Myrmecia incisa is a green coccoid freshwater microalgae, which is rich in arachidonic acid (ArA, C20: 4?-6, Δ5, 8, 11, 14), a long chain polyunsaturated fatty acid (PUFA), especially under nitrogen starvation stress. A cDNA library of M. incisa was constructed with λ phage vectors and a 545 nt expressed sequence tag (EST) was screened from this library as a putative elongase gene due to its 56% and 49% identity to Marchantia polymorpha L. and Ostreococcus tauri Courties et Chrétiennot-Dinet, respectively. Based upon this EST sequence, an elongase gene designated MiFAE was isolated from M. incisa via 5′/3′ rapid amplification of cDNA ends (RACE). The cDNA sequence was 1 331 bp long and included a 33 bp 5′-untranslated region (UTR) and a 431 bp 3′-UTR with a typical poly-A tail. The 867 bp ORF encoded a predicted protein of 288 amino acids. This protein was characterized by a conserved histidine-rich box and a MYxYY motif that was present in other members of the elongase family. The genomic DNA sequence of MiFAE was found to be interrupted by three introns with splicing sites of Introns I (81 bp), II (81 bp), and III (67 bp) that conformed to the GT-AG rule. Quantitative real-time PCR showed that the transcription level of MiFAE in this microalga under nitrogen starvation was higher than that under normal condition. Prior to the ArA content accumulation, the transcription of MiFAE was enhanced, suggesting that it was possibly responsible for the ArA accumulation in this microalga cultured under nitrogen starvation conditions.
cDNA library of Myrmecia incisa H4301 was constructed using λ phage vectors.The library consisted of 1.5×10 6 clones with a recombination rate of 90%,and 1942 clones were randomly sequenced.All 1854 readable expressed sequence tags(ESTs) were clustered into 596 non-redundant sequences(NRSs),among which 126 NRSs were from 1384 ESTs,showing a high redundancy.Among the 596 NRSs,30 were ribosomal RNA,and 152 significantly matched with those available in NCBI database and JGI Genome Portal,the latter were divided into nine subcategories.Overall,59 NRSs were involved in photosynthesis,the respiratory electron transport chain,ATP synthesis,oxidation reduction,fatty acid biosynthesis,glucose metabolism,protein metabolism,and small molecular metabolism,suggesting that these genes were abundantly transcribed during energy and substance metabolism.Acyl-carrier protein,ferrodoxin and fatty acid elongase genes obtained from this cDNA library enabled presumption of a possible biosynthesis pathway of ArA in M.incisa.Codon usage analysis of 142 NRSs with 17798 codons in the predicted coding regions showed that the average G+C content level of the total codons was 55.39%,and that of the third position in base trimers was 66.42%,indicating a strong bias toward cytosine and/or guanosine in this algal genome.Among all synonymous codons,NAG was most favored,while NUA was most avoided.Phylogenic trees inferred from ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit genes and the extra partial sequences of 18S rRNA obtained from this library demonstrated that M.incisa belonged to Trebouxiophyceae and was significantly clustered with M.incisa SAG 2007,Lobosphaera tirolensis,M.bisecta,and Parietochloris incisa,but was clearly distant from P.pseudoalveolaris and P.alveolar.Transmission electron microscopy revealed pyrenoids traversed by many parallel thylakoids membranes,while starch grains were only clearly observed when cells were grown under nitrogen stress.Based on combined investigation of the phylogeny and morphological
