基于sRNA的氧化葡萄糖酸杆菌基因调控的研究  

The Translational Regulation of Gene Expression in Gluconobacter oxydans by Small RNAs

作　　者：李真真 赵佳晨 马昱澍[1] LI Zhen-zhen;ZHAO Jia-chen;MA Yu-shu(State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 20023)

机构地区：[1]生物反应器工程国家重点实验室华东理工大学,上海200237

出　　处：《生物技术通报》2018年第3期59-66,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31370080);生物反应器工程国家重点实验室开放课题

摘　　要：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。This study is aimed to establish an effective tool to control gene expression in Gluconobacter oxydans. Cis-or trans-actingnon-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 insertedinto the RBS upstream bound with RBS to form a stem-loop structure in m RNA,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 tore-initiate translation. In a trans-acting configuration,the complementary double strands of small non-coding RNA transcribed from a separatepromoter and a RBS were able to specifically repress the reporter gene expression by blocking the binding of ribosome and m RNA. Based on theabove results,a series of trans-acting small RNAs were designed and imported,by which the endogenous gene pst I 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. oxydansother than gene knockout.

关 键 词：氧化葡萄糖酸杆菌 非编码小RNA 翻译调控 核糖体结合位点 基因敲除 

分 类 号：Q78[生物学—分子生物学]