文献类型：期刊文献

中文题名：Streptomyces rimosus M4018中氧化应激转录抑制因子Rex的克隆表达及与rex操纵子的体外结合活性

英文题名：Cloning and expression of the redox-sensing transcriptional repressor Rex and in vitro DNA-binding assay of the Rex and rex operator in Streptomyces rimosus M4018

作者：沈晶[1];唐振宇[1];肖慈英[1];郭美锦[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2012

卷号：52

期号：1

起止页码：38

中文期刊名：微生物学报

外文期刊名：Acta Microbiologica Sinica

收录：MEDLINE(收录号：22489458);CSTPCD;;Scopus;北大核心:【北大核心2011】；CSCD:【CSCD2011_2012】；PubMed;

基金：国家重点实验室开放课题经费(2060204);国家"863计划"(2007 BAI26B02)~~

语种：中文

中文关键词：龟裂链霉菌;氧化应激;转录抑制因子Rex;基因克隆和表达;凝胶迁移分析

外文关键词：Streptomyces rimosus; redox-sensing; transcriptional repressor Rex; gene cloning and expression;electrophoretic mobility shift assay

摘要：【目的】为了研究龟裂链霉菌Streptomyces rimosus M4018中的rex基因对自身rex operator(ROP)的调控机制。【方法】根据天蓝色链霉菌Streptomyces coelicolor A3(2)中rex基因的同源序列设计引物进行PCR,从S.rimosus M4018中获得其rex基因(Sr-rex)。同时,通过染色体步移的方法,获得其上游的ROP序列。采用体外凝胶迁移的方法,分析了Sr-Rex对ROP的调控作用。【结果】获取的Sr-rex基因核苷酸序列长度为846 bp,预测的编码氨基酸序列与S.coelicolor A3(2)中Rex的同源性为84%,获得GenBank登录号:GQ849479。圆二色光谱显示Sr-Rex的结构以α螺旋和β折叠为主,与软件预测相符。凝胶迁移实验表明,Sr-Rex能与S.rimosus M4108中扩增到的ROP片段特异性结合。同时,以Rex:ROP的最小结合序列为基础,设计了一条22 bp的单链DNA片段,和Sr-Rex的最大结合摩尔浓度比约为5:1。高浓度的NADH抑制两者的结合活性,而NAD+对结合没有影响。【结论】在S.rimosus M4108中,Rex是通过响应胞内NAD(H)水平的方式来调控ROP的表达的。
[ Objective] The aim is to explore the self-regulation mechanism of the rex in Streptomyces rimosus M4018. [ Methods] We cloned the rex of S. rimosus M4018 （Sr-rex） based on its homologoussequence in Streptomyces coelicolor A3 （2） and its upstream rex operator （ROP） fragment using PCR and genome walking. An electrophoretic mobility shift assay （EMSA） was applied to analyze the regulation of rex to ROP in vitro. [ Results] Sr-rex is 846 bp in length and has a 84% identity with the one in S. coelicolor A3 （2） in amino acid sequence. It was deposited in Genbank under the accession number GQ849479. The expressed Sr-Rex by E. coli was mainly composed of alpha-helixes and beta-sheets, which was in compliance with the prediction. An Electrophoretic Mobility Shift Assay （EMSA） confirmed the specific binding activity of Sr^Rex with ROP. Meanwhile, we synthesized a 22 bp DNA fragment （ROP1） based on the minimal binding site of ROP. The maximal binding ratio of this fragment to Sr-Rex was 5：1 （molar）. NADH negatively affected the binding activity, however, NAD＋ had no impact on it. [ Conclusion] In S. rimosus M4018, the Rex regulated the gene expression of ROP via sensing the intracellular level of NAD （H）.