鳜鱼肥胖基因的克隆与组织表达  

Molecular cloning and tissue expression analysis of obese gene in Chinese perch(Siniperca chuatsi)

作　　者：李光照[1] 梁旭方[1] 陈亮[1] 王琳[1] 

机构地区：[1]暨南大学生物工程学系,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2010年第3期318-323,330,共7页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：广东省海洋渔业科技推广专项项目(A200899D02);广东省自然科学基金项目(031886;980702)

摘　　要：为了研究脊椎动物肥胖基因(obese gene,ob)结构与功能关系,利用PCR和RACE方法克隆得到鳜鱼肥胖基因全长序列:鳜鱼ob基因全长1 398 bp,由2个外显子和1个内含子构成,其完整的开放阅读框由486 bp组成,编码161个氨基酸.应用Genome Walker方法克隆得到一段长为357 bp的鳜鱼ob基因5′侧翼区序列,并利用相关软件预测其中具有多个保守的顺式调控元件.我们将克隆得到的鳜鱼ob基因编码氨基酸序列leptin与其他物种leptin序列分别进行同源性比较,并构建系统进化树,发现虽然不同物种间leptin序列差异非常大,但进化树分析显示所有的leptin序列,包括哺乳动物、两栖动物和真骨鱼类,各自聚集成簇.最后通过荧光实时定量PCR方法研究鳜鱼不同组织ob基因的表达水平,与哺乳动物ob基因主要在脂肪组织表达分布不同,鳜鱼ob基因在所有被检测组织中均有表达,在肝脏组织中大量表达,其次是脑,在肠道、脂肪、脾和肌肉中只有微量表达,说明鱼类ob基因的表达分布及其调控机制可能与哺乳动物存在差别.In order to study the structural and functional relationship of vertebrate obese gene,full-length obese gene was isolated from Chinese perch by PCR and RACE methods.The Chinese perch ob gene consists of 2 exons and 1 intron and was 1 398 bp in length,which contained a complete open reading frame of 486 bp encoding a 161 amino acid peptide.Using genome walker method,the 5′-flanking region of Chinese perch ob gene was obtained and several potential regulatory elements were identified.Despite the significantly low amino acid sequence identity of leptin between different species,phylogenetic analysis revealed that teleostean leptins branch off before the separation of the amphibian and mammalian leptin cluster.Using Real-Time PCR,we analysed the ob gene tissue expression patterns of Chinese perch.The mRNA expression of Chinese perch ob gene was detected in all the examined tissues： highest in liver,followed by brain,and at low levels in other tissues including adipose tissue,intestine,spleen,and muscle,indicating that the ob gene expression and regulatory mechanism of fish might be different from mammal.

关 键 词：肥胖基因 基因克隆 序列分析 组织表达 鳜鱼 

分 类 号：Q786[生物学—分子生物学]