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为研究草鱼(Ctenopharyngodon idella)miR-33在固醇调节元件结合蛋白-1(Sterol regulatory element binding protein 1,SREBP-1)调节脂代谢中的作用,构建了草鱼SREBP-1基因3’端非翻译区(Untranslated region,UTR)含miR-33靶序列的双荧光素酶报告基因载体。首先用PCR法获得SREBP-1 mRNA含miR-33结合位点的3’UTR序列(379 bp),与双荧光素酶报告载体pmir GLO重组后转染DH5α感受态细胞。然后经筛选和双酶切鉴定,获得含有SREBP-1基因3’-UTR的双荧光素酶报告载体:pmir GLO-SREBP-1-3’-UTR。最后在草鱼肝细胞L8824共转染miR-33mimics和pmir GLO-SREBP-1-3’-UTR,明确miR-33与SREBP-1基因3’UTR区的靶向关系。结果表明,含有SREBP-1基因3’-UTR序列的双荧光素酶报告基因载体构建成功,PCR、双酶切和基因测序证明序列与目标一致;转染pmir GLO-SREBP-1-3’-UTR的L8824细胞可表达荧光素酶,单独转染载体组的荧光素酶活性显著高于共转染载体和miR-33 mimics组(P<0.05)。本研究证实草鱼SREBP-1是miR-33的直接靶基因,miR-33通过和SREBP-1 mRNA 3’UTR结合,调控SREBP-1基因的转录后表达。
To investigate the role of miR-33 in the regulation of lipid metabolism by steroid regulatory element binding protein 1 (SREBP-1) in grass carp (Ctenopharyngodon idella), a 3 ’untranslated (Untranslated region, UTR) dual luciferase reporter vector containing the miR-33 target sequence. First, the 3’UTR sequence (379 bp) of miR-33 binding site of SREBP-1 mRNA was obtained by PCR and transfected into DH5α competent cells after recombination with dual luciferase reporter vector pmir GLO. Then screened and double digested to obtain the dual luciferase reporter vector containing the 3’-UTR of SREBP-1 gene: pmir GLO-SREBP-1-3’-UTR. Finally, miR-33mimics and pmir GLO-SREBP-1-3’-UTR were cotransfected in L8824 cells of grass carp, and the targeting relationship between miR-33 and the 3’UTR of SREBP-1 gene was clarified. The results showed that the dual luciferase reporter gene vector containing 3’-UTR sequence of SREBP-1 gene was successfully constructed, and the sequence was consistent with the target by PCR, double enzyme digestion and gene sequencing. The pmir GLO-SREBP-1-3’- UTR of L8824 cells could express luciferase. The luciferase activity of vector transfected group was significantly higher than that of co-transfection vector and miR-33 mimics group (P <0.05). This study confirmed that SREBP-1 is a direct target of miR-33. MiR-33 regulates the post-transcriptional expression of SREBP-1 by binding to the 3’UTR of SREBP-1 mRNA.