鲤JAK2基因分子克隆及组织表达分析  

Cloning and Expression Analysis of JAK2 Gene in Common Carp, Cyprinus carpio

作　　者：付友[1,2] 高谦[2] 文春根[1] 吴平[2,3] 刘德立[3] 

机构地区：[1]南昌大学生命科学与食品工程学院,江西南昌330031 [2]中国科学院水生生物研究所淡水生态与生物技术国家重点实验室,湖北武汉430072 [3]华中师范大学生命科学学院,湖北武汉4300790

出　　处：《广东海洋大学学报》2015年第1期8-17,共10页Journal of Guangdong Ocean University

基　　金：国家自然科学基金面上项目(31272666);国家自然科学基金面上项目(30871937);国家科技支撑计划课题(2012BAD25B0202)

摘　　要：采用同源克隆策略和RACE-PCR技术,克隆得到可能的鲤(Cyprinus carpio)两面神激酶2(JAK2)基因的c DNA全长序列,包括3 378 bp的开放阅读框,732 bp的5'-非编码区,529 bp的3'-非编码区,总长度达4 639bp。开放阅读框可编码1 125个氨基酸,推测分子质量和理论等电点分别为129.33 ku和6.99。同源性分析显示,克隆的鲤鱼基因与斑马鱼(Danio rerio)JAK2同源性最高,其氨基酸序列同一性和相似度分别达89%和95%。结构域预测表明,所编码氨基酸序列包含FERM、SH2及两个酪氨酸激酶结构域,这4个结构域也保守地存在于其他脊椎动物的JAK分子中。高级结构预测显示,鲤JAK2主要包括α-螺旋(α-helix)、β-折叠(β-sheet)和连接环(loop)等3类结构元件。脊椎动物JAK分子系统进化树显示,JAK1、JAK2、JAK3和TYK2等4类JAK分子分别聚类,鲤JAK2处于JAK2支系中,与所有其他的鱼类JAK2聚为一大支,并与两栖类和哺乳类组成的另一大支构成姊妹群,表明它们具有共同的祖先基因,为直系同源关系。实时荧光定量PCR检测结果表明,鲤JAK2在皮肤中表达量最高,其次是肠、血液和脑,而在肌肉、鳃、头肾、心、肝、脾中表达量较低;鲤JAK3在脾中表达量最高,在其他组织中表达量均很低,甚至未检出。The full-length c DNA sequence of JAK2 gene from common carp, Cyprinus carpio is obtained by using homology cloning and RACE（rapid amplification of c DNA ends） PCR. The full c DNA sequence of common carp JAK2 in length is 4 639 bp, containing a 3 378 bp complete open reading frame（ORF） which encodes 1 125 amino acids, a 732 bp 5＇-untranslated region（5＇-UTR）, and a 529 bp 3＇-UTR, with corresponding molecular mass of 129.33 ku and the theoretical isoelectric point of 6.99. The identity and similarity of its amino acid sequence with the corresponding sequence of zebrafish are 89% and 95%, respectively. Bioinformatics analysis indicated that common carp JAK2 had a FERM domain, an SH2 domain, a pseudokinase domain, and a tyrosine kinase domain. These functional domains are conservative among JAKs from fish, amphibians and mammals, showing that they share similar functional mechanism. The spatial structural elements of carp JAK2 are predicted to include α-helixes, β-sheets, and loops. Based on the phylogenetic tree of JAK2 generated by neighbor joining method in MEGA 4.0, it shows that each kind of JAKs forms a large monophyletic group, and that the JAK2 main-clade is a sister group to the JAK3 one, which means JAK2 is more closely related to JAK3. Moreover, all JAK2 s from the teleost species, including common carp JAK2 in the present study, cluster together, then form the sister group with the second cluster consisting of all from amphibians and mammals. Thus, it is suggested that they should share a common ancestral gene of JAK2 and have remarkable orthologous relationships. The results of real-time quantitive PCR reveal that JAK2 and JAK3 genes are differentially expressed in various tissues of common carp at the level of m RNA. JAK2 is expressed in skin with the highest level, secondly in intestine, blood and brain, and expressed lowly in muscle, head kidney, heart, liver, gill and spleen.The expression level of carp JAK3 in spleen is particularly high, but relatively lower in other tissues. Th

关 键 词：鲤 JAK2 JAK3 基因克隆 组织表达分析 

分 类 号：Q78[生物学—分子生物学] Q959.468