Abstract
Fatty acyl desaturase 2 (fads2) is a rate-limiting enzyme in long chain polyunsaturated fatty acids (LC-PUFAs) biosynthesis. In mammals, the lipid metabolism is modulated by a transcription factor, peroxisome proliferator-activated receptor alpha β (pparαβ); however, the detailed mechanism via pparαβ regulates fads2 remains unclear in fish. In the present study, we identified the sequence features of Trachinotus ovatus fatty acyl desaturase 2a (Tofads2a) and fatty acyl desaturase 2b (Tofads2b), which both encoded 442 amino acid polypeptides containing cytochrome-b5-like domains and three representative histidine-rich domains. The Phylogenetic and genome organization analyses revealed characteristic phylogeny: the majority of fads2s exhibited a highly conserved exon/intron architecture. Tissue expression patterns by quantitative real-time PCR (qRT-PCR) showed that the two Tofads2s were prominently expressed in the brain. A nutritional study indicated that the transcription of the two Tofads2s was significantly implicated by treatment with a 1: 1 ratio of fish oil: soybean oil (FO:SO) in the liver and brain. Furthermore, functional characterization in yeast demonstrated that both Tofads2a and Tofads2b possessed Δ4/Δ5/Δ8 desaturation activity. Furthermore, promoter activity assays showed that the expressions of the two Tofads2s were actively regulated by pparαβ. Moreover, mutation analyses showed that the M1 and M4/M5 binding sites of pparαβ were functionally vital for binding to Tofads2a and Tofads2b promoters, respectively. Transcriptional activities of the two Tofads2s promoters were significantly reduced after targeted mutation of M1 or M4/M5. Electrophoretic mobile shift assays (EMSAs) verified that pparαβ interacted with the M1 binding site in Tofads2a promoter to accommodate Tofads2a transcription. Briefly, pparαβ plays an important role in Tofads2 expression and may promote the LC-PUFAs biosynthesis by regulating the expression of two Tofads2s.
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