小鼠节律基因clock,bmal1 3'UTR重组质粒构建与应用  

Constructing and applying recombinant plasmid of clock and bmal1 3'UTR

作　　者：王艳利[1] 吕柯[2] 陈海龙[2] 冀国华[2] 王婷梅[1] 张永亮[1] 毕蕾[2] 钟萍[2] 曲丽娜[2] 李莹辉[1,2] 

机构地区：[1]西北工业大学生命科学学院,西安710072 [2]中国航天员科研训练中心航天医学基础与应用国家重点实验室,北京100094

出　　处：《生物学杂志》2016年第4期15-19,共5页Journal of Biology

基　　金：国家科技重大专项项目(2012ZX09J12201);中国航天员科研训练中心航天医学基础与应用国家重点实验室自主项目(Grant No.SMFA09A06;SMFA13B02;SYFD130051864)

摘　　要：为探讨miRNA与节律基因的相互作用,首先通过NCBI查找小鼠节律基因clock,bmal1 3'UTR序列,PCR扩增节律基因clock,bmal1 3'UTR全长后将目的片段定向克隆到PGL3-promoter质粒上并测序验证,构建节律基因clock,bmal1 3'UTR重组质粒。然后通过Target Scan软件预测可能与clock,bmal1 3'UTR相互作用的miRNAs,利用脂质体将miRNA模拟物与重组质粒共转293T细胞进行荧光活性检测,初步分析可能调控clock,bmal1表达的miRNAs。结果显示,采用RVP4引物进行的重组质粒反向测序结果与NCBI上查找的序列反向互补,成功构建小鼠节律基因clock,bmal1 3'UTR重组质粒,为进一步研究节律基因转录后调控机制奠定基础。miR-199a-5p(P<0.05)与miR-142-3p(P<0.01)下调重组质粒活性约33%和45%,初步证实miR-199a-5p能够与clock 3'UTR,miR-142-3p能够与bmal1 3'UTR作用调控基因转录活性。To investigate the effects of miRNAs on the post-transcriptional regulation of circadian genes,we first constructed the clock and bmal1 3＇UTR recombinant plasmid. The 3＇UTR sequence of mouse clock and bmal1 genes searched from the NCBI was awplified,and then cloned into the p GL3- promoter plasmid. Plasmid DNA was subsequently sequenced to ensure the authenticity and direction of the inserted clock and bmal1 3＇UTR. Next,the miRNAs modulation clock and bmal1 expression was analyzed by detecting the luciferase activity. The miRNAs targeting clock,bmal1 gene 3＇ UTR was predicted by Target Scan. The luciferase activity was analyzed in293 T cells which were cotransfection with recombinant plasmid and miRNA mimic by LP2000. Result showed that the sequences analyzed by RVP4 primer were inversely complementary to the sequences in NCBI. The mouse clock and bmal1 3＇UTR recombinant plasmids were successfully constructed,which may lay a foundation for further study of post-transcriptional regulation mechanism of circadian genes. Also the results showed that the luciferase activity of clock and bmal1 3＇ UTR recombinant plasmid was reduced 33% and45% by miR-199a-5p（ P〈0. 05） and miR-142- 3p（ P〈0. 01）,respectively. The activity of clock and bmal1 gene was modulated by miR-199a-5p and miR-142- 3p,respectively.

关 键 词：CLOCK BMAL1 双荧光素酶报告基因 miR-199a-5p miR-142-3p 

分 类 号：Q782[生物学—分子生物学]