内蒙古绒山羊角蛋白5基因启动子克隆及序列分析

Cloning and Sequence Analysis of the Inner Mongolia Cashmere Goat Keratin 5 Gene Promoter

试验旨在了解角蛋白5(keratin 5,K5)可能的调控序列。本研究根据UCSC公布的牛K5基因5′侧翼区设计PCR引物,扩增了内蒙古绒山羊K5基因部分启动子序列。通过产物纯化、连接、转化,并对测序结果进行了生物信息学分析。结果扩增得到内蒙古绒山羊K5基因启动子序列长度为1452bp(GenBank登录号为:JQ277735),与牛和人相应序列的相似性分别为91.5%和74%。转录起始位点位于翻译起始密码子ATG上游-101bp位置;含有两个TATA盒,分别位于翻译起始位点上游-129—-124bp(ATAAAA)和-178—-174bp(TTAAT)位置;通过在线分析软件预测发现(按5′→3′)SRY,MZF1,v-Myb,SRY,AP-1,CDP CR,HNF-4,AML-1a,HSF2,AP-4,AP2,AP2,Sp1,Nkx-2,Sp1和GATA-1转录因子结合位点。其中,转录因子SRY(TGTGTTT),和CDP CR(GATTGATGGC)是绒山羊特有的;转录因子HNF-4,AML-1a,HSF2,AP-4,AP2,Sp1,Nkx-2和GATA-1(AGCCATCATG)在绒山羊、牛和人K5启动子上的结合位点高度保守。两个最小增强子分别位于翻译起始位点ATG上游-140—-91bp和-114—-67bp位置,含有24bp(GCGGCTCCCAGGTAACAGAGC-CGC)重叠区,预测其与绒山羊K5基因的转录调控有关。试验确定了内蒙古绒山羊K5基因启动子的转录起始位置、转录因子结合位点及最小增强子序列,为进一步研究绒山羊K5基因的表达调控机制奠定了理论基础。

This experiment was conducted to study the potential regulatory sequence of Keratin 5（K5）. This study accord- ing to the UCSC bovine K5 gene 5＇ flank regions designed the PCR primers, and Inner Mongolia Cashmere goat K5 gene pro- moter was amplified. Analysis the K5 gene promoter sequence after product purification,ligation,transformation and sequence. It was found that 1452 hp Inner Mongolia Cashmere goat K5 promoter sequence was confirmed, which showed 91.5% and 74 %homology with that of bovine and human respectively. The transcription start site was mapped to -101 bp of translation initiation site and two TATA boxes located in -129 to -124 bp （ATAAAA） and-178 to -174 bp （TTAAT） of translation initiation site respectively. The potential transcription factor binding motifs were predicted after analysis of promoter online software, including （5r to 3＇）SRY, MZF1, v-Myb, SRY, AP-1, CDP CR, HNF-4, AML-la, HSF2, AP-4, AP2, AP2, Spl, Nkx-2, Spl and GATA-1, in which SRY（TGTGTTT） and CDP CR（GATTGATGGC） were specific for Cashmere goat, and HNF-4, AML-la, HSF2, AP-4, AP2, Spl, Nkx-2 and GATA-I（AGCCATCATG） were conserved in Cashmere goat, bovine and human. Moreover, the two minimal enhancer reside from --140 to -91 hp and -114 to -67 bp of translation ini- tiation site respectively, and contained 24 bp （GCGGCTCCCAGGTAACAGAGCCGC） overlap, which might be related with the Cashmere goat K5 gene transcriptional regulation. In this experiment, we inferred the transcription start site, transcription factor binding motifs and minimal enhancer elements in Inner Mongolia Cashmere goat K5 gene promoter, which may be help- ful for exploring the mechanism of gene expression of cashmere goat K5 gene.