褐牙鲆中miRNAs与甲状腺激素受体TRβ的相互作用研究  

Interaction between miRNAs and Thyroid Hormone Receptor TRβin Bastard Halibut Paralichthys olivaceus

作　　者：刘素萍[1] 付元帅[1] 喻杰[1] 张俊玲[1] 施志仪[1] LIU Suping;FU Yuanshuai;YU Jie;ZHANG Junling;SHI Zhiyi(Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Engineering Research Center of Aquaculture, National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University, Shanghai 201306, China)

机构地区：[1]上海海洋大学,农业农村部淡水水产种质资源重点实验室,上海水产养殖工程技术研究中心,水产科学国家级实验教学示范中心,上海201306

出　　处：《水产科学》2020年第3期299-305,共7页Fisheries Science

基　　金：国家自然科学基金资助项目(41676138).

摘　　要：甲状腺激素通过与其受体结合对褐牙鲆变态起重要的调控作用,而miRNAs通过结合靶基因3′-UTR在转录后水平调节靶基因的表达。为探讨miRNAs在褐牙鲆变态中的作用,通过生物信息学方法预测褐牙鲆TRβ基因3′-UTR存在的miRNAs结合位点,并利用3′-RACE方法克隆TRβ基因的3′-UTR序列,通过体外DNA重组技术构建应用于miRNAs靶向基因检测的双荧光素酶重组报告载体,利用细胞转染和双荧光素酶报告技术分析多个miRNAs与TRβ基因的作用关系。研究结果显示,克隆得到了一段长为437 bp的褐牙鲆TRβ基因的3′-UTR序列,发现3′-UTR序列上存在pol-miR-125a、pol-miR-214、pol-miR-460-5p、pol-miR-138和pol-miR-125a*的结合位点,成功构建了双荧光素酶重组报告载体,命名为pmirGLO-TRβ-3′-UTR。双荧光素酶报告分析结果表明,pol-miR-125a、pol-miR-214、pol-miR-460-5p和pol-miR-138均为作用于褐牙鲆TRβ的靶向miRNAs,而pol-miR-125a*对TRβ基因无直接的调节作用。研究结果将为后续深入研究miRNAs与TRβ在褐牙鲆变态发育中的功能及其作用机制提供基础。Thyroid hormone(TH)plays an important role in regulating bastard halibut Paralichthys olivaceus metamorphosis through binding to its receptors(TRs),which is one of the key members of TRs,while miRNAs regulate the expression of target genes at the post-transcriptional level by binding to the 3′-untranslated region(3′-UTR)of target genes.We predicted that the binding site of multiple miRNAs might is in the 3′-UTR of TRβgene in bastard halibut by bioinformatics.In order to further explore the relationship between miRNAs and TRβ,in this study,total RNAs from bastard halibut adults were extracted by the Trizol method.The 3′-UTR sequence of TRβgene was cloned from the total RNA by 3′-RACE method;then the PCR primers for the desired restriction sites SacⅠand XbaⅠrequired for the recombinant plasmid containing dual-luciferase reporter gene were designed and synthesized according to the cloned 3′-UTR sequence of TRβgene.After PCR amplification,the target 3′-UTR cDNA sequence of TRβgene containing specific restriction enzyme site was obtained,whose gene fragment and pmirGLO vector were digested by double enzymes.The ligation reaction was performed with T4 DNA Ligase and transformed into DH5αcompetent cells.The recombinant plasmid was extracted,and then double enzyme digestion,agarose gel electrophoresis and sequencing validation were performed.The results showed that the dual-luciferase recombinant reporter vector was successfully constructed and named as pmirGLO-TRβ-3′-UTR;finally five predicted miRNAs(pol-miR-125a,pol-miR-214,pol-miR-460-5p,pol-miR-138 and pol-miR-125a*)were selected to examine the interaction with TRβ,five miRNA mimics and the recombinant plasmid pmirGLO-TRβ-3′-UTR constructed above were transfected into 293T cells,and the luciferase reporter gene system was used to detect the luminescence value.The analysis revealed that bastard halibut TRβis target gene of pol-miR-125a,pol-miR-214,pol-miR-460-5p and pol-miR-138,but it is not a target gene of pol-miR-125a*.These findings

关 键 词：褐牙鲆 MIRNA TRβ pmirGLO-TRβ-3′-UTR 双荧光素酶报告基因技术 

分 类 号：S917[农业科学—水产科学]