扬子鳄胃蛋白酶原C基因克隆、序列分析及其表达

摘要

目的 探讨扬子鳄胃蛋白酶原C (PGC)基因的分子克隆和结构特征,研究其系统进化关系和组织定位表达,分析成年扬子鳄在不同生命活动时期胃黏膜组织中PGC基因表达的变化规律.方法 采用反转录PCR和cDNA末端快速扩增技术,从扬子鳄胃黏膜中克隆PGC基因cDNA全长序列;利用生物信息学方法和工具预测其蛋白的理化参数和高级结构;通过Clustal X软件对扬子鳄和其他脊椎动物PGC氨基酸序列进行序列比对,使用MEGA6软件构建系统进化邻接树;应用免疫组织化学法对扬子鳄胃黏膜组织中PGC进行组织定位;用实时定量PCR法检测PGC mRNA在不同生命活动时期胃黏膜组织中的表达水平.结果 从扬子鳄胃黏膜中克隆并获得扬子鳄PGC基因cDNA序列,该序列全长1568 bp,其中开放阅读框1167 bp,编码388个氨基酸;递交GenBank数据库,获得登录号为KY799383.扬子鳄PGC蛋白相对分子质量为41 998,理论等电点为4.16.同源建模法预测该蛋白具有2个天冬氨酸催化活性位点及6个必需半胱氨酸残基位点.系统进化关系表明,扬子鳄PGC氨基酸序列与密河鳄紧密聚为一支,然后与其他鳄类聚在一起.扬子鳄PGC特异性地表达于胃黏膜组织,且繁殖期与冬眠期表达差异有统计学意义(P＜0.05).结论 扬子鳄PGC基因具有高度保守性,其蛋白是胃特有的一种具有消化功能的天冬氨酸蛋白酶.%Objective To investigate the molecular clone and structural features of pepsinogen C (PGC) gene in the stomach of Alligator sinensis,explore the phylogenetic relationships and tissue distribution,and analyze the variation of PGC expression in the stomachs of adult Alligator sinensis at different life stages.Methods The full-length cDNA of PGC gene of Alligator sinensis was cloned by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends and then sequenced.The physical and chemical parameters and advanced structures of the PGC protein were predicted by bioinformatics methods and tools.The PGC amino acid sequences of the Alligator sinensis and other vertebrates were compared by Clustal X software.The neighbor-joining phylogenetic tree was built by MEGA 6 software.Immunohistochemistry was used to locate PGC in the gastric mucosa of Alligator sinensis.The variation of the PGC mRNA levels in the stomach at different life stages was detected by quantitative real-time polymerase chain reaction.Results Reverse transcription polymerase chain reaction and rapid amplification of cDNA ends revealed a 1568 bp cDNA full-length sequence containing 1167 bp open reading frame,which encoded 388 amino acids.The PGC gene of Alligator sinensis had been deposited in the GenBank Data Libraries under the accession number of KY799383.Bioinformatics analysis predicted that the Alligator sinensis PGC had a theoretical relative molecular mass of 41 998 with a theoretical isoelectric point of 4.16.In addition,the three-dimensional structure of the PGC was constructed by homology modeling to predict its active site with two essential aspartyl residues and six essential cysteine residues involved in forming three disulphide bonds.The neighbor-joining phylogenetic tree of vertebrates from the amino acids sequences of PGC showed all crocodiles were clustered as a group,and the PGC of Alligator sinensis was the closest to Alligator mississippiensis.Alligator sinensis PGC was specifically expressed in the gastric mucosa,and its expressions significantly differed during reproduction and hibernation significantly (P ＜ 0.05).Conclusions Alligator sinensis PGC gene is highly conserved in evolution.Its protein is a gastric specific digestive proteinase that belongs to a aspartic proteinase family.