[1] 孙地 陈芝[1] 文莹[1] 

机构地区：[1]中国农业大学生物学院,农业生物技术国家重点实验室,北京100193

出　　处：《微生物学报》2016年第3期471-484,共14页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31170045)~~

摘　　要：【目的】研究阿维链霉菌中ECF-σ^5子对阿维菌素合成、形态分化和环境胁迫的调控,为揭示阿维菌素生物合成的调控机制和ECF-σ因子的调控网络提供依据。【方法】构建sig5基因缺失、回补和过表达菌株,通过形态观察和摇瓶发酵实验初步确定σ^5形态分化、菌体生长和阿维菌素合成的影响。进一步通过RT-q PCR、EMSA和Ch IP实验寻找确定σ^5靶基因,再通过胁迫实验揭示σ^5能参与的胁迫反应。【结果】对sig5相关突变株的摇瓶发酵和形态观察结果表明,σ^5阿维菌素合成具有抑制作用,但不影响菌体生长和形态分化。sig5基因缺失导致阿维菌素生物合成途径特异性正调控基因ave R和结构基因ave A1的转录水平提高,但σ^5不与ave R和ave A1的启动子区结合。σ^5结合在自身基因及附近基因SAV612、SAV615、SAV618的启动子区,正调控这些基因及所在操纵子的表达。胁迫实验暗示σ^5能参与渗透压引起的胁迫反应。【结论】ECF-σ^5子在转录水平间接负调控阿维菌素的合成。[Objective] We investegated the role of extracytoplasmic function（ECF） σ factor, σ^5 in avermectin biosynthesis, morphological differentiation and stress response in S. avermitilis. [Methods] We constructed sig5 gene deletion, complementation and overexpression strains and determined the role of σ^5in avermectin production and morphological differentiation by shaking flask fermentation and morphological observation of these strains. We used RT-q PCR, EMSA and Ch IP assays to identify the target genes of σ^5 We used stress tests to reveal the stress response that σ^5may be involved in. [Results] Determination of avermectin production and morphological observation in sig5 related strains implied that σ^5inhibits avermectin production, but has no effect on growth or morphology. Deletion of sig5 increased transcription levels of pathway-specific activator gene ave R and structural gene ave A1, but σ^5did not bind to the promoter regions of ave R and ave A1. RT-q PCR and Ch IP assays showed that σ^5positively regulates the transcription of itself and adjacent genes by binding to the promoter regions of sig5, SAV612, SAV615 and SAV618.Stress tests suggested that σ^5is involved in responding to osmotic stress. [Conclusion] Our findings indicated that σ^5ndirectly inhibits avermectin production by affecting transcription of ave genes.

关 键 词：阿维链霉菌 阿维菌素 ECF-σ因子 σ^5 

分 类 号：Q936[生物学—微生物学]