鸭CD8α基因启动子的分析及其转录活性

摘要

[目的]对鸭CD8α基因启动子活性区域进行分析,为鸭CD8α基因功能和表达调控机理研究提供依据.[方法]利用前期基因组步移技术获得的鸭CD8α基因的启动子区序列,制备一系列启动子缺失突变体(-625/-1 bp,-1 110/-1 bp,-1 413/-1 bp,-2 151/-1 bp),定向亚克隆至荧光素酶表达载体pGL3-Basic中,构建荧光素酶报告基因重组载体,采用Lipofectamine 2000将重组质粒瞬时转染DT40细胞,分析CD8α基因启动子系列缺失突变体在细胞内的转录活性.[结果]鸭CD8α基因5'侧翼区长片段具有较强的启动子活性,-1110--625启动子活性最强,且-625--1和-625--1 110 bp区域均存在正调控元件.[结论]成功构建了荧光素酶报告基因真核表达载体,确定了鸭CD8α基因调控区,为进一步研究其转录调控机制奠定了基础.%[Objective] This experiment was conducted to explore the function determination and expression regulation of CD8α gene in ducks by analysis of transcriptional activity of its promoter. [Method] The promoter missing mutants were constructed (-625/-1 bp, -111 0/-1 bp, -1413/-1 bp, -2151/-1 bp) accroding to the sequence of CD8α gene promoter, and then subcloned into pGL3 basic vectors to construct luciferase reporter gene vectors, respectively. The recombinant vectors were transfected into DT40 cells with Lipofectamine 2000, and the transcriptional activities were detected. [ Result] Results of the study indicated that CD8α gene promoter had obviously promoter activity. The sequence from -1 110 to -625 bp of 5' flanking region had the strongest promoter activities, including two positive (-625/-1 bp and -1 110/-625 bp) regulatory domains. [Conclusion] The luciferase reporter gene eukaryotic expression vector with CD8α gene promoter sequences was constructed successfully using deletion mutation and its major regulatory regions were found, which play an important role for analyzing the promoter activity and transcriptional regulation mechanism.