鳜小肽转运载体PepT1基因分子特征及其表达研究

MOLECULAR CHARACTERIZATION AND EXPRESSION RESEARCH OF OLIGOPEPTIDE TRANSPORTER PEPT1 IN SINIPERCA CHUATSI

小肽转运载体(PepT1)是低亲和力、高容量的肽转运载体,在小肽的吸收过程中发挥着重要的作用。研究采用同源克隆和RACE技术克隆了鳜鱼(Siniperca chuatsi)PepT1基因全长cDNA序列,其cDNA序列全长为2480 bp,包含43 bp的5′UTR序列,232 bp的3′UTR序列,以及2205 bp开放阅读框,编码735个氨基酸。氨基酸序列同源性分析结果显示,鳜鱼与石斑鱼(Epinephelus aeneus)、鲈鱼(Dicentrarchus labrax)PepT1间同源性均为89%,与其他非鱼类物种的同源性则在46%—56%。经预测,鳜鱼PepT1编码蛋白的分子量为64.8 kD,等电点为8.97,该蛋白具有与哺乳动物同源蛋白相似的12个螺旋跨膜结构,并且在跨膜区9和10之间有一个大的外环;跨膜区氨基酸高度保守,并存在有5个膜外N-糖基化位点和3个膜内含蛋白激酶C基序的相同区域。实时荧光定量表达分析表明,鳜鱼PepT1基因在前肠和中肠中表达量显著高于后肠(P<0.05),这说明前、中肠是鳜鱼肠道吸收小肽的主要部位;在胚后不同发育阶段鳜鱼前肠均能检测到PepT1基因的表达,并且在10 g个体中表达量最高,之后随着体重的增加其表达量维持在一个稳定水平。本研究结果首次报道了鳜鱼PepT1基因全序列及其分子表达特征,为鱼类营养及生理学的研究提供有价值的参考资料。

Small peptide transporter（PepT1） is a transporter with low-affinity and high capacity peptide,which plays an important role in the peptide absorption.In order to reveal the molecular mechanism of the small peptide transporter PepT1-mediated protein digestion and absorption,the small peptide transporter PepT1 gene of the Siniperca chuatsi was cloned by using RT-PCR and RACE techniques.The full-length cDNA sequence of the PepT1 was 2480 bp,including 43 nucleotides at 5′UTR and 232 nucleotides at 3′UTR and 2205 nucleotides as an open reading frame encoding a 735-amino-acid peptide.The PepT1 amino acid sequence was determined and it shared a similarity to those of other mammalian homolog protein,which included 12 helix trans-membrane regions and existed a large outer-ring between 9th and 10th of the transmembrane region.Homologous analysis of the PepT1 showed that the amino acid of the PepT1 was highly homologous to those of other vertebrates and it showed high percentages of similarities at 89% with Epinephelus aeneus and Dicentrarchus labrax.In addition,the PepT1 amino acid sequence varied in similarity to other vertebrates from 46% to 56%.The encoded protein molecular weight was predicted at 64.8 kD with pI at 8.97.The quantitative RT-PCR analysis demonstrated that the foregut and midgut may be the key parts of intestinal absorption of small peptides as the PepT1 expression in the foregut and midgut was significantly higher than the hindgut（P＆lt;0.05）.The PepT1 gene expression was detected in all post-embryonic developmental stages,and it showed a peak expression the growing to about 10g in weight,then the expression remained stable in late developmental stages.The significance of the work first reported the PepT1 cDNA structure and its expression pattern,furthermore,it provided a valuable reference for research on fish nutrition and physiology.