The microalgae Myrmecia incise is rich in arachidonic acid (20:4 006, 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 co6 pathway by the catalysis of △12, A6, A5 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 corresponding DNAs of the △12, A6, and AS FAD genes (GenBank accession numbers: JN205757, JN205755, and JN205756, respectively). The full-length cDNAs for the A12, A6, and AS 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 △12, △6, and △5 FAD genes were interrupted by 4, 5, and 7 introns, respectively, with all the splice sites matching the "GT-AG" rule. The encoded A12, A6, and A5 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 △5 FAD and △12 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 △12, △6, △5, and ω3 FADs in M. incisa were clustered within their corresponding clades, the △12 and ω3 FADs were genetically closer, and the A6 and A5 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 A6 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.%为探讨缺刻缘绿藻(Myrmecia incisa)花生四烯酸(20:4ω6,AA)合成过程中一系列脂肪酸去饱和酶(FAD)的作用,本研究克隆了△12、△6及△5 FAD基因的cDNA全长序列(GenBank登录号分别为JN205757、JN205755和JN205756)及其相应的DNA序列.它们的cDNA全长分别为1 806 bp、2 674 bp及2 318 bp,其中ORF长为1 137bp、1 443 bp和1 446 bp,分别编码由378、480和481个氨基酸组成的蛋白；通过其cDNA与DNA序列的比对,发现△12、△6及△5 FAD基因分别具有4、5和7个内含子,其剪切位点均符合“GT-AG”规则.△12、△6和△5 FAD编码蛋白均富含疏水性氨基酸,约占各自氨基酸组成的52.8％、46.6％及50.9％.密码子的偏好性与缺刻缘绿藻其他基因保持一致.推测这3个FAD基因编码蛋白都存在4个跨膜区,而△12和△5 FAD还各有一个明显不跨膜的疏水区；都具有FAD家族各自相对保守的3个组氨酸簇基序.基于缺刻缘绿藻和其他物种相应的FAD基因编码蛋白序列所构建的Neighbor-j oining(NJ)系统进化树,表明△12、△6、△5及ω3 FAD分别隶属于各自的分支,其中,△12与ω3 FAD的亲缘关系较近,而△6与△5 FAD具有较近的亲缘关系.利用实时荧光定量PCR(quantitative real-time PCR,Q-RT-PCR)分析这3个FAD基因在缺刻缘绿藻缺氮培养96 h后又恢复氮培养72 h过程中的相对转录量,结果表明这3个基因的相对转录量都受到氮信号的调控,它们随着氮饥饿培养时间延长明显上升,而氮恢复培养后迅速并显著地下降到氮饥饿胁迫前的水平.结合已知脂肪酸成分在此过程中的变化,推测这3个基因对缺刻缘绿藻AA的合成和积累都起着重要的作用,而△6 FAD的作用可能更关键.
展开▼
