草鱼APN基因的克隆及表达特征  

Clone of aminopeptidase N gene and its expression analysis in Ctenopharyngodon idellus tissues

作　　者：冯军厂[1,2] 聂国兴[1] 刘臻[2] 张建社[2] 王赏初[2] 鲁双庆[2] 

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

出　　处：《水产学报》2012年第6期859-867,共9页Journal of Fisheries of China

基　　金：国家自然科学基金项目(31001114);湖南省高校产业化培育项目(10CY017);河南省高校科技创新杰出人才项目(2010HASTIT020)

摘　　要：氨肽酶N(APN)是肽酶M1家族的成员之一,在蛋白质的消化中发挥重要作用。采用同源克隆和RACE技术首次克隆草鱼APN基因的全长cDNA序列。该cDNA全长为3258bp,包含27bp的5UTR序列,552bp的3UTR序列,2679bp开放阅读框,编码892个氨基酸;草鱼与斑马鱼基因同源性和编码氨基酸同源性分别为81.5%和75.4%,与其他动物同源性分别为58.8%～61.2%和54.3%～60.2%。经预测,其编码蛋白的分子量为100.61ku,等电点为5.14,该蛋白具有与哺乳动物十分相似的1个螺旋跨膜结构,但跨膜区氨基酸同源性较低;系统进化分析表明,草鱼APN基因与斑马鱼的亲缘关系最近;利用Real-timePCR技术检测了该基因的发育表达,结果显示草鱼出膜4d后APNmRNA表达量相对稳定;APN在草鱼前肠、中肠和后肠均有较高的表达量,以前肠组织表达量最高;昼夜节律研究发现,肠道APN基因06:00-18:00的表达量较18:00-06:00高。Aminopeptidase N is a member of the peptidase M1 family, and plays an important role in the digestion of protein. In this study, a full-length cDNA of APN gene was cloned from Ctenopharyngodon idellus with RT-PCR and RACE techniques, and its mRNA expression profile at different tissues was ana-lyzed by real-time PCR method. The full-length of cDNA sequence of APN had 3 258 nucleotides, includ-ing 27 nucleotides at 5’UTR and 552 nucleotides at 3’UTR. Its open reading frame（ORF） had 2 679 nu-cleotides encoding a 892-amino-acid peptide. The deduced amino acid sequences of APN gene from C. idellus displayed the highest similarity with Danio rerio （75.4%）, but varied to other animals from 61.2% to 58.8%. The encoded protein molecular weight was predicted at 100.61 ku with pI at 5.14. Phylogenetic analysis showed that the sequence of APN gene was clustered with D. rerio as its closest neighbor, which shared a sequence similarity of 81.5%, and had lower similarity with other animals from 60.2% to 54.3%. The APN protein had one helix trans-membrane region, but its amino acid sequence of the region demon-strated a low homology relationship to other vertebrates. The abundances of APN mRNA assayed by real-time PCR were differentially expressed at different tissues with a gradient from higher to lower among the tissues of fore-intestine, hind-intestine, liver, mid-intestine, kidney, muscle, spleen and heart, respec-tively. However, the APN mRNA expression was relatively stable after incubation for 4 days. The effects of the circadian rhythms on APN expression of C. idellus showed that there was a time-dependent pattern at higher rhythm during 06：00-18：00 and lower rhythm during 18：00-06：00. Therefore, our study could serve as an important research tool to study the relationship between APN gene’s function and its structure, and investigate its molecular mechanism for protein degradation in vivo.

关 键 词：草鱼 APNcDNA 分子特征 mRNA表达模式 

分 类 号：Q785[生物学—分子生物学] S917[农业科学—水产科学]