Robo1基因miR-218靶位点序列克隆及其突变体构建  

Cloning of miR-218 target sequences and its mutant of Robo1 gene

作　　者：章倩倩[1] 丁一[1] 王丽京[1] 

机构地区：[1]广东药学院血管生物学研究所,广东广州510006

出　　处：《中国老年学杂志》2013年第15期3646-3648,共3页Chinese Journal of Gerontology

基　　金：国家自然科学基金(31200896);广东省自然科学基金(S2012040007658);广东省医学科研基金(B2012181)

摘　　要：目的克隆Robo1基因3'UTR的miR-218靶位点区域序列并构建"seed"区位点突变体,以便进一步研究miR-218对Robo1基因的调控作用。方法通过targetscan 6.2对Robo1 3'UTR进行分析找出miR-218靶位点,根据NCBI GenBank中Robo1 mRNA序列设计引物,利用RT-PCR技术从人类细胞总RNA中对包含miR-218靶位点的序列进行克隆;并设计一对突变引物,通过重叠PCR法获得"seed"区点突变基因序列;通过双荧光报告基因检测miR-218对Robo1基因的调控。结果经测序验证,成功克隆了目的基因序列,并成功构建了"seed"区点突变体,并且通过报告基因检测发现miR-218对Robo1表达有显著抑制(P<0.05)。结论成功克隆了含有miR-218靶位点的Robo1 3'UTR基因序列及构建了"seed"区点突变体,并阐明miR-218直接作用于Robo1基因3'UTR区,为进一步研究miR-218对Robo1基因的转录后调控机制奠定了基础。Objective To clone the miR-218 target sequence of Robol gene and construct the mutant of ＇ seed＇ region for further researching the regulatory function of miR-218 on Robol. Methods It was found that Robol 3＇ UTR containing miR-218 target site analy- sing by targetscan 6.2, and designed the primers according to the mRNA sequence of Robol from the gene database of NCBI GenBank. Then the miR-218 target sequences of Robol gene was cloned from human cells using RT-PCR assay, and construced the mutant of ＇seed＇ region by overlapping PCR. Therefore, luciferase reporter was performed to determine whether Robol was directed target of miR-218. Results The miR-218 target sequences and its mutant of Robol gene were cloned and verified by sequencing, and Robol was significantly inhibited by miR-218. Conclusions miR-218 target sequences and its mutant of Robol gene are cloned, and Robol is direct target of miR-218, which provides the foundation for investigating the regulatory mechanism of miR-218 on Robol.

关 键 词：ROBO1 MIR-218 靶位点 突变体 

分 类 号：R73-3[医药卫生—肿瘤]