大鼠睾丸组织uPA基因启动子区的克隆与分析

Cloning and Analysis of the Promoter Region of Rat uPA Gene

目的:克隆大鼠睾丸组织uPA基因的启动子区并对该序列进行分析。方法:从大鼠睾丸组织中提取基因组DNA,以其为模板设计uPA基因引物,运用降落PCR法扩增uPA基因5'端上游的真核转录调控序列。测序得到的PCR产物用启动子区的分析软件分析,并与其DNA序列进行比对。结果:得到的uPA基因长度为1572bp(登录号X65651)。经软件分析:该序列包含uPA基因完整的开放阅读框(ORF)、21bp的外显子部分,1551bp区域为转录起始的上游部分在其5'端UTR区的-30bp位置有一个不典型的TATA盒,其上游有非常明显的GC盒及启动子区常见的AP1(activeprotein1)、SP1等结合位点。结论:成功克隆了大鼠睾丸组织uPA基因启动子区。分析表明,该片段包含真核转录调控区域、不典型的TATA盒、典型的GC盒及启动子区所常见的AP1、SP1等结合区域。

Objective：To clone and analyze the promoter sequence of rat urokinase plasminogen activator （uPA） protein gene. Methods： The genomic DNA was extracted from rat testicle tissue. According to uPA, the gene upper and lower primer of uPA gene were designed and synthesized, then touch-down PCR were performed.Alter proper purification,the PCR product was sequence,analyzed with the promoter prediction software and compared with the DNA sequence of rat uPA. Results：The cloned uPA gene was about 1 572 bp in length, Which contained a full open-reading frame with 21 bp in length exons, and the upper region of transcriptional start isl 551 bp in length which is eucaryou transcriptional control area. The 5′UTR has a promoter region including a non-conspicuous TATA-box. Not only the GC-box binding region was found in this gene, but also active protein 1（AP1） and SP1 were seen in other regions. Conclusion： A 1572 bp uPA gene fragment （GenBank accession NO. X65651） was obtained from rat genomic DNA library, containing eucaryou transcriptional control area with a promoter region, non-conspicuous TATA- box, GC-box and 1 extrons.A TATA-box was located at the upper -30 region.