KLF3基因3′-UTR区双荧光素酶报告载体及其突变载体的构建与活性鉴定  

Construction and Activity Identification of Dual Luciferase Reporter Vector and Mutation Vector of KLF3 Gene 3′-UTR

作　　者：翟博[1] 李旭[1] 王春昕[1] 赵云辉[1] 孙铭[2] 张立春[1] 张明新[1] ZHAI Bo LI Xu WANG Chun-xin ZHAO Yun-hui SUN Ming ZHANG Li-chun ZHANG Ming-xin(Sheep Breeding Laboratory, Branch of Animal Husbandry, J ilin Academy of Agricultural Sciences, Gongzhuling 136100, China Tonghua Teachers College Branch, Tonghua 134001, China)

机构地区：[1]吉林省农业科学院畜牧科学分院养羊研究室,公主岭136100 [2]通化师范学院分院,通化134001

出　　处：《中国畜牧兽医》2017年第3期644-650,共7页China Animal Husbandry & Veterinary Medicine

基　　金：国家863资助项目(2013AA102506);国家绒毛用羊产业技术体系(CARS40-16);国家科技支撑计划子课题(2011BAD28B05-1-5);吉林省博士后科研人员科研项目(0010424);吉林省农业科学院博士后科研项目(2060599)

摘　　要：试验旨在构建锌指蛋白3(KLF3)基因3′-UTR区双荧光素酶基因报告载体及其突变载体,初步分析可能调控KLF3基因表达的miRNAs。首先通过PCR方法扩增KLF3基因的3′-UTR序列,将其克隆到经XhoⅠ、NotⅠ双酶切的双荧光素酶报告载体中;运用Targetscan软件预测可能与KLF3基因3′-UTR相互作用的miRNA;使用脂质体2000转染试剂将miRNAs mimics与构建好的KLF3基因3′-UTR段双荧光素酶报告载体或突变载体共转染于常规培养的293T细胞中,检测荧光素酶活性。结果表明,KLF3基因3′-UTR可能是miR-21的作用靶位点;双荧光报告显示,miR-21 mimics组(0.6900±0.0144)比突变组(1.000±0.0688)和空白对照组(1.000±0.0159)KLF3基因3′-UTR双荧光素酶基因报告载体和突变载体的活性降低了31%(P<0.01)。本试验成功构建了含有KLF3基因3′-UTR段双荧光素酶基因报告载体与突变载体,初步证实miR-21对KLF3基因有调控作用。This study was aimed to construct the dual luciferase reporter vector which contained KLF3 gene 3′-UTR and its mutation vector,and analyze the possible miRNAs which could regulate KLF3 gene expression.In this study,The KLF3 gene 3′-UTR was amplified by polymerase chain reaction（PCR）and inserted in the dual luciferase reporter vector which was digested by Xho Ⅰ and Not Ⅰ;With Targetscan to predict the miRNAs which might interact with KLF3 gene 3′-UTR;293Tcells was treated with recombinant vector or its mutation vector or empty vector and miRNA mimics or control by Lipofectamine 2000.The dual luciferase reporter assay system was used to compare the activity of luciferase.The results showed that KLF3 gene 3′-UTR might be the action points of miR-21;Double fluorescent report results showed that miR-21 mimics group（0.6900±0.0144）was decreased 31% than the mutation vector group（1.000±0.0688）and the blank control group（1.000±0.0159）with the luciferase activity of 293 Tcells.This study successfully constructed KLF3 gene 3′-UTR dual luciferase reporter vector and its mutation vector;Preliminary evidence showed that miR-21 could regulate the expression of KLF3 gene.

关 键 词：KLF3基因 双荧光素酶基因报告载体 MIR-21 3′-非编码区 

分 类 号：Q782[生物学—分子生物学]