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Alternative TitleMolecular cloning and characterization of Fas-associated death domain (FADD) from amphioxus Branchiostoma belcheri
Thesis Advisor聂品
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
KeywordFadd 凋亡 文昌鱼 Hela细胞
Abstract细胞凋亡,又称细胞程序性死亡,是多细胞有机体中广泛存在的生物现象,在机体的发育,免疫应答,清除受损细胞以及维持内环境稳定方面起到了重要的作用。目前有大量的研究阐述细胞凋亡的机制,尤其是在哺乳动物中,然而关于头索动物细胞凋亡方面的研究报道却非常有限。本研究克隆得到了文昌鱼Fas 死亡结构域相关蛋白FADD,并对其做了功能分析。 FADD (Fas-associated death domain protein),又称MORT1,是死亡信号转导中的一个连接蛋白,参与Fas或某些肿瘤坏死因子受体 (tumor necrosis factor receptor,TNFR) 超家族成员介导的细胞凋亡通路。在脊椎动物中,FADD的结构和功能都很保守,为了探讨低等物种中FADD的特性及其在进化中是否保守,我们采用RACE-PCR和Genome Walking 的方法分别克隆得到白氏文昌鱼Branchiostoma belcheri FADD (bbFADD) 的cDNA序列和基因组DNA序列。bbFADD cDNA全长1239 bp,编码217个氨基酸。与脊椎动物的FADD一样,bbFADD同样含有2个结构域,即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)。已报道的人类和鼠的FADD的基因编码区都含有两个外显子,但是bbFADD却含有3个外显子,与海胆FADD基因组结构相似。基于各物种FADD氨基酸序列构建的系统进化树显示,虽然文昌鱼处在无脊椎动物进化到脊椎动物的中间过渡阶段,但是文昌鱼与海胆的亲缘关系更近。bbFADD在HeLa细胞中的超表达能够引起HeLa细胞发生凋亡,证明了我们克隆得到的bbFADD是有生物学功能的,它可能能够参与到人类细胞的凋亡通路中引起细胞发生凋亡,这也从侧面说明了凋亡系统在进化过程中是保守的。
Other AbstractApoptosis, or programmed cell death, is a widespread biological phenomenon in multicellular organisms that plays important roles during development, immune response, elimination of damaged cells, and maintenance of homeostasis. The mechanism of apoptosis was well illustrated, but the knowledge of apoptosis in the species between vertebrate and invertebrate is rather limited. Here we have characterized FADD from amphioxus Branchiostoma belcheri. FADD (Fas-associated death domain), also termed MORT1, plays a critical role in both CD95 (Fas/APO-1) or certain members of the tumor necrosis factor receptor (TNFR) superfamily (called ‘death receptors’) apoptotic signaling pathway, is well conserved in vertebrate species. To reveal the features of FADD in lower species, we have cloned and identified bbFADD (Branchiostoma belcheri FADD) cDNA and genomic DNA sequences by using the methods of RACE-PCE and Genomic Walking. The full length cDNA of bbFADD is 1239 base pairs (bp), encoding 217 amino acids (aa). bbFADD contains two protein interaction domains that are structurally similar to those of vertebrate FADDs, a death effector domain (DED) and a death domain (DD) at its N-terminus and C-terminus, respectively. The reported FADD genomic structure in both human and mouse contain two exons while bbFADD contains three ones. Phylogenetic relationship analysis showed that although amphioxus is at the evolutional intermediate stage from invertebrate to vertebrate, its FADD is more similar to its sea urchin orthologue at the amino acid level. Over-expression of bbFADD in human HeLa cells was able to induce apoptosis indicating that the bbFADD may perform its function by participating in the human apoptotic signaling pathway and the apoptosis system is evolutionarily conserved from amphioxus to mammal.
Document Type学位论文
Recommended Citation
GB/T 7714
张晶. 白氏文昌鱼Fas死亡结构域相关蛋白FADD的克隆及功能研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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