中华绒螯蟹(Eriocheir sinensis)延伸因子EF-2基因全长cDNA克隆及表达

THE FULL LENGTH cDNA CLONING AND EXPRESSION OF ELONGATION FACTOR 2 FROM THE CHINESE MITTEN CRAB ERIOCHEIR SINENSIS

采用RT-PCR和RACE方法克隆了中华绒螯蟹延伸因子EF-2全长cDNA,序列分析表明EF-2全长2752bp,编码846个氨基酸。经BLASTX分析表明,EF-2核苷酸序列与镶边拟蠢蟹EF-2序列的同源性最高,其相似性为88%;所编码的氨基酸序列与甲壳动物EF-2的氨基酸序列相似性都在90%以上。聚类分析显示,中华绒螯蟹EF-2的氨基酸序列与镶边拟蠢蟹EF-2聚为一支,并与其它甲壳动物聚为一体。荧光定量PCR结果显示,EF-2在正常成熟中华绒螯蟹肌肉中表达量最高,精巢、肝胰腺中有少量表达,心脏、卵巢、胃、肠、鳃中有微量表达。检测了不同发育状态的幼蟹、早熟蟹和正常成熟蟹EF-2在肝胰腺、鳃以及肌肉中的相对表达情况;同时也检测了在不同pH处理下幼蟹EF-2在肝胰腺和鳃中随时间变化的相对表达情况,结果显示pH胁迫对幼蟹EF-2表达有一定的诱导效果。

In this study, we cloned EF-2 gene from Eriocheir sinensis using reverse transcriptase polymerase chain reaction （RT-PCR） and rapid-amplification of eDNA ends （RACE）, the full-length eDNA sequence of EF-2 was 2752bp which coded 846 amino acid residues. Comparation results which was used by BLASTX software showed that the nucleotide homology of EF-2 was 88% similar to Libinia emarginata＇s and amino acid homology of EF-2 was higher than 90% similar to others＇ crustaceans. The phylogenetic analysis based on amino acid sequence showed that EF-2 had highest similarity with EF-2 of L. emarginata and clustered with other crustaceans. The expression ofEF-2 gene in different tissues and stage of normal mature crab were analyzed by real-time fluorescent quantitative PCR. The result showed that EF-2 mRNA was mainly detected in muscle and small amount in testis, hepatopancreas and trace in heart, ovary, stomach, intestine and gill. We examined the expression of the EF-2 in hepatopancreas, gills and muscles in different developmental status of crablet, precocious crab and normal mature crab; and also examined the expression of EF-2 in hepatopancreas and gills in crablet which were exposed to different pH, the results showed that pH stress can induce the expression of EF-2.