猪PLE1基因启动子克隆及序列分析

Cloning and analysis of porcine PLE1 promoter

通过巢式PCR方法获得猪PLE1基因5′端上游启动子序列,通过T/A克隆法对PLE1基因的启动子进行克隆,并对PCR鉴定为阳性的克隆子进行测序,参考人、啮齿类的羧酸酯酶家族的启动子结构,并利用启动子在线分析软件对其进行生物信息学分析。结果成功克隆得到PLE1基因5′端上游1 153bp的调控片段,分析表明该调控区没有CpG岛,也不存在典型的TATA盒结构;有2个转录起始位点分别位于翻译起始密码子ATG上游-39bp和-37bp处,潜在的转录因子结合位点有C/EBP、Sp1、USF、CdxA、GATA-X、GATA-1、GATA-2、GATA-3、MZF1、AML-1a、SRY、Nkx-2、Lyf-1、deltaE等。另外还发现了7种基序分别为EGF_1、INTEGRIN_BETA、CTCK_1、ANAPHYLATOXIN_1、THIOLASE_3、TUBULIN、VWFC_1。研究结果可为进一步揭示PLE1基因转录调控机制提供理论参考。

This experiment was conducted to clone and analyze the promoter sequences of pig liver carboxylesterase 1 （PLE1）. The 5＇ upstream promoter sequence of PLE1 gene was amplified by nested PCR,then was cloned by T/A clone method. The positive clones identified by PCR were se quenced. In addition,considering the confirmed promoter structure of human and rat carboxyles terase family and using promoter online software,the promoter sequence was analyzed. A 1 153 bp 5＇ upstream promoter sequence of PLE1 gene was cloned. It was found that there is no CpG island or typical TATA box existing in the region. Meanwhile, the result showed that two transcription start sites were mapped to --39 bp and --37 bp of translation start site. And the potential tran- scription factor binding sites were predicted by hioinformatic methods, including C/EBP, Spl, USF, CdxA, GATA-X, GATA-1, GATA-2, GATA-3, MZF1, AMLqa, SRY, Nkx-2, Lyf-1, deltaE etc. Moreover,7 motifs were found, including EGF, INTEGRIN_BETA, CTCK_1, ANAPHY- LATOXIN_1, THIOLASE_3, TUBULIN, VWFC 1. In this experiment, the promoter of PLE1 gene was successfully cloned,which may be helpful for further exploring the transcription mecha nism of PLE1 gene.