Robo1基因miR-218靶位点序列克隆及其突变体构建

摘要

Objective To clone the miR-218 target sequence of Robo1 gene and construct the mutant of ‘seed' region for further researching the regulatory function of miR-218 on Robo1.Methods It was found that Robo1 3' UTR containing miR-218 target site analysing by targetscan 6.2,and designed the primers according to the mRNA sequence of Robo1 from the gene database of NCBI GenBank.Then the miR-218 target sequences of Robo1 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 Robo1 was directed target of miR-218.Results The miR-218 target sequences and its mutant of Robo1 gene were cloned and verified by sequencing,and Robo1 was significantly inhibited by miR-218.Conclusions miR-218 target sequences and its mutant of Robo1 gene are cloned,and Robo1 is direct target of miR-218,which provides the foundation for investigating the regulatory mechanism of miR-218 on Robo1.%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.