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)

  • 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-1δ cDNA全长933 bp, 编码263个氨基酸,经BLASTN和BLASTX软件分析表明, 此EF-1δ cDNA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)。

     

    Abstract: 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-1δ 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-1 δ 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-1 δ has highest similarity with EF-1δ of Lepeophtheirus 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.

     

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