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

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 P0.05.It also has significant difference at P0.05 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.