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

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

摘要:

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。

Abstract:

Abstract:［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) ．