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p53基因3′UTR双荧光素酶报告质粒的构建及其与miRNA-2127靶向关系的验证

Construction of Dual-Luciferase Reporter Plasmids by 3′UTR Region of p53 Gene and Their Targeted Relationship with miRNA-2127

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【作者】 张玉霞袁小远王友令孟凯

【Author】 Zhang Yuxia;Yuan Xiaoyuan;Wang Youling;Meng Kai;Institute of Poultry Sciences, Shandong Academy of Agricultural Sciences;

【通讯作者】 孟凯;

【机构】 山东省农业科学院家禽研究所

【摘要】 为了验证miRNA-2127与p53基因的靶向关系,利用生物信息学软件预测p53与miRNA-2127的结合位点,全基因合成法合成该结合位点的野生型与突变型模板,并将其克隆到pmiR-RB-REPORTTM双荧光素酶报告载体中,构建野生型与突变型重组双荧光素酶报告质粒。将293T细胞分为4组,分别共转染野生型报告质粒+阴性对照(NC)、野生型报告质粒+miRNA-2127、突变型报告质粒+NC、突变型报告质粒+miRNA-2127。检测各组细胞中荧光素酶活性差异,结果显示:共转染了野生型报告质粒+miRNA-2127的293T细胞与共转染了野生型报告质粒+NC组相比,荧光素酶活性显著降低(P<0.05);且突变型报告质粒+miRNA-2127组的相对荧光素酶活性显著高于野生型报告质粒+miRNA-2127(P<0.05),说明miRNA-2127能够靶向调控p53基因,且结合位点位于3′UTR区369—375间。

【Abstract】 To verify the targeted relationship between miRNA-2127 and p53, the binding sites of p53 and miRNAs-2127 were predicted by bioinformatics software. The wild-type and mutant templates of the binding sites were synthesized by the whole gene synthesis method, and were cloned into pmiR-RB-REPORTTM dual-luciferase reporter vector to construct the wild-type and mutant-type recombinant dual-luciferase reporter plasmids. The cultured monolayer 293 T cells were divided into 4 groups including co-transfected wild-type reporter plasmid + NC control, co-transfected wild-type reporter plasmid + miRNA-2127, co-transfected mutant reporter plasmid + NC control and co-transfected mutant reporter plasmid + miRNA-2127, respectively. The difference of luciferase activity in each group was detected. The results showed that compared with co-transfected wild-type plasmid + NC control, the relative luciferase activity of 293 T cells in the group with co-transfected wild-type plasmid + miRNA-2127 showed a significant decrease(P<0.05), and that of co-transfected mutant reporter plasmid + miRNA-2127 group was significantly higher than co-transfected wild-type reporter plasmid + miRNA-2127 group. The results indicated that miRNA-2127 could targeted regulate p53 and the binding sites were located between 369 and 375 in the 3′ UTR region.

【关键词】 p53miRNA-2127双荧光素酶报告系统
【Key words】 p53miRNA-2127Dual-luciferase reporter system
【基金】 山东省自然科学基金项目(ZR2017BC039);山东省农业科学院高层次人才及创新团队引进计划项目;山东省农业科学院青年科研基金项目(2015YQN61)
  • 【文献出处】 山东农业科学 ,Shandong Agricultural Sciences , 编辑部邮箱 ,2019年01期
  • 【分类号】Q78
  • 【被引频次】3
  • 【下载频次】704
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