中文摘要：

目的：从变形链球菌基因组中筛选反应调控蛋白ComE结合的核酸序列。方法：⑴提取变形链球菌UA159基因组,进行超声剪切处理。以基因组片段为模板,用随机引物进行延伸,切胶纯化和PCR扩增后获得基因组文库。⑵应用基因组SELEX技术,以ComE蛋白为靶物质,与变形链球菌基因组文库共同孵育,循环筛选8轮。筛选产物TA克隆后送去测序,测序结果进行生物信息学分析。⑶将分析得到的2个序列分别克隆入pFW5-luc载体中,然后分别重组到变形链球菌UA159野生株和comE突变株的基因组中,采用RT-PCR的方法检测luc片段的表达。结果：⑴获得了变形链球菌UA159的基因组文库,片段范围约为100～300bp。⑵随机挑取54个克隆送去测序,经生物信息学分析和RT-PCR初步验证,最终获得1个ComE蛋白可能的结合序列。结论：采用基因组SELEX技术,筛选反应调控蛋白ComE可能的结合序列,并进行了初步验证,为后续进行变形链球菌反应调控蛋白ComE调控机制的研究奠定了基础。

英文摘要：

Objective：To screen the binding sequences of ComE from the genome of Streptococcus mutans.Methods：⑴The genome of Streptococcus mutans UA159 was extracted and sheared by sonication,and then incubated with two different random primers successively.Finally,the extention product was purified from gel extraction and amplified by PCR,and the genomic library of Streptococcus mutans was constructed.⑵Genomic SELEX for eight times to obtain the binding sequences of ComE.Selected DNA fragments were cloned into the pGEM-T Easy Vector and sent to sequence analysis.The sequences were further analyzed by bioinformatics programs.⑶Two obtained sequences were cloned into pFW5-luc,and then the corresponding recombinant plasmids were transformed into Streptococcus mutans UA159 and Streptococcus mutans comE mutant respectively.Finally,the expression of luc were tested by RT-PCR.Results：⑴The genomic library of Streptococcus mutans UA159 was obtained with the sequences ranging from 100 bp to 300 bp.⑵54 clones were selected randomly and sent to sequencing.Two candidates were identified through bioinformatics analysis.Through primary validation,one possible binding sequence was obtained.Conclusion：According to genomic SELEX,eight selection cycles were finished and one possible binding sequence was obtained,which laid the basis for the explanation of the regulation mechanism of ComE in Streptococcus mutans.