Molecular cloning and analysis of fatty acid desaturase and elongase genes in orange-spotted grouper (Epinephelus coioides)

Full-length cDNAs of fatty acid desaturase(FAD)and fatty acid elongase(ELO)genes,which were involved in the biosynthesis of highly unsaturated fatty acids(HUFA),were isolated from orange-spotted grouper(Epinephelus coioides)by RT-PCR(reverse transcription polymerase chain reaction) and RACE(rapid-amplification of cDNA ends)methods.The results revealed that the FAD cDNA was 1979 bp,including an ORF(open reading frame)of 1338 bp specified a protein of 445 amino acids.The deduced amino acid sequence shared 66.5%-90.8% identity with Sparus aurata,Oncorhynchus keta,Cyprinus carpio and Danio rerio.Analysis online showed that its secondary structure was mainly composed helix and extended-beta,and phylogenetic tree revealed that it clustered closely with Sparus aurata and Dicentrarchus labrax.The protein sequence contained all the characteristics of microsomal FADs,including three histidine boxes,two transmembrane regions,and a cytochrome b5 domain.The 1228 bp cDNA of ELO included 885 bp ORF encoding a 294 amino acid peptide that contained a single histidine box redox centre motif,a canonical ER retention signal and multiple transmembrane regions similar with other ELOs.This protein sequence had 71.1%-85.7% identity with Sparus aurata,Oncorhynchus keta,Oreochromis niloticus and Danio rerio.Analysis online showed that its secondary structure was rich with helix and extended-beta,and phylogenetic tree revealed that it clustered closely with Sparus aurata.These results will help future study on the HUFA biosynthesis pathway in orange-spotted grouper.