应用于miRNA靶标检测的双荧光素酶报告载体的构建及鉴定  

The Construction and Identification of Dual-luciferase Reporter Plasmids Used in miRNA Target Detection

作　　者：印翠[1] 张俊玲[1] 施志仪[1] 孙文慧[1] 孙近近 

机构地区：[1]上海海洋大学水产与生命学院农业部淡水水产种质资源重点实验室,上海201306

出　　处：《生物技术通报》2015年第12期180-185,共6页Biotechnology Bulletin

基　　金：国家自然科学基金青年项目(41306128);国家自然科学基金面上项目(30271017)

摘　　要：以牙鲆空通气孔同源框2基因(empty spiracles homeobox 2,emx2)为例,构建包含emx2 3'UTR区的野生型和突变型双荧光素酶重组报告表达载体,以期应用于mi RNA靶标的检测。利用Trizol法提取牙鲆成鱼精卵巢混合组织总RNA,参照已克隆出来的emx2基因c DNA序列,设计并合成emx2 3'UTR片段的引物并进行PCR扩增,将得到的基因片段和psi CHECK-2载体双酶切后,用T4 DNA Ligase酶进行连接反应,并转化入DH5α感受态细胞,筛选后得到野生型重组质粒;同时采用定点诱变法对emx2基因进行体外定点诱变并采用同样的方法形成突变型重组质粒。对野生型和突变型重组质粒进行双酶切、琼脂糖凝胶电泳鉴定及测序分析。成功克隆了emx2 3'UTR区,并将emx2 3'UTR区的mi RNA靶点序列GACTTGA突变为AGTCCAG,成功构建了野生型和突变型包含emx2 3'UTR区的mi RNA靶标检测载体。通过RT-PCR、基因重组及定点诱变技术成功构建了应用于mi RNA靶标验证的野生型和突变型双荧光素酶报告载体psi CHECK-emx2-3'UTR和psi CHECK-mutated-emx2-3'UTR。Taking Paralichthys olivaceus empty spiracles homeobox 2（emx2）as an example, we constructed the wild-type and mutant luciferase reporter plasmids containing emx2 3＇UTR region for being utilized in mi RNA target detection. The total RNA was extracted from the mixtures of testis and ovarian in adult fish with Trizol. Using previously-cloned c DNA sequences of emx2 as a reference, a pair of specific primers for emx2 3＇UTR fragment were designed and synthetized, then amplified genes by RT-PCR and psi CHECK-2 vector were treated with double restriction enzyme digestion, and then ligated with T4 DNA Ligase. The ligated fragment was transformed into the competent cells of DH5α, then the wild-type recombinant plasmid were obtained by screening. In vitro site-directed mutagenesis of emx2 was carried out, and a site-directed mutant plasmid was generated by the same methods. The results indicated that the emx2 3＇UTR of P. olivaceus was successfully cloned, and GACTTGA, the sequence of mi RNA target sites in it was mutated to AGTCCAG. The wild-type and mutant luciferase reporter plasmids used in mi RNA target detection were constructed successfully. In conclusion, with the techniques of RT-PCR, gene recombination and site-directed mutagenesis, the wild-type luciferase reporter plasmids psi CHECK-emx2-3＇UTR and mutant one of psi CHECK-mutated-emx2-3＇UTR used in mi RNA target detection were successfully constructed, which lays the foundation for further researches on identification and function of mi RNA target emx2.

关 键 词：牙鲆 MIRNA emx2 定点诱变 psiCHECK-emx2-3'UTR psiCHECK-mutated-emx2-3'UTR 

分 类 号：Q78[生物学—分子生物学] Q55