腐生型眼虫Astasia longa两种TIM同工酶cDNA的克隆和分析  

Cloning and analysis of two triosephosphate isomerases(TIM)cDNA of saprophytic euglenoid Astasia longa

作　　者：申望[1] 文建凡[1] 孙桂玲[1] 董锦润[1] 

机构地区：[1]中国科学院昆明动物研究所细胞与分子进化重点实验室

出　　处：《动物学报》2004年第3期414-419,共6页ACTA ZOOLOGICA SINICA

基　　金：中国科学院生物特支费 (STZ 0 0 2 3 );中国科学院知识创新工程重要方向 (KSCX2 SW 10 1C);云南省中青年学术和技术带头人培养经费 ( 2 0 0 0YP19)资助项目~~

摘　　要：利用 3′ RACE和 5′ RACE技术 ,从腐生型眼虫长变胞藻 (Astasialonga)克隆了磷酸丙糖异构酶(TIM)的两个同工酶cDNA全序列。分析表明 :它们分别编码定位于细胞质的胞质型TIM (cTIM)和定位于质体的质体型TIM (pTIM) ;后者的N端具有引导该酶定位到质体中去的典型“前导序列”。根据这些事实我们推测腐生型眼虫A .longa质体中可能存在功能性的TIM ,并进一步认为该质体可能不只是一般意义上的“叶绿体退化的残迹” 。Astasia longa is a colourless,nonphotosynthetic flagellate protist that is phylogenetically related to E.gracilis ,a photoautotrophic euglenoid.It does not have chloroplasts but plastids.Its plastid genome has lost most photosynthetic related genes,and is only half in size of the chloroplast genome of E.gracilis .In this study,two cDNAs,encoding the isoenzymes of triose phosphate isomerases(TIM),were cloned and sequenced from A.longa by 3′ RACE and 5′ RACE.Both of them have a polyA sequence in the 3′ terminal and a spliced leader sequence(SL sequence,which is a hallmark of englenoids matured mRNA)in the 5′ terminal.These means we have got two complete cDNA(mRNA)sequences.The two sequences are different in length,one is 857 bp and the other 1 273 bp(both without polyA sequence).Sequence alignment showed that the two deduced amino acid sequences were closely homologous to known TIMs from other species(for example,to Arabidopsis thaliana cytoplasmic TIM,amino acid identities are 61% and 55%,respectively),and had the common active site amino acids and important sequence motifs which are conserved in other known TIMs.All of these proved that the two sequences are cDNAs of two TIM isoenzymes.Comparison of the two deduced amino acid sequences indicated that one of them has a longer N terminal.Analyses of the longer N terminal sequence by SignalP,ChloroP and DAS showed that it contained a signal peptide,a transit peptide and two hydrophobic domains,which resembled the bipartite leader sequence structure of chloroplast proteins encoded by nuclear genes in E.gracilis .Thus,the TIM with the bipartite leader sequence must target to plastids,and is a plastid TIM(pTIM),and the other TIM without a leader sequence must target to cytoplasm,and is a cytoplasmic TIM(cTIM).According to these,we argue that the plastid of A.longa might not just be a “chloroplast vestige” but a functional organelle related to TIM at least.

关 键 词：腐生型眼虫 磷酸丙糖异构酶 质体 克隆 同工酶 CDNA 

分 类 号：Q785[生物学—分子生物学]