草鱼小肽转运载体PepT1基因的克隆与表达特征  

Cloning of the Pept1 gene and analysis of its expression in Ctenopharyngodon idellus tissues

作　　者：冯军厂[1,2] 刘臻[1,3] 鲁双庆[1,3] 聂国兴[2] 周玲[1,3] 孙浪[1,3] 

机构地区：[1]长沙学院生物技术与营养研究所,湖南长沙410003 [2]河南师范大学生命科学学院,河南新乡453007 [3]长沙学院生物工程与环境科学系,湖南长沙410003

出　　处：《中国水产科学》2013年第2期276-285,共10页Journal of Fishery Sciences of China

基　　金：国家自然科学基金资助项目(31001114);湖南省高校产业化培育项目(10CY017)

摘　　要：采用同源克隆和RACE技术克隆草鱼(Ctenopharyngodon idellus)PepT1基因的全长cDNA序列。该cDNA全长为2 762 bp,包含141 bp的5′UTR序列,479 bp的3′UTR序列,2 142 bp开放阅读框,编码713个氨基酸;草鱼与鲫(Carassius auratus)、斑马鱼(Danio rerio)的核苷酸同源性分别为77.6%和74.0%,氨基酸同源性分别为78.0%和76.7%;与其他物种的核苷酸同源性为53.9%～59.1%,而氨基酸的同源性为57.2%～61.8%。经预测,其编码蛋白的分子量为79.29 kD,等电点为5.87,该蛋白具有与哺乳动物十分相似的11个螺旋跨膜结构,跨膜区氨基酸高度保守;系统进化分析表明,草鱼PepT1基因与鲫鱼和斑马鱼的亲缘关系最近;利用Real-time PCR技术检测了该基因的时空表达,结果显示,PepT1在草鱼前肠组织表达量最高,其次是肌肉组织;草鱼出膜7 d后PepT1 mRNA表达量相对稳定;昼夜节律研究发现,肠道PepT1基因夜间的表达量较白天高。本研究旨在为小肽转运载体PepT1介导肠道转运小肽调控草鱼对饲料蛋白消化吸收的分子机理提供理论基础。To study the molecular mechanism of the small peptide transporter PepTl-mediated protein digestion and absorption, a full-length cDNA sequence of the PepT1 gene was cloned from Ctenopharyngodon idellus using RT-PCR and RACE techniques. The full-length cDNA sequence of PepT1 had 2762 nucleotides, including 141 nucleotides at 5＇UTR and 479 nucleotides at YUTR. Its open reading frame had 2142 nucleotides encoding a 713-amino-acid peptide. The PepTl gene sequences from C idel[us were most similar to those of Carassius au- ratus and Danio rerio at 77.6% and 74.0%, respectively; the deduced amino acid similarities were 78.0% and 76.7%, respectively; but the PepT1 gene sequence varied in similarity to other animals from 53.9% to 59.1%, and the deduced amino acid similarities were 60.5%--61.6%. The encoded protein molecular weight was predicted at 79.29 kD with pI at 5.87. The PepT1 protein had 11 helix trans-membrane regions; its amino acid sequence was highly homologous to those of other vertebrates. Phylogenetic analysis showed that the PepT1 gene sequence clustered with C. auratus and D. rerio as its closest neighbor. The abundances of PepT1 mRNA assayed by real-time PCR were differentially expressed by tissue type; the highest expression was in the foregut tissue and the second was in the muscle. However, PepT1 mRNA expression was relatively stable after incubation for 7 days. The PepT1 gene expressed rhythmically in the small intestine of C idellus. Its expression was higher during the night and lower during the day. This work provides a theoretical basis for the molecular mechanism of protein digestion and absorption by the small peptide transporter PepT1, which mediates intestinal transport of small pep- tides in vivo.

关 键 词：草鱼 PepT1cDNA 分子特征 mRNA表达丰度 

分 类 号：S965[农业科学—水产养殖]