cDNA cloning, sequence structural analysis and tissue expression of tail-interacting protein 47 gene (TIP47) in xinong saanen dairy goat | 西农萨能羊47 kD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

Chinese. 47 kD尾部相互作用蛋白基因(tail-interacting protein 47, 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1 906 bp(GenBank收录号为：HQ846827), 包括5'UTR 82 bp，完整编码区 1 314 bp及3'UTR 510 bp，编码蛋白质的氨基酸数为437，分子量47.36 kD，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TIP47 mRNA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。

English. 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 with bovine, 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.