Expressed sequence tags analysis revealing the taxonomic position and fatty acid biosynthesis in an oleaginous green microalga, Myrmecia incisa Reisigl (Trebouxiophyceae, Chlorophyta)

Abstract

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 characteristics, it is proposed that M. incisa be kept in the genus Myrmecia in which there might be two parallel groups, one living freely and another symbiotic species.

Transcriptome analysis reveals unique C4-like photosynthesis and oil body formation in an arachidonic acid-rich microalga Myrmecia incisa Reisigl H4301

“…The latter is consistent with the unicellular morphology without any flagella during the vegetative growth of M . incisa [11]. The molecular functional distribution indicated the top two assigned categories are binding processes (about 26%) and catalytic activity (about 24%).…”

“…In addition, carotenoids content increased as well under nitrogen starvation in Parietochloris incisa (synonym with M . incisa [11,12]) [13]. Thus, M .…”

“…Some common carotenogenesis genes have been identified in microalgae [20-22], whereas the molecular information for carotenoids biosynthesis in M . incisa is rather limited [10,11]. …”

“…By using Sanger sequencing, a cDNA library from M. incisa generated only 595 unique sequences from 1,854 readable ESTs with little information of interest [11]. In contrast to this sequencing method, next-generation sequencing (NGS) technologies have led to a revolution in genomics and genetics and provided cheaper and faster delivery of sequencing information [23,24].…”

Identification and characterization of three genes encoding acyl-CoA: diacylglycerol acyltransferase (DGAT) from the microalga Myrmecia incisa Reisigl

“…Zhang, Ji'nan University, China. This alga was cultivated in BG-11 medium in 800-mL glass flasks, which were placed in a temperature-regulated photoincubator at 25°C and illuminated from the side with a light: dark regime of 12 h:12 h by Phillips (Amsterdam, Holland) cool-white fluorescent tubes (36 W) at a light irradiance of 115 μmol photons m −2 s −1 [11]. During the cultivation, flasks were shaken several times per day at a regular interval.…”