猪胰岛素样生长因子-Ⅰ的克隆及生物信息学分析  

Cloning and Bioinformatics Analysis IGF-I Gene in Pig

作　　者：王锐[1] 赵跃[1] 张永云[2] 李卫真[3] 王淑燕[3] 霍海龙[1] 刘丽仙[1] 苗永旺[3] 霍金龙[3] 

机构地区：[1]云南农业职业技术学院畜牧兽医系,云南昆明650031 [2]云南农业大学农科专业基础实验教学示范中心,云南昆明650201 [3]云南农业大学动物科学技术学院,云南昆明650201

出　　处：《西南农业学报》2014年第2期864-869,共6页Southwest China Journal of Agricultural Sciences

基　　金：云南省教育厅科学研究基金项目(2011Y058;2011Y219);国家自然科学基金项目(31160439);云南省应用基础研究计划面上项目(2010ZC241)

摘　　要：为了克隆猪胰岛素样生长因子-Ⅰ(IGF-I)以及检测该因子在各组织中的转录水平,分别提取猪13种组织RNA并反转录为cDNA,利用RT-PCR方法扩增IGF-I编码区全长序列,将纯化的片段与pMD18-T载体连接,转化大肠杆菌DH 5α,筛选阳性克隆并测定其序列,利用生物信息学方法分析其序列特征,并与牛等5个物种构建系统进化树。同时采用半定量RT-PCR方法分析IGF-I在猪13种组织中的表达情况。结果显示,猪IGF-I编码区全长462 bp(GenBank登录号:JX827417),编码153个氨基酸。氨基酸二级结构预测表明无规卷曲占的比例最大,为57.52%。氨基酸序列同源性分析表明,与牛、马、羊、鹿、鸡等5个物种的相似性分别为98.0%、97.4%、96.1%、96.1%、83%,多组织半定量RT-PCR研究表明IGF-I mRNA在猪的13个组织中均有表达,其中在肌肉、肝脏、脾脏、卵巢、大肠中表达量较高,其他组织略低。In order to clone the pig insulin-like growth factor Ⅰ （IGF-Ⅰ） and detect the transcription level of the factor in different tissues,the RNA of 13 kinds of tissues was extracted,then the RNA was subjected to reverse transcription to form cDNA,and the gene encoding region entire sequence of the IGF-Ⅰ was amplified through a RT-PCR method,then the purified product of PCR was ligated with a pMD18-T and transferred into the bacterium DH5α for replication.The positive clones were screened and sequenced.The sequence character analyzed by using bioinformatics method and phylogeny evolution tree was constructed with other five species,such as cattle.Meanwhile,the semi-quantitative RT-PCR was used to analyze the expression profile of IGF-Ⅰ gene in 13 kinds tissues.The result showed the coding region of IGF-Ⅰ gene was 462 bp （GenBank accession number：JX827417）,and coded 153 amino acids.The prediction of secondary structure indicated that random coils were percent 57.52.The amino acid homology analysis showed that pig IGF-Ⅰ had high homology with the IGF-Ⅰ of five species：cattle （98.0 ％）,horse （97.4 ％）,sheep （96.1％）,deer （96.1％）,chicken （83 ％）.Semi-quantitative RT-PCR analysis of mRNA from different tissues indicted that IGF-Ⅰ mRNA expressed in 13 tissues,and the expression level was higher in muscle,liver,spleen,ovary and large intestine than other tissues.

关 键 词：胰岛素样生长因子 基因克隆 生物信息学分析 组织表达 

分 类 号：S828[农业科学—畜牧学]