Fatty acid desaturases are rate-limiting enzymes in long-chain polyunsaturated fatty acid biosynthesis. The transcription factor peroxisome proliferator-activated receptor alpha b (PPARαb) regulates lipid metabolism in mammals, however, the mechanism whereby PPARαb 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 Δ4/Δ5/Δ8 Fad activity. Promoter activity assays indicated that ToFads6 desaturase transcription was positively regulated by PPARαb. Similarly, PPARαb RNA interference decreased ToPPARαb and ToFads6 expression at the mRNA and protein levels in a time-dependent manner. Mutation analyses showed that the M2 binding site of PPARαb 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 PPARαb interacted with the binding site of the ToFads6 promoter region, to regulate ToFads6 transcription. In summary, PPARαb played a vital role in ToFads6 regulation and may promote the biosynthesis of long-chain polyunsaturated fatty acids by regulating ToFads6 expression.
Keywords: PPARαb and Fads6; Trachinotus ovatus; fatty acid desaturases; heterologous expression; transcriptional activity.