Characterization of fatty acid desaturase (FAD) genes in Myrmecia incisa and the effect of nitrogen starvation on their transcription

Abstract

The microalgae Myrmecia incise is rich in arachidonic acid(20:4 ω6,AA).The synthesis of AA is thought to begin with the conversion of oleic acid(18:1,oleic acid) to linoleic acid(18:2,linoleic acid,LA) and then proceeding along the ω6 pathway by the catalysis of Δ12,Δ6,Δ5 and other fatty acid desaturases(FADs),based on the fatty acids that have been detected in M.incisa.We cloned the full length cDNAs and the corre-sponding DNAs of the Δ12,Δ6,and Δ5 FAD genes(GenBank accession numbers: JN205757,JN205755,and JN205756,respectively).The full-length cDNAs for the Δ12,Δ6,and Δ5 FAD genes were 1806 bp,2674 bp,and 2318 bp,respectively,and their corresponding ORFs were 1 137 bp,1 443 bp,and 1446 bp in length encoding putative proteins consisting of 378,480,and 481 amino acids,respectively.Compared to their corresponding DNA sequences,the coding sequences of the Δ12,Δ6,and Δ5 FAD genes were interrupted by 4,5,and 7 introns,respec-tively,with all the splice sites matching the "GT-AG" rule.The encoded Δ12,Δ6,and Δ5 FAD proteins were rich in hydrophobic amino acids which accounted for 52.8,46.6,and 50.9% of total amino acids,respectively.The codon preference of these FAD genes was identical to that deduced from other genes using an M.incisa cDNA library.Prediction of transmembrane domains revealed that these three FADs had four transmembrane domains,while Δ5 FAD and Δ12 FAD had another hydrophobic but not transmembrane region.These FADs were conserved,having three histidine boxes similar to the FAD family.The neighbor-joining(NJ) phylogenetic tree inferred from the putative proteins of the FAD genes indicated that the Δ12,Δ6,Δ5,and ω3 FADs in M.incisa were clustered within their corresponding clades,the Δ12 and ω3 FADs were genetically closer,and the Δ6 and Δ5 FADs had a closer genetic relationship.The relative transcription of these three FAD genes was estimated by quantitative real-time PCR(Q-RT-PCR) in M.incisa during culture under nitrogen starvation for 96 h followed by nitrogen replenishment for 72 h.The relative transcription of these three genes increased under nitrogen starvation,then declined rapidly and significantly after M.incisa were transferred to the nitrogen rich medium,suggesting that these FAD genes were subject to regulation by nitrogen.Taking the known variation of fatty acids together,our results suggest that the increased transcription of these FAD genes may play an important role in the synthesis and accumulation of AA when M.incisa is cultured in nitrogen deficient conditions,and that Δ6 FAD is more critical than the remaining FADs.Our observations provide a foundation for investigation of the metabolic pathway and molecular regulation mechanism for AA biosynthesis and accumulation in M.incisa during nitrogen starvation.