小鼠Reelin基因启动子克隆鉴定及生物信息学分析

Cloning and bioinformations analysis of promoter sequences of mice Reelin

目的克隆小鼠Reelin基因启动子区并分析其潜在DNA甲基化位点和转录因子结合位点。方法根据NCBI上小鼠Reelin 5'非翻译区序列设计引物,利用高保真PCR方法克隆昆明小鼠Reelin启动子区序列,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Reelin启动子-636 bp至-135bp序列甲基化情况分析。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。结果成功克隆了小鼠Reelin基因启动子区0至-450片段。甲基化测序分析小鼠Reelin启动子区-636 bp至-135 bp序列没有CpG二核苷酸被甲基化。小鼠Reelin翻译起始点0至-450片段利用CpG岛序列分析软件分析显示,该区域C+G含量高达78.71%,CpG含量为14.44%。该区域有120多个潜在转录因子结合位点。结论在小鼠Reelin基因启动子区0～-450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Reelin基因表达调控起重要作用。

Objective： To clone the mice Reelin gene promoter and to analyse the potential DNA methylation site and transcription binding sites. Methods： Sequence of the 5 flanking region of mouse Reelin gene was searched out and downloaded from NCBI. According to the targeted sequence, primers were designed and synthesized for the PCR amplification. The 450bp （ -450 -0 bp） fragment was amplified by PCR. The PCR product was cloned into PCR 2.1 vector and was sequenced. Bisulfite-Modified DNA Sequencing was used to identify the DNA methylation state of Reelin promoter （ - 636 bp - 135 bp）. Methyl Primer Express software V1.0 and TFSEARCH software were used to analyse the DNA methylation site and transcription factor binding site. Results ： Result of DNA sequencing showed that the 450 bp fragment of mouse Reelin 5 promoter was successfully cloned. No CpG dinucleotide was found in -636 bp - 135bp fragment. The 450bp upstream sequence of mouse Reelin displayed a very high G ＋ C content （78.71%） and CpG content（ 14.44% ）. More than 120 potential transcription factor binding sites were found in this sequence. Conclusion： One potential CpG island and some potential transcription factor binding sites are identified in 450bp upstream sequence of mouse Reelin.