靶向奶牛Lepr基因的miR-30d报告基因载体构建及靶向验证

Construction of dairy cow Lepr targeting miR-30d reporter gene vector and verification of its targeting

构建靶向奶牛Lepr基因的miR-30d荧光素酶报告基因载体,验证奶牛乳腺中瘦素受体基因(Lepr)是否为miR-30d的生物学靶基因。将microRNA荧光素酶表达报告载体特异性改造,使其含有TargetScan预测的与miR-30d靶向结合的奶牛Lepr基因序列,对miR-30d与重组荧光素酶载体质粒共转染后的细胞裂解液进行荧光素酶活性检测分析。结果表明,靶向奶牛Lepr基因的miR-30d荧光素酶表达报告载体构建成功,奶牛Lepr基因是miR-30d的靶基因,旨为miR-30d与Lepr基因的相互作用及与此基因相关的乳腺生物网络调控研究奠定实验基础。

To establish luciferase reporter vector of dairy cow Lepr targeting miR-30d, and identify if Lepr is a target gene of mitk-30d in dairy cow mammary gland. The hiciferase expression reporter vector was rebuilt with the sequence which Lepr could combine with miR-30d. The cells which miR-30d and the rebuilt vector were cotransfected in were fragmentated and the luciferase activity was detected. The luciferase expression reporter vector is constructured successfully, Lepr is a target gene of miR-30d. This study establishes the experimental foundation for both the interaction between miR.-30d and Lepr and the regulation of miR-30d on the biological network associated with Lepr.