per1,per2 3′UTR双荧光素酶报告基因重组质粒的构建

Construction of per1,per2 3′UTR Dual-Luciferase Reporter Gene Recombinant Plasmid

构建了包含目的片段小鼠节律基因per1,per2 3′UTR全长的重组质粒,为初步分析转录后可能调控per1,per2基因的miRNAs提供了有效手段。用反转录试剂盒将从小鼠肝脏组织提取的RNA反转录得到cDNA,以获得的cDNA为模板PCR扩增per1,per2 3′UTR全长,经回收纯化、酶切后将目的片段定向克隆到PGL3-promoter质粒上,再经转化扩增挑取克隆进行菌落PCR,对获得的阳性克隆进行酶切鉴定并测序。PCR产物鉴定结果表明,目的片段per1,per2 3′UTR序列扩增成功;菌落PCR及单酶切鉴定结果表明,per1,per2 3′UTR已插入PGL3-promoter载体中;测序结果表明,per1,per2 3′UTR插入序列,插入方向正确。per1,per2 3′UTR双荧光素酶报告基因重组质粒的成功构建为进一步研究节律基因转录后的调控机制奠定了基础。

To analyze the miRNAs regulated per1,per2 expression at post-transcriptional level,per1,per23′UTR dual-luciferase reporter gene recombinant plasmid was constructed.The 3′UTR sequence of mouse per1,per2 genes with the template cDNA reversed from RNA of mouse liver tissue was amplified by PCR,and then cloned into a PGL3-promoter plasmid after recovery,purification and enzyme digestion.The positive clone was selected and further identified by sequencing to ensure the authenticity and direction of the inserted per1,per2 3′UTR.PCR Product identification results suggested that the target fragment per1,per2 3′UTR sequence was amplified successfully.Single colony PCR and restriction enzyme analysis results concluded that per1,per23′UTR had been inserted into PGL3-promoter vector.DNA Sequence results showed that per1,per2 3′UTR was cloned into PGL3-promoter dual-luciferase reporter gene vector with right direction.The per1,per2 3′UTR dual-luciferase reporter gene recombinant plasmid was successfully constructed,which might lay the foundation for a further study of post-transcriptional regulation mechanism of circadian genes.