vicR反义长链非编码RNA 协同RNase III调控变异链球菌生物膜致龋性效果和机制的研究

Antisense long non-coding RNA has widely regarded as a crucial regulator in bacterial metabolism and virulent gene expressions in the pathogens. An antisense RNA which is non-encoded RNAs is complementary to a messenger RNA (mRNA) strand transcribed and results in duplex (double-stranded RNA) formation which may inhibit the translation of a complementary mRNA. The double-strand-specific endonuclease RNase III which is encoded by rnc gene has a critical role in specific double-stranded RNA structures cleavage. In the preliminary study of Streptococcus mutans，our research group have found that VicR is directly involved in regulating the transcription level of rnc gene. Then, we detected a vicR antisense long non-coding RNA transcript (named as vicR antisense lncRNA) upstream of the rnc gene for the first time. Furthermore study showed that RNase III which encoded by rnc gene can regulate the expression of transcription factor vicR through vicR antisense lncRNA, and then interfere the post transcriptional modification level of Streptococcus mutans. Therefore, the applicant hereby put forward a new hypothesis that the potential mechanism between the vicR antisense lncRNA and endonuclease RNase III in post-transcriptional control in S.mutans. In order to verify the hypothesis, the applicants will adopt synthetically the methods of microbiological, molecular biological techniques, and bioinformatics. The goal of this study is to define the interaction of vicR antisense lncRNA and endonuclease RNase III, and to investigate the potential roles in the regulation in post-transcriptional controls. This project would provide a new perspective of gene regulation in prokaryotes and pursue the new valid management for caries prevention and therapy.

反义长链非编码RNA是细菌代谢和毒力基因表达的重要调节因子；RNase III能降解它与编码蛋白基因形成的互补双链RNA，干扰mRNA的转录。课题组在变异链球菌前期研究中已经发现VicR直接参与调控rnc基因转录水平，而后又首次验证一条vicR反义长链非编码RNA。通过进一步研究证实，由rnc基因编码的RNase III可经其调控转录因子VicR的表达，从而干预变异链球菌转录后水平修饰。据此申请者提出RNase III与vicR反义长链非编码RNA交互作用共调控变异链球菌转录后修饰水平的新假说。课题拟采用微生物学、分子生物学、生物化学、生物信息学等技术阐明：①vicR反义长链非编码RNA与RNase III之间交互作用关系；②二者交互作用关系共调控变异链球菌转录后修饰水平。课题以原核生物反义长链非编码RNA调控作用模式为研究主题，以期为龋病预防及病原微生物感染控制提供理论依据。

反义长链非编码RNA是细菌代谢和毒力基因表达的重要调节因子；RNase III能降解它与编码蛋白基因形成的互补双链RNA，干扰mRNA的转录。课题组在变异链球菌前期研究中已经发现VicR直接参与调控rnc基因转录水平，而后又首次验证一条vicR反义长链非编码RNA。通过进一步研究证实，由rnc基因编码的RNase III可经其调控转录因子VicR的表达，从而干预变异链球菌转录后水平修饰。课题采用突变菌株构建、大鼠动物试验、蛋白构建与纯化等方法，采用CLSM、多糖成分与结构分析、定量PCR、western blot以及染色质免疫共沉淀等技术，研究发现rnc基因编码的RNase III可切割asvicR高表达菌株的asvicR RNA，而rnc缺失时，asvicR RNA表达较对照组高1.5倍，同时vicR蛋白可以与RNase III切割产生的msRNA1657生成复合物（约为对照组10倍），变异链球菌中asvicR高表达明显抑制多糖合成相关基因gtfB/C/D及其蛋白的表达，减少细菌形成的生物膜量以及多糖量并改变多糖组分与结构，经过进一步大鼠体内致龋性试验以及临床高龋ECC与无龋菌株验证，确定asvicR高表达菌株致龋性降低。进而结合查阅文献以及课题组试验结果，归纳其机制为asvicR RNase III切割产生的msRNA1657相互作用，asvicR高表达时asvicR与vicR基因结合，导致vicR蛋白表达降低，vicR蛋白与多糖相关基因如gtfB/C启动子区域结合减少，抑制了GtfB/C蛋白的表达，而msRNA1657与vicR蛋白可进一步减少其结合，从而减少细菌产生多糖量并改变多糖组分与结构，从而降低了asvicR高表达菌株的致龋性。课题通过对原核生物变异链球菌反义长链非编码RNA调控作用模式的研究，以期该机制的阐述可以为龋病预防及病原微生物感染控制提供理论依据。

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