鹅垂体特异性转录因子荧光素酶报告基因重组体的构建和鉴定

Construction and Identification of Luciferase Reporter Recombinant Driven by Pit1 Promoter of Goose

为研究垂体特异性转录因子(Pituitary transcription factor 1,Pit1)启动子转录调控机制,将获得的Pit1基因的启动子,插入荧光素酶报告基因载体中,构建Pit1基因启动子调控的报告基因重组表达质粒。本研究利用染色体步移技术扩增鹅Pit1基因的启动子,定向亚克隆至荧光素酶表达载体pGL3-Basic中,构建含有正确目的基因的报告基因重组体pGL3-Pit1,并通过限性内切酶酶切、PCR及测序进行鉴定。通过酶切鉴定及基因测序证明,所克隆的基因产物与预期结果一致,序列无碱基突变。结果,成功构建了含有Pit1启动子基因序列的荧光素酶报告基因真核表达载体,为下一步分析该启动子活性及转录调控机理奠定基础。

To study the possible regulation mechanism of Pit1 gene expression,promoter sequence of Pit1 gene was subcloned and a Pit1 promoter-luciferase reporter vector was constructed.The promoter of Pit1 gene was cloned by genome walking and subcloned into the luciferase expression vector pGL3-Basic directly.The result showed that the recombinant reporter gene pGL3-Pit1 was constructed including correct target gene,and identified by restrictive endonuclease enzyme cutting,PCR and sequencing.The result indicate that the luciferase reporter gene eukaryotic expression vector containing Pit1 promoter sequences was constructed successfully,furthermore,the result will play an important role for analyzing the promoter activity and transcriptional regulation mechanism.