中华绒螯蟹延伸因子EF-1δ基因全长cDNA克隆及表达  

The full length cDNA cloning and expression of elongation factor 1δ from the Chinese mitten crab(Eriocheir sinensis)

作　　者：郭子好[1] 杨志刚[1] 刘志伟[1] 王瑶[1] 杨筱珍[1] 成永旭[1] 

机构地区：[1]上海海洋大学农业部淡水水产种质资源重点实验室,上海201306

出　　处：《水产学报》2012年第7期1026-1033,共8页Journal of Fisheries of China

基　　金：国家"八六三"高技术研究发展计划(2012AA100809);上海市教育委员会科研创新项目(11YZ151)

摘　　要：根据非洲爪蟾延伸因子-1δ(elongation factor-1δ,EF-1δ)基因的保守序列设计引物,采用逆转录聚合酶链反应(RT-PCR)以及cDNA末端快速扩增(RACE)技术克隆出中华绒螯蟹EF-1δ基因并进行各组织间的表达分析。序列分析表明,中华绒螯蟹EF-1δcDNA全长933 bp,编码263个氨基酸,经BLASTN和BLASTX软件分析表明,此EF-1δcDNA核苷酸序列与非洲爪蟾EF-1δ核苷酸序列的同源性最高,其相似性为70%;所编码的氨基酸序列与大红斑蝶EF-1δ的氨基酸序列相似性为54%。聚类分析表明,中华绒螯蟹EF-1δ的氨基酸序列与鱼虱EF-1δ聚为一支。荧光定量PCR结果显示,EF-1δ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。不同发育状态的中华绒螯蟹EF-1δ在肌肉组织中表达量均显著高于肝胰腺和鳃组织中表达量(P<0.05);3个不同发育状态蟹EF-1δ在肌肉中的表达量也呈显著性差异(P<0.05),在早熟蟹肌肉中表达量最高,正常成熟蟹肌肉中次之,幼蟹肌肉中最低;不同发育状态蟹EF-1δ在肝胰腺和鳃组织中的表达没有显著差异(P>0.05)。Elongation factor （EF） is a protein factor which plays roles in the peptide chain elongation in the process of protein synthesis, including elongation factor 1（EF-1） and elongation factor 2（EF-2）. Elongation factor 1 consists of four subunits α, β, γ and δ, and plays a key role in protein translation process. In this study, we cloned EF-1 ~ gene from Eriocheir sinensis using reverse transcriptase polymerase chain reaction （RT-PCR） and rapid-amplification of cDNA ends （RACE）, and primers were designed according to the conserved sequence of elongation factor-1δ from Xenopus laevis. The full-length cDNA sequence of EF-1δ is 933 bp which codes 263 amino acid residues. And comparison results showed that the nucleotide homology of EF-1δ was 70% similar to Xenopus laevis and amino acid homology of EF-1δ was 54% similar to Danaus plexippus, using BLASTN and BLASTX software. The phylogenetic analysis based on amino acid sequence shows that EF-1δ has highest similarity with EF-18 ofLepeophtheirus salmonis. The expression of the gene in different tissues and stages of E. sinensis was analyzed by real-time fluorescent quantitative PCR. The result showed the EF-1δ mRNA was mainly detected in muscle and small amount in testis, hepatopancreas and trace in heart, ovary, stomach, intestine, gill. EF-1δ mRNA was detected with high level in muscles compared to hepatopancreas and gill in different developmental states of the crab, and displays significant difference at P〈0.05. It also has significant difference at P〈0.05 between muscles in different developmental states, and it was detected the highest expression in precocious crab, followed by mature crab and by a lower expression of crablet; And there is no significant expression difference between hepatopancreas and gill tissues in different developmental states.

关 键 词：中华绒螯蟹 EF-1δ 基因 克隆 表达 

分 类 号：Q785[生物学—分子生物学] S917.4[农业科学—水产科学]