西农萨能羊胰岛素诱导基因2(INSIG2)的克隆及组织表达分析

摘要

Insulin induced gene 2 (INSIG2) plays an important role in regulating the biogenesis and metabolism of lipid, while the synthesis and metabolism of lipid in goat mammary gland is closely related to milk quality. In order to clarify the relationship between them and provide further scientific support to improve the goat dairy flavor, we used RT-PCR to clone INSIG2 from Xinong Saanen goat {Copra hircus) mammary gland total RNA and analyze its expression in ten tissues. The results showed that we got a sequence of 915 bp (GenBank accession No. JQ361767), including 678 bp of open reading frame, which encoded a protein of 225 amino acids with a predicted molecular weight of 24.8730 kD and pI8.77. Nucleotides and amino acid (aa)sequences alignment showed that the nucleotides and aa of goat INSIG2 had the highest score similarity with bovine (Bos taurus) in GenBank. Protein structure analysis showed that INSIG2 contained six transmembrane helices; hydrophobicity analysis indicated that both N-terminal and C-terminal of the protein were hydrophilic; the genetic distance analysis showed that goat had the closest relationship with bovine, followed by pig (Sus scrofa). RT-qPCR was used to examine the mRNA expression of INSIG2 in ten tissues, and the result showed that INSIG2 was expressed in all 10 tissues, the lung had the highest expression of INSIG2, followed by muscle, there was moderate expression in mammary gland, and the minimal expression was in heart. The study of INSIG2 gene cloning and tissue expression analysis will provide a basis for function studies of the gene in goat mammary gland.%胰岛素诱导基因2(insulin induced gene 2,INSIG2)是参与脂质形成和代谢调控的重要基因,而山羊乳腺中脂质合成和代谢与乳品质密切相关.为进一步明确它们之间的关系进而为改善山羊乳品风味提供科学支持.本实验以西农萨能羊(Capra hircus)乳腺组织为研究材料,采用RT-PCR技术克隆山羊INSIG2基因,并采用实时荧光定量(RT-qPCR)技术在山羊10个组织中对INSIG2基因进行了组织表达分析.研究获得了915 bp的cDNA序列(GenBank登录号:JQ361767),其中编码区678 bp,该基因编码225个氨基酸,预测蛋白质分子量为24.8730kD,等电点(pI)为8.77.通过核苷酸和氨基酸序列比对分析发现,山羊的INSIG2与GenBank中牛Bos taurus)的相似性最高.蛋白质结构分析显示,INSIG2存在6个跨膜螺旋；疏水性分析发现,其N端和C端均具有亲水性；遗传距离分析发现,山羊与牛的亲缘关系最近,其次是猪(Sus scrofa).INSIG2基因的组织表达分析表明,INSIG2基因在10个被检测组织中均有表达,且肺组织中表达量最高,肌肉次之,乳腺组织的表达量居中,心脏组织中最低.研究结果为该基因在山羊乳腺组织中的功能研究提供依据.