大口黑鲈胰岛素基因cDNA全序列的克隆及其编码蛋白的结构分析

Full-length cDNA cloning of insulin gene and its encoding protein structure analysis in largemouth bass(Micropterus salmoides)

胰岛素在鱼体内是一种重要的内分泌激素,其主要的生理功能是调控鱼体的代谢和血糖稳定。用RT-PCR和cDNA末端快速扩增法(RACE)技术克隆了大口黑鲈(Micropterus salmoides)胰岛素基因cDNA全序列,并对其编码的氨基酸序列和蛋白结构进行了分析。结果显示,该基因cDNA序列全长665 bp,其中包括5'端非翻译区101 bp、3'端非翻译区213 bp和开放阅读框351 bp。该开放阅读框编码包括信号肽、B链、C肽和A链的116个氨基酸,其分子量约为12.7 ku,理论等电点为5.44。在线分析表明:推测的大口黑鲈前胰岛素原氨基酸序列存在一段跨膜区;并存在信号肽序列,信号肽分裂位点在Ala24-Phe25处。大口黑鲈与其他硬骨鱼类及脊椎动物的前胰岛素原氨基酸序列的比对结果显示,大口黑鲈前胰岛素原A链和B链氨基酸序列均高度保守,与其他硬骨鱼类的前胰岛素原氨基酸序列相似度较高,为75%～94%,但与其他脊椎动物的相似度较低,为59%～62%。用邻接法构建的系统进化树显示,大口黑鲈前胰岛素原与同为鲈形目的岩钝鲈、红甘鲹和尼罗罗非鱼的亲缘关系较近。研究亮点:采用RT-PCR和RACE技术首次克隆了大口黑鲈胰岛素基因的cDNA全序列,并对其基因结构特征以及相应的蛋白质序列进行了分析,为进一步研究大口黑鲈营养与内分泌的关系,鱼类系统进化和胰岛素在基因工程中的应用等奠定了基础。

Insulin is one of the most important endocrine hormones,playing a critical physiological role in regulating metabolism and glucostasis in fish.In this paper,the complete cDNA of largemouth bass（Micropterus salmoides） insulin gene was cloned by reverse transcription-PCR（RT-PCR） and rapid amplification of cDNA ends（RACE） methods,and its encoding amino acid sequence as well as the protein structure was analyzed.The insulin full-length cDNA sequence consists of 655 bp,comprising a 101 bp 5′-untranslated region,a 213 bp 3′-untranslated region and an open reading frame（ORF） of 351 bp in length.The ORF encodes a preproinsulin of 116 amino acids with a molecular weight of about 12.7 ku and a theoretical isoelectric point of 5.44,from which a signal peptide,a B-chain,a C-peptide and an A-chain could be derived.Online analysis suggests that the deduced preproinsulin contains a transmembrane region,and that it has a signal peptide whose cleavage site occurs between Alanine24 and Phenylalanine25.Comparative analysis of preproinsulin amino acid sequences related indicates that A-chain and B-chain sequences of propreinsulins are highly conserved between teleost and other vertebrate species,and that the preproinsulin amino acid sequence of largemouth bass shares high similarity of 75%-94% to those of other teleosts,but low similarity of 59%-62% to those of other vertebrates.The phylogenetic tree by the Neighbor-joining method shows that the preproinsulin of largemouth bass has high homology with that of some of the other Perciformes fishes,such as rock bass,greater amberjack and tilapia.