小鼠PP1基因启动子克隆鉴定及潜在甲基化位点分析

Cloning and bioinformation analysis of promoter sequences of mice PP1

目的 PP1分子是一种与学习记忆相关的分子,目前对其表达调控尚未有系统的研究。为了研究小鼠PP1基因启动子区甲基化潜在的位点和转录因子结合位点而克隆小鼠PP1启动子区。方法本实验利用NCBI上小鼠PP15'非翻译区序列设计引物,使用高保真Taq酶,利用PCR方法扩增昆明小鼠PP1启动子0至-320序列,并进行TA克隆,测序鉴定。利用亚硫酸氢钠处理基因组DNA,通过PCR技术和测序技术对PP1启动子0至-320序列甲基化情况分析。然后再应用Methyl Primer Express software V1.0和TFSEARCH软件分析该区域甲基化位点和转录因子结合位点。利用CpG岛序列分析软件分析。结果成功克隆得到PP1基因启动区0到-320 bp片段。甲基化测序分析该片段只有一个CpG位点被甲基化。软件分析表明小鼠PP1翻译起始点到-320 bp区域C+G含量高达70.25%,CpG含量为11.75%,含有70个潜在的转录因子结合位点。结论在小鼠PP1基因启动子区0～-320发现有一个CpG岛和多个转录因子结合位点,该区域可能在小鼠PP1基因表达调控起重要作用。

Objective： To clone the mice PP1 gene promoter and to analyze the potential DNA methylation site and tran- scription binding sites. Methods ： Sequence of the 5′flanking region of mouse Reelin gene was searched out and downloaded from NCBI. Based on the targeted sequence, primers of PP1 promoter were designed and synthesized for the PCR amplifica- tion. The 320bp （ -320 bp～0 bp） fragment was amplified by PCR using high fidelity taq polymerase. The PCR product was cloned into PCR2.1 vector and was sequenced. Bisul.flte-Modified DNA Sequencing was used to identify the DNA methylation state of PP1 promoter （ -320 bp ～0 bp）. Methyl Primer Express software V1.0 and TFSEARCH software were used to ana- lyze the DNA methylation site and transcription factor binding site. Results： Results of DNA sequencing showed that the 320 bp fragment of mouse PP1 5′promoter was successfully cloned. A CpG dinuele0tide was found in this fragment. The 320bp up- stream sequence of mouse Reelin displayed a very high G ＋ C content （70.25%） and CpG content（ 11.75% ）. More than 70 potential transcription factor binding sites were found in this sequence. Conclusion： One potential CpG island and some poten- tial transcription factor binding sites are identified in 320bp upstream sequence of mouse PP1, which may play an important role in the expression and manipulation of PP1.