Identification of Fatty Acid Desaturase 6 in Golden Pompano <i>Trachinotus Ovatus</i> (Linnaeus 1758) and Its Regulation by the PPARαb Transcription Factor

Fatty acid desaturases are rate-limiting enzymes in long-chain polyunsaturated fatty acid biosynthesis. The transcription factor peroxisome proliferator-activated receptor alpha b (PPAR&#945;b) regulates lipid metabolism in mammals, however, the mechanism whereby PPAR&#945;b regulates fatty acid desaturases is largely unknown in fish. In this study, we report the full length cDNA sequence of Trachinotus ovatus fatty acid desaturase, which encodes a 380 amino acid polypeptide, possessing three characteristic histidine domains. Phylogenetic and gene exon/intron structure analyses showed typical phylogeny: the T. ovatus fatty acid desaturase contained a highly conserved exon/intron architecture. Moreover, functional characterization by heterologous expression in yeast indicated that T. ovatus desaturase was a fatty acid desaturase, with &#916;4/&#916;5/&#916;8 Fad activity. Promoter activity assays indicated that ToFads6 desaturase transcription was positively regulated by PPAR&#945;b. Similarly, PPAR&#945;b RNA interference decreased ToPPAR&#945;b and ToFads6 expression at the mRNA and protein levels in a time-dependent manner. Mutation analyses showed that the M2 binding site of PPAR&#945;b was functionally important for protein binding, and transcriptional activity of the ToFads6 promoter was significantly decreased after targeted mutation of M2. Electrophoretic mobile shift assays confirmed that PPAR&#945;b interacted with the binding site of the ToFads6 promoter region, to regulate ToFads6 transcription. In summary, PPAR&#945;b played a vital role in ToFads6 regulation and may promote the biosynthesis of long-chain polyunsaturated fatty acids by regulating ToFads6 expression