昆明犬IGF-1基因CDS区克隆、生物信息学分析及组织表达研究

试验旨在系统研究昆明犬胰岛素样生长因子1（insulin-like growth factor-1，IGF-1）基因的结构和功能，揭示该基因的组织表达规律，为探讨工作犬体型及生长发育性状提供基本数据。以昆明犬为试验材料，运用分子克隆技术扩增IGF-1基因CDS区，应用生物信息学方法进行序列同源性比对并构建系统进化树；获得对应的氨基酸序列并分析蛋白理化特性及蛋白亚细胞结构、亲疏水性和磷酸化位点，利用SOPMA和SWISS-MODEL软件预测蛋白二级结构并构建蛋白质三级结构模型；同时利用实时荧光定量方法检测该基因在昆明犬各组织的表达情况。结果显示，昆明犬IGF-1基因CDS区长462 bp，编码153个氨基酸，其核苷酸序列与家犬的同源性较高（100%），与小鼠和鸡的同源性较低（82.6%和81.2%）。IGF-1蛋白分子质量和理论等电点分别为16.97 ku和9.36，属亲水性分泌型蛋白，无跨膜结构，存在信号肽区域（第1-48位氨基酸）；亚细胞定位分析表明，IGF-1蛋白主要分布于细胞核（56.5%）及线粒体（17.4%）中；磷酸化位点分析发现，IGF-1蛋白存在15个磷酸化位点；该蛋白的二级结构以α-螺旋和无规则卷曲为主。组织表达显示，IGF-1基因在昆明犬不同组织中均有表达，尤其在脾脏、肝脏及肾脏中呈现高表达。本试验结果为今后深入研究IGF-1基因功能提供了理论参考，并为深入探讨IGF-1基因对昆明犬生长发育性状研究提供一定的基础资料。

Cloning,Bioinformatics Analysis and Tissue Expression Detection of IGF-1 Gene CDS Regions in Kunming Dogs

This study was aimed to systematically analyze the gene structure and function of insulin-like growth factor-1 (IGF-1) in Kunming dogs,in order to better understand the gene tissue expression pattern,and provide basic data for the study of body size and growth characteristics of working dogs.Kunming dogs was taken as the research object to amplify the CDS region of IGF-1 gene by molecular cloning technique.Sequence homology alignment and phylogenetic tree of IGF-1 gene were constructed by bioinformatics method.The physical and chemical property,subcellular structure,hydrophilicity and hydrophobicity and phosphorylation site of the corresponding amino acid sequence were analyzed by online predicition software.The secondary and tertiary structures of IGF-1 protein were predicted by SOPMA and SWISS-MODEL software.Meanwhile,the expression of IGF-1 gene in different tissues of Kunming dogs was detected by Real-time quantitative PCR.The results showed that the CDS region of IGF-1 gene in Kunming dogs was 462 bp which encoding 153 amino acids.IGF-1 gene CDS in Kunming dogs had higher homology with Canis lupus familiaris (100%),but lower homology with Mus musculus and Gallus gallus (82.6% and 81.2%).The theoretical molecular weight and isoelectric point of IGF-1 protein was 16.97 ku and 9.36,respectively.It was a hydrophilic and secretory protein with no transmembrane structure and its signal peptide region was 1-48 amino acids.The results of subcellular localization showed that IGF-1 protein was mainly located in the nucleus (56.5%) and mitochondria (17.4%).There were 15 phosphorylation sites.The secondary structure of the protein was mainly composed of alpha helix and random coil.Tissue expression showed that IGF-1 gene was expressed in different tissues of Kunming dogs,especially in spleen,liver and kidney.The results laid a theoretical reference for a better study of IGF-1 gene functions,and provided certain basic data for further exploration of growth and development characters of IGF-1 gene in Kunming dogs.