msRNAs介导rnc基因调控变异链球菌胞外多糖合成代谢的机制研究

Extracellular polysaccharide anabolism, as the target of caries prevention, plays critical roles in stability and cariogenic ability of dental biofilm microrganisms. The vicRKX signal pathway is one of the most important extracellular polysaccharide regulatory pathways in streptococcus mutans. In our previous study, rnc gene significantly affected extracellular polysaccharide synthesis gene, vicRKX signal pathway and the fate of some msRNAs. Based on this, the applicant hereby proposed a new hypothesis that rnc gene encodes RNase Ⅲ, RNase Ⅲ acts on the msRNAs and msRNAs regulate posttranscript level of extracellular polysaccharide synthesis genes and vicRKX pathways, then finally modulate extracellular polysaccharide synthesis and metabolism. To confirm this mechanism, this project applies a combination of recombinant plasmids, hybridization, nuclear magnetic resonance and so on, to solve the following problems:①Screen specific functional msRNAs which have relationship with the anabolism of extracellular polysaccharide, then clear their binding sites;②Study how msRNAs modulate the expression, activity and biological changes of extracellular polysaccharide synthase;③Analysis the function and mechanism of how rnc gene encodes RNase Ⅲ and subsequently acts on the production of msRNAs;④Clarify the mode and mechanism of rnc → RNase Ⅲ → msRNAs → mRNA of target genes →biological effects. This study makes valuable contributions to the delicate molecular mechanism of extracellular polysaccharide anabolism in the dental biofilm, and so does to its application value in anticaries explorations.

胞外多糖合成代谢作为目前龋病防治靶点是影响菌斑微生态稳定性、调节细菌致龋力的关键路径。vicRKX通路是变异链球菌中最重要的多糖调控途径。申请者在前期 “rnc基因明显调控vicRKX通路及多糖合成代谢且导致msRNA发生变化”的基础上，率先提出：rnc基因编码RNA调控酶（RNase Ⅲ），经msRNAs调控多糖合成基因和vicRKX通路转录后水平，调节胞外多糖合成代谢的新机制。通过综合使用质粒重组、分子杂交、核磁共振等技术①筛选与多糖合成相关的功能性msRNAs，明确其碱基作用位点；②研究msRNAs对vicRKX通路、多糖合成酶表达及细菌生物学的影响；③分析rnc基因编码RNase Ⅲ对msRNAs生成的作用及机制；④阐明rnc→RNase Ⅲ→msRNAs→目标mRNA→生物学效应的模式及机制。本研究对阐明菌斑微生态中多糖合成调控新机制、探索防龋新途径具有重要的科学意义和应用价值。

胞外多糖合成代谢作为目前龋病防治靶点是影响牙菌斑微生态稳定性、调节细菌致龋力的关键路径。vicRKX通路是变异链球菌中最重要的多糖调控途径。课题组在前期 “rnc基因明显调控vicRKX通路及多糖合成代谢且导致msRNA发生变化”的基础上，展开了本课题的研究。在研究过程中，课题组首先找到了rnc-基因启动子，明确了 rnc基因不与vicRKX共转录；随后通过测序分析找到了3个相关的msRNAs分子(msRNA 1657/1701/3405),并对其进行了结构预测，而后通过实验证实其中msRNA 3405最有可能与vicR mRNA-150~+50区段结合，证明rnc对vic-基因启动子(operon)的调节可通过msRNAs进行，从而调节vicR蛋白的表达；干扰变异链球菌生物膜胞外多糖的合成，扰乱生物膜结构，导致细菌致龋性降低；最后课题组采用调取变异链球菌rnc基因在以pET-21a(+)为载体构建重组质粒，导入大肠杆菌进行Rnc 蛋白 (核酸内切酶Ribonuclease III)的提取的方法，顺利完成了RNase Ⅲ的构建和提取，并通过体外试验确定RNase Ⅲ且具有活性,能够切割RNA产生msRNAs。、.综合上述研究，本课题基本完成了rnc基因→RNase Ⅲ→msRNAs→vicRKX通路→多糖合成基因→胞外多糖合成→致龋性这一调控过程的研究。从分子机制层面进一步剖析菌斑生物膜形成的过程中多糖合成的调控过程，加深了牙菌斑生物膜致龋机制的理论依据，可以为防治龋病提供潜在作用靶点，具有重要的科学理论价值和临床应用前景。同时，本课题将msRNAs功能机制引入细菌微生物研究，对微生物调节机制的研究提供了新思路。