草鱼SREBP-1-3'-UTR双荧光素酶报告载体构建及miR-33对其表达的影响  

Construction of dual luciferase recombinant vector containing SREBP-1-3'-UTR of Ctenopharyngodon idella and effect of miR-33 on its expression

作　　者：王俊丽[1] 卢荣华[2] 秦超彬 常志光[2] 孙君君[2] 杨峰[2] 聂国兴[2] WANG Junli LU Ronghua QIN Chaobin CHANG Zhiguang SUN Junjun YANG Feng NIE Guoxing(College of Life Sciences, Henan Normal University, Xinxiang 453007, Henan, China College of Fisheries, Henan Normal University, Xinxiang 453007, Henan, China)

机构地区：[1]河南师范大学生命科学学院,河南新乡453007 [2]河南师范大学水产学院,河南新乡453007

出　　处：《上海海洋大学学报》2017年第1期17-22,共6页Journal of Shanghai Ocean University

基　　金：国家自然科学基金(31372545;31402311);河南省高校科技创新团队支持计划(14IRTSTHN013);河南省科技创新杰出青年支持计划(154100510009);河南省基础与前沿技术研究项目(142300410158);河南省教育厅科学技术研究重点项目(13A240548)

摘　　要：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 e binding protein xplore the role of miR-33 in the regulation of fat metabolism by sterol regulatory element 1 （SREBP-1）, the dual-luciferase reporter assay vector containing SREBP-1 gene 3＇- untranslated region of Ctenopharyngodon idella has been constructed. The 3＇-UTR of SREBP-1 with miR-33 combining site was first amplified （with the length of 379 bp） and transfeeted into competent cells （DH5α） after being reeombined with dual-lueiferase reporter assay vector （pmirGLO）. Screening and identifying with XbaI and SaeI to digest the recombinant vector were then conducted. Finally, the relationship between miR- 33 and the 3＇-UTR of SREBP-1 was analysed by the co-transfeetion of the recombinant vector and miR-33 mimics into L8824 cells. The eleetrophoretogram of PCR products, the restriction map of recombinant vector, and the result of gene sequencing comfirmed that the dual-lueiferase reporter assay vector containing 3＇-UTR of SREBP-1 was successfully constructed. In addition, the activity of lueiferase expressed by transfected 1B824 cells was detected and the expression level of the control was significantly higher than that of the co- transfected group including the recombinant vector and miRNA-33 mimics. The results suggested SREBP-1 is the target gene of miR-33. By binding to the 3＇-UTR region of SREBP-1, miR-33 plays an important role in the regulation of post-transcription of SREBP-1.

关 键 词：草鱼 SREBP-1 miR-33 3'非翻译区 pmirGLO报告基因载体 荧光素酶活性 

分 类 号：S917[农业科学—水产科学]