Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

Abstract

BackgroundPseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions.ResultsThrough microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions.ConclusionOur results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important neurotoxin that affects human health as well as ecosystem function.

Transcriptome sequencing of three Pseudo-nitzschia species reveals comparable gene sets and the presence of Nitric Oxide Synthase genes in diatoms

“…S3 and Table S7) 25 in the toxic P. multistriata with respect to the non-toxic P. arenysensis and did not find substantial differences. In a study comparing P. multiseries cells in the stationary phase (high DA production) versus the exponential phase (low DA production) a number of genes resulted upregulated 26 . We searched for homologs of these genes and found similar FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values for each given transcript in the three species except for SLC6 (Sodium and Chloride-dependent amino acid transporter) for which the mean P. multistriata FPKM value was 7 and 11 fold higher than in P. arenysensis and P. delicatissima , respectively (35.16 FPKM versus 4.92 and 3.15 FPKM), indicating that this transcript might be more expressed in a toxin-producing species.…”

Physiological adjustments and transcriptome reprogramming are involved in the acclimation to salinity gradients in diatoms

“…After a first‐strand cDNA synthesis (RT) with random primers using SuperScript III Reverse Transcriptase (Invitrogen) following manufacturer's protocol, we tested the specificity of each primer via PCR followed by electrophoresis analysis. The histone H2 was chosen as the reference, as proposed previously (Boissonneault et al ., ). qPCR analyses were performed according to the conditions described by the manufacturer for the Brilliant II SYBR® Green QPCR Master Mix (Agilent), using a Stratagene Mx3000p system (Agilent).…”

Transcriptomic responses to grazing reveal the metabolic pathway leading to the biosynthesis of domoic acid and highlight different defense strategies in diatoms

“…Gene expression in Pseudo-nitzschia multiseries at low and high DA production induced by silicate limitation has been investigated [28]. When comparing exponential vs. stationary phase, the study suggests 12 genes to be involved in DA production.…”