树鼩Mfsd2a基因的克隆分析和不同组织表达量的检测

Cloning and Analysis of Tree Shrew Mfsd2a Gene and Detection of Its Expression in Different Tissues

目的对树鼩主要促进因子超家族成员2a(Mfsd2a)基因进行克隆、测序和生物信息学分析,并定量检测其组织表达量。方法从树鼩脑和脊髓中提取总RNA,RT-PCR扩增Mfsd2a基因片段,测序后利用生物信息学软件对其序列特征、系统进化、编码蛋白的结构和理化性质进行分析;以树鼩β-actin为内参,qPCR检测树鼩Mfsd2a基因在不同组织中的表达情况。结果克隆获得Mfsd2a基因片段全长1 796 bp,与NCBI上公布的树鼩Mfsd2a基因预测序列高度一致,比对显示其与MFS转运蛋白超家族同源。其编码区全长1 464 bp,编码488个氨基酸。预测结果显示树鼩Mfsd2a蛋白具有明显的疏水性区域,无信号肽,二级结构元件由α螺旋、β折叠、无规卷曲和延伸链组成。OCTOPUS分析发现Mfsd2a存在12个跨膜结构,修饰结构预测发现Mfsd2a蛋白存在两处N糖基化位点,亚细胞定位发现其可能主要分布于细胞膜和内质网。qPCR检测树鼩不同组织的Mfsd2a表达,结果显示各组织均有表达,在大脑、脊髓中的表达水平相对较高。结论成功克隆了树鼩Mfsd2a基因,建立了该基因表达定量检测方法,为今后利用树鼩神经系统疾病模型深入开展Mfsd2a基因功能研究提供了理论和技术支持。

Objective To clone and sequence the major facilitator superfamily domain containing 2 a(Mfsd2 a) gene of tree shrew and analyze its bioinformatics characteristics, and quantitatively detect its expression in different tissues. Methods Total RNA was extracted from the brain and spinal cord of tree shrews, Mfsd2 a gene was amplified by RT-PCR. Bioinformatics software was used to analyze its sequence characteristics, system evolution, phylogenetics, structure and physicochemical properties of the encoding proteins. The expression of Mfsd2 a gene in different tissues were detected by qPCR usingβ-actin as an internal reference. Results The full-length of Mfsd2 a gene fragment was 1 796 bp, which was highly consistent with the tree shrew Mfsd2 a gene sequence published on NCBI. The coding sequence was 1 464 bp, which encoded 488 amino acids. It was also homologous to the MFS transporter superfamily. The tree shrew Mfsd2 a protein had a distinct hydrophobic region, but no signal peptide, and its secondary structural elements were consist of alpha helix, beta sheet, random coil and extended strand. Twelve transmembrane structures were found by OCTOPUS analysis. Two N-glycosylation sites were predicted by modified structure. Subcellular localization revealed that it may be mainly distributed in cell membrane and endoplasmic reticulum. qPCR results showed that Mfsd2 a were expressed in different tissues of tree shrew, and the it was relatively high in brain and spinal cord.Conclusion The Mfsd2 a gene was successfully cloned, and a quantitative detection method for the expression of Mfsd2 a was established, which might provide theoretical and technical support for further study of Mfsd2 a gene function using tree shrew neurological disease model in the future.