白氏文昌鱼FADD的克隆及功能研究  

MOLECULAR CLONING AND CHARACTERIZATION OF FAS-ASSOCIATED DEATH DOMAIN(FADD) FROM BRANCHIOSTOMA BELCHERI

作　　者：张晶[1,2] 黄贝[1,2] 高谦[1] 聂品[1] 

机构地区：[1]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,武汉430072 [2]中国科学院研究生院,北京100049

出　　处：《水生生物学报》2009年第6期1175-1184,共10页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金委联合资助基金项目(U0631010)资助

摘　　要：Fas死亡结构域相关蛋白(Fas-associated death domain protein,FADD)是死亡信号转导通路中的连接蛋白,在脊椎动物中其结构和功能都很保守。本文首次克隆了头索动物白氏文昌鱼(Branchiostoma belcheri)FADD(bbFADD)的cDNA和基因组DNA序列。bbFADDcDNA全长1239 bp,编码217个氨基酸。与脊椎动物的FADD一样,bbFADD含有N端的死亡效应结构域(Death Effector Domain,DED)和C端的死亡结构域(Death Domain,DD)。bbFADD氨基酸序列的第33位氨基酸苯丙氨酸在进化过程中相对保守,此苯丙氨酸在FADD自我相互作用中具有重要作用。哺乳类的FADD基因编码区含有两个外显子,而bbFADD基因含有3个外显子。一般认为头索动物处在无脊椎动物进化到脊椎动物的中间过渡阶段,但基于FADD氨基酸序列的系统进化树和同源性分析显示,文昌鱼与海胆的亲缘关系更近。bbFADD在HeLa细胞中超表达能够引起HeLa细胞的凋亡,暗示bbFADD可能能够在人类细胞凋亡通路中起作用,推测凋亡系统在生物进化过程中相当保守。FADD（Fas-associated death domain protein）,also termed MORT1,plays a critical role in the apoptotic signaling pathways of both CD95（Fas/APO-1） and certain members of the tumor necrosis factor receptor（TNFR） superfamily,and is well conserved in vertebrates,especially in mammals.To reveal the features of FADD in lower animals,we have characterized FADD from amphioxus,Branchiostoma belcheri（Chordata,Cephalochordata）,which is a model animal,and have an insight into the origin and evolution of apoptotic signaling system of the vertebrate.Branchiostoma belcheri FADD（bbFADD） cDNA and genomic DNA sequences were obtained by using RACE-PCR and Genomic Walking,respectively.The full-length cDNA of bbFADD consisted of 1239 base pairs（bp） encoding 217 amino acid residues（aa） with a death effector domain（DED）（12— 95th;84 aa） near the N-terminal and a death domain（DD）（129— 211st;83 aa） by the C-terminal.bbFADD genomic sequence,the length of which was 2840 bp,consisted of three exons and two introns in the gene coding region.The structure of bbFADD gene was different from that of its vertebrate counterparts,with two exons.The result of multiple alignments among FADDs from various species supported that DED and DD regions were more conservative than other regions.Furthermore,the 33rd phenylalanine residue（F33） of bbFADD amino acid sequence,which was equivalent to the 25th phenylalanine residue（F25） in human FADD.This site,which was crucial to mediate FADD self-association,was well conserved through sea urchin and human.Homology analysis showed that the percent identity and the percent similarity between bbFADD and vertebrate FADDs were 27.5%— 30.0% and 44.7%— 55.3%,while 35.1%— 36.9% and 51.1%— 52.0% between bbFADD and sea urchin FADD,respectively.In NJ phylogenetic tree based on amino acid sequences of FADDs,B.belcheri was grouped with sea urchin with the suggestion that comparing with lower vertebrate,fish,amphioxus was closely relative to sea urchin although amphiox

关 键 词：FADD 白氏文昌鱼 基因克隆 超表达 细胞凋亡 

分 类 号：Q781[生物学—分子生物学]