西农萨能羊47kD尾部相互作用蛋白基因(TIP47)的cDNA克隆、序列结构分析与组织表达  

cDNA Cloning, Sequence Structural Analysis and Tissue Expression of Tail-interacting Protein 47 Gene (TIP47) in Xinong Saanen Dairy Goat

作　　者：郝娟[1] 罗军[1] 王紫骞[1] 赵旺生[1] 孙雨婷[1] 钟瑜[1] 李君[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2011年第5期864-872,共9页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育项目(No.2009ZX08009-162B);陕西省重大科技创新项目(No.2009ZKC07-01-1);省部级公益性行业(农业)科研专项(No.201103038)共同资助

摘　　要：47kD尾部相互作用蛋白基因(tail-interacting protein47,TIP47)是影响脂滴形成和代谢的重要基因。本实验以西农萨能羊(Capra hircus)的乳腺组织为研究材料,采用RT-PCR和RACE,克隆了山羊TIP47基因的cDNA全长序列。结果得到TIP47基因cDNA序列全长1906bp(GenBank收录号为:HQ846827),包括5'UTR82bp,完整编码区1314bp及3'UTR510bp,编码蛋白质的氨基酸数为437,分子量47.36kD,等电点(pI)5.14。对TIP47基因的核苷酸和氨基酸序列分析发现,山羊的TIP47与GenBank中牛(Bos taurus)、人(Homo sapiens)、小鼠(Mus musculus)和猪(Sus scrofa)的TIP47相似性较高。蛋白质结构分析显示,TIP47不存在信号肽序列和跨膜结构;疏水性分析发现,其N端疏水性较强,中间靠近C端部分亲水性较强;三级结构分析发现,其C端呈大写的L形;该基因的遗传距离分析发现,山羊与牛的亲缘关系最近,其次是猪和人。通过RT-qPCR技术对TIP47基因进行了组织表达分析,发现所检测的12个组织中均有TIP47mRNA的表达,其中乳腺组织中表达最丰富,且远远高于其它组织,脾脏、肺脏次之,肌肉相对最少。研究结果提示,TIP47基因可能在乳腺组织中对脂滴的形成代谢过程有重要作用。Tail-interacting protein 47 gene（TIP47） is important for lipid droplet biogenesis and metabolism, to study the function and expression of goat TIP47 gene, we cloned TIP47 from Xinong Saanen goat（Capra hircus） mammary gland total RNA using RT-PCR and RACE PCR. The results showed that the cDNA sequence of goat TIP47 was 1 906 bp in length （GenBank accession No. HQ846827）, including 82 bp of 5＇UTR, 1 314 bp of CDS, and 510 bp of 3＇UTR, encoding a protein of 437 amino acids with a molecular weight of 47.36 kD and pI 5.74. Nucleotides and amino acid（aa） sequences alignment showed that the nucleotides and aa of goat TIP47 had high score similarity with bovine （Bos taurus）, human （Homo sapiens）, mouse （Mus musculus） and pig （Sus scrofa） in GenBank. Primary protein structure analysis showed that TIP47 did not contain signal peptide sequence and transmembrane structure; the N-terminal of the protein was more hydrophobic than that of the C-terminal; the C-terminal of the protein was shaped like an L in tertiary structure. Goat TIP47 had the closest relationship withbovine, followed in pig and human, which was generated by phylogenetic tree analysis. TIP47 was expressed in all 12 tissues examined by Real-time fluorescence quantitative PCR, the result showed that the TIP47 mRNA expression in mammary gland was the highest and far higher than those of all other tissues examined, followed in spleen and liver; TIP47 expression in muscle was the lowest. The results suggest that TIP47 plays a potentially role in biogenesis and metabolism of lipid droplet in goat mammary gland, setting the base for function studies of this gene in lipid droplet activites.

关 键 词：西农萨能羊 47kD尾部相互作用蛋白(TIP47) 脂滴形成 基因克隆 序列结构分析 组织表达 

分 类 号：Q78[生物学—分子生物学]