坝上长尾鸡pmel基因核心启动子的鉴定

Identification of the core promoter of the pmel gene of Bashang long-tail chickens

摘要:

为探明坝上长尾鸡的前黑素小体蛋白 (Pre-melanosomal protein，Pmel) 基因核心启动子区，首先构建了双荧光素酶表达载体，通过脂质体瞬时转染鸡胚成纤维细胞DF1，并利用双荧光素酶检测试剂盒进行启动活性检测。成功克隆了坝上长尾鸡pmel基因5￠侧翼区片段1 268 bp，预测启动子区 (?1 200—+68) 含有2个CpG岛和多种转录因子结合位点，构建了9个含有不同长度pmel基因启动子片段的表达载体及1个核心启动子区突变载体，说明鸡pmel基因启动子的核心区域为?840—+68 bp，其中?840—?590 bp和?525—?266 bp区域为正调控区，?590—?525 bp区域为负调控区，多态位点 (?456、?435、?410、?374和?341) 对pmel基因启动子活性有较大影响。

Abstract:

To explore the activity of the pmel core promoter of Bashang long-tail chickens, we constructed dual-luciferase expression vectors and transiently transfected into DF1 cells with Lipofectamine 2000. We measured the luciferase activity with the dual-luciferase detection kit. The 1 268 bp fragment in 5￠-flanking region of the pmel gene in Bashang long-tail chickens was cloned. The region from ?1 200 bp to +68 bp included 2 CpG islands and multiple transcription factor binding sites. We constructed 9 expression vectors with different promoter regions and a mutant vector of the core promoter region of the pmel gene of Bashang long-tail chickens. The core promoter region from –840 bp to +68 bp was identified in the pmel gene. The region from ?590 to ?525 bp negatively regulated the pmel gene during the transcription process. The ?840—?590 bp and ?525—?266 bp regions were positive regulatory regions. The polymorphic sites (?456, ?435, ?410, ?374 and ?341) had a significant effect on the promoter activity of the pmel gene.