差异显示技术对变异链球菌生物膜基因表达差异的分析  

Detection of differential gene expression in biofilm-forming versus planktonic populations of Streptococcus mutans using RFDD-PCR analysis

作　　者：黄正蔚[1] 刘正[1] 

机构地区：[1]上海第二医科大学附属第九人民医院,200011

出　　处：《中华微生物学和免疫学杂志》2005年第9期777-780,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家十五科技攻关计划(2004BA720A24);国家自然科学基金(编号30400497);上海市科委重点科研基金资助(编号04JC14066)

摘　　要：目的对生物膜和浮游状态下的变异链球菌细胞进行比较研究,采用限制性酶切差异显示技术比较分析两者的基因表达谱。方法分别收集变异链球菌浮游菌细胞与贴壁的生物膜细胞,分离纯化总RNA,对于逆转录获得的cDNA进行限制性酶切差异显示PCR技术(RFDD-PCR)比较分析两者的表达谱,对于获得的差异表达片段通过克隆测序并于BLAST比对获得这些片段的基因信息,进而推断其功能。结果通过对差异片段的分析,确证其中4条片段分属结构基fruA、SMU.438c、SMU.751与adhB/C。结论限制性酶切差异显示技术可用于分析生物膜形成过程中基因表达的差异。Objective Microbial proliferation and biofdm formation on tooth surface are characteristics of Streptococcus mutans which is associated with some phenotypic alterations such as reduced antimicrobial susceptibility. To identify genes which are typically expressed in biofilms, a restrictive fragment differential display PCR （RFDD-PCR） analysis was adapted for gram-positive bacteria and used with cDNA derived from populations S. mutans Ingbritt growing in a biofilm or in a planktonic state. Methods Difference display PCR（DD-PCR） has become a method of choice to observe differences in the expression levels of a large number of expressed genes at one time. In comparison with previously described DD-PCR protocol, RFDD-PCR has the advantages that only minimal quantities of total RNA are needed and, most importantly, that total RNA can be used since the large amount of rRNA in total RNA does not interfere with the RFDD-PCR procedure. Results Using a series of high stringency PCR amplification steps, four genes were identified as being differentially expressed in biofilm versus planktonic cultures. These genes are coded for a fructan hydrolase, a 2-hydroxyglutaryl-CoA dehydratase, a transcriptional accessory protein and an acetoin dehydrogenase. Conclusion RFDINPCR is a sensitive and specific method to detect transcripts differentially expressed as a function of different S. mutans growth conditions.

关 键 词：变异链球菌 限制性酶切差异显示技术 生物膜基因表达差异 差异显示技术 生物膜形成 基因表达差异 变异链球菌 差异表达片段 限制性酶切 基因表达谱 PCR技术 膜细胞 

分 类 号：R780.2[医药卫生—口腔医学]