摘要
目前在细菌中已经鉴定了大量小RNA参与调控许多重要的生命活动,然而小RNA在沙雷氏菌属(Serratia)中的作用还了解得很少。其中RsmB属于CsrB家族的小RNA,是翻译阻遏蛋白RsmA的拮抗剂。普城沙雷氏菌(Serratia plymuthica)G3分离自小麦内茎,是一种能产多种抗菌因子和植物激素的内生细菌。该研究旨在以G3菌株为模式细菌,构建RsmB突变体,为进一步研究RsmB介导的转录后全局调控机制奠定基础。使用基于sacB的自杀性载体pDM4,采用基因替换和同源重组策略,构建了G3菌株的rsmB基因删除插入突变体△rsmB∶∶Gm。进一步以基因组DNA为模板,通过PCR和测序对突变体进行了验证,并构建了互补菌株。突变分析结果表明RsmB正向控制细菌几丁质酶活性和泳动运动性,这与RNA结合蛋白RsmA过量表达的表型一致;而互补菌株完全恢复了几丁质酶活性和运动性,再次证明△rsmB∶∶Gm突变体构建成功。RsmB突变体成功构建,将为深入研究Rsm系统对生防菌普城沙雷氏菌的调控作用及机制奠定基础。
Noncoding regulatory RNA molecules（ncRNAs）,also known as small RNAs（sRNAs） have been identified in a wide range of bacteria and found to play critical regulatory roles in many biological processes.Although most of these sRNAs seem to function by base pairing with mRNAs,the CsrB sRNA family acts by sequestering an RNA-binding regulatory protein CsrA（carbon storage regulator） or RsmA（regulator of secondary metabolism）.It has been known that the Rsm system participates in global regulatory circuits that control central carbon flux,the production of extracellular products,cell motility,biofilm formation,quorum sensing and pathogenesis depending on the particular bacterium.However,its function in the genus of Serratia remains less understood.Serratia plymuthica G3 was a endophytic strain isolated from the stems of wheat in Taian（Shangdong Province） with a potential to produce a variety of antifungal factors,such as the antibiotic pyrrolnitrin,extracellular chitinase and protease,and siderophores,as well as plant hormone indole-3-acetic acid for plant growth promotion.Using gene replacement and homologous combination strategy with the sacB-based suicide vector pDM4,a G3 chromosome mutant △rsmB ∶ ∶ Gm was constructed,which was deficient in rsmB,a small RNA regulator belonging to CsrB family and serving as an antagonist of the translational repressor RsmA.The RsmB mutant was verified through PCR-based cloning and DNA sequencing.Mutation in RsmB abolished chitinolytic activity and significantly reduced the swimming motility,when compared with the wild type G3.This is consistent with the overexpression of the RNA-binding protein RsmA.Complementary plasmid pUCP26-rsmB in the mutant fully restored the chitinase activity and the swimming motility to the level of wild type,reconfirming the construction of △rsmB ∶ ∶ Gm mutant.This will help further investigation of the global post-transcriptional regulatory mechanisms of the Rsm system involved in the biocontrol-related strains of Serratia plymut
出处
《江苏农业学报》
CSCD
北大核心
2011年第5期969-973,共5页
Jiangsu Journal of Agricultural Sciences
基金
国家自然科学基金项目(30670030
30811130218)
江苏大学启动基金项目(07JDG030)
山东省科技发展计划项目(2010GNC10911)
