This study is aimed to establish an effective tool to control gene expression in Gluconobacter oxydans.Cis-or trans-acting non-coding small RNAs,complementary to ribosomal binding site (RBS),were designed and constructed to modulate gene expression in G.oxydans at translational level.Using green fluorescent protein as reporter gene,in a cis-acting configuration,a cis repressor sequence inserted into the RBS upstream bound with RBS to form a stem-loop structure in mRNA,which repressed the reporter gene expression.Such a stemloop was disrupted by an anti-repressor sequence that had higher affinity than a cis repressor sequence,thus this allowed the reporter gene to re-initiate translation.In a trans-acting configuration,the complementary double strands of small non-coding RNA transcribed from a separate promoter and a RBS were able to specifically repress the reporter gene expression by blocking the binding of ribosome and mRNA.Based on the above results,a series of trans-acting small RNAs were designed and imported,by which the endogenous gene pstⅠ expression inhibition in G.oxydans was realized.In summary,this study offers an alternative approach to study gene functions and regulate gene expression in G.oxydans other than gene knockout.%在氧化葡萄糖酸杆菌中建立一种调控基因表达的工具.通过顺式/反式作用的非编码小RNA与核糖体结合位点(RBS)的相互作用,在翻译水平上对氧化葡萄糖酸杆菌中目标基因的表达进行调控.以绿色荧光蛋白作为报告基因,在所构建的顺式调控系统中,插入RBS上游的顺式阻遏序列,能够在转录后与RBS形成茎环结构,从而抑制报告基因的表达.这一茎环结构能够被与顺式阻遏序列具有更高亲和力的抗阻遏序列打开,从而重启报告基因的翻译;在所构建的反式调控系统中,由独立启动子控制转录的非编码小RNA与RBS互补形成双链,通过阻碍核糖体与mRNA的结合抑制报告基因的表达.在此基础上,设计并导入了一系列反式作用的小RNA,实现了对氧化葡萄糖酸杆菌中内源基因pstⅠ表达的抑制.本研究提供了一种在氧化葡萄糖酸杆菌中不同于传统基因敲除的调控基因表达的方法.
展开▼
