中文摘要：

目的克隆Robol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Robol基因的调控作用。方法通过targetscan6．2对Robol3’UTR进行分析找出miR，218靶位点，根据NCBIGenBank中RobolmRNA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Robol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Robol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Robol3’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Robol基因3’UTR区，为进一步研究miR~18对Robol基因的转录后调控机制奠定了基础。

英文摘要：

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.