八肋游仆虫Rab家族新成员Eo-rab-1N基因的克隆与序列分析

Rab蛋白家族属于小分子GTP结合蛋白家族Ras超家族中最大的亚家族，主要在囊泡运输中起作用。本实验运用PCR、RT-PCR等技术，从八肋游仆虫中克隆到一种新的rab基因。序列分析结果表明：在大核中，该基因全长884bp，除去两端的端粒与非编码区，该基因在大核中由723bp组成。从小核中克隆相应的基因片段，此基因片段序列与大核中序列一致，表明该基因在小核中无内部删除序列的存在。通过RT-PCR，从mRNA获得的该基因的开放读框为663bp，表明该基因在转录过程中有内含子的删除。大核基因序列和cDNA序列比较，发现60bp的内含子序列位于大核基因的153~212bp之间，并符合一类内含子GU-AG剪切规则。在遗传密码使用上，该基因内部含有2个TGA，在游仆虫中编码半胱氨酸。同时首次发现，八肋游仆虫基因使用TAG作为终止密码子。NCBI上序列比对表明该基因翻译的蛋白与其它物种Rab1蛋白的同源性达49%~52%，因此我们将它命名为Eo-rab-1N，GenBank登录号为DQ105562。Eo-rab-1N与其他物种的Rab1蛋白构建进化树，发现该蛋白的进化与物种的进化保持一致，表明该基因在细胞中具有重要功能。

Cloning and Sequence Analysis of a Novel Member of the Rab Gene Family from Euplotes octocarinatus

Rab proteins belong to a subfamily of small GTP-binding proteins of the Ras superfamily,which play an important role in intracellular vesicular traffic.In this study,a rab gene was obtained from Euplotes octocarinatus by polymerase chain reaction(PCR) and RT-PCR.The rab gene from macronucleic DNA was 884 bp in length,including non-coding regions and telomeric sequences at both ends.The rab gene from micronuclear DNA(723 bp),lacking of internal eliminated sequences,was identical to rab gene from macronuclear DNA.RT-PCR showed that the opening reading frame of the rab gene was 663 bp long.The rab gene from macronuclear DNA contained an intron of 60 bp at the position from 153 bp to 212 bp of macronuclear DNA.The rab gene had two in-frame TGAs encoding for cysteine in Euplotes octocarinatus.The rab gene used TAG as stop codon,which was the first report in Euplotes octocarinatus.The result of BLAST in NCBI demonstrates that the Rab shares a homology of 49%~52% at the amino acid level with Rab1 proteins from a number of other eukaryote,which suggesting that the Rab is a Rab1 homolog.The rab gene was therefore designated Eo-rab-1N(GenBank accession number: DQ105562).The evolution of Eo-rab-1N was analyzed using phylogenetic tree of amino acids sequences of Rab1 obtained from GenBank.