中国医学科学院学报2018，Vol.40Issue(2)：201-210,10.DOI:10.3881/j.issn.1000-503X.2018.02.010
扬子鳄胃蛋白酶原C基因克隆、序列分析及其表达
Molecular Cloning, Sequence Analysis, and Expression of Pepsinogens C Gene from Adult Alligator Sinensis Stomach
黄小梅 1孙龙 2吴孝兵2
作者信息
- 1. 皖南医学院基础医学院病理教研室,安徽芜湖241000
- 2. 安徽师范大学生命科学院安徽省重要生物资源保护和利用重点实验室,安徽芜湖241000
- 折叠
摘要
Abstract
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.关键词
扬子鳄/胃蛋白酶原/基因克隆/序列分析/基因表达Key words
Alligator sinensis/pepsinogen/gene cloning/sequence analysis/gene expression分类
生物科学引用本文复制引用
黄小梅,孙龙,吴孝兵..扬子鳄胃蛋白酶原C基因克隆、序列分析及其表达[J].中国医学科学院学报,2018,40(2):201-210,10.
