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 Pitl gene expression, promoter sequence of Pitl gene was subcloned and a Pitl promoter-luciferase reporter vector was constructed. The promoter of Pitl 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-Pitl was constructed including correct target gene, and identified by restrictive endonucle-ase enzyme cutting, PCR and sequencing. The result indicate that the luciferase reporter gene eu-karyotic expression vector containing Pitl promoter sequences was constructed successfully, furthermore, the result will play an important role for analyzing the promoter activity and transcrip-tional regulation mechanism.
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