Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries
1 Department of Biological Sciences, Plymouth State University, MSC 64, 17 High St., Plymouth, NH 03264, USA
2 Koch Institute, Massachusetts Institute of Technology, 76-553, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
3 Present address: Mascoma Corporation, 67 Etna Road Suite 300, Lebanon, NH 03766, USA
4 Fisheries and Oceans Canada, Gulf Fisheries Centre, P.O. Box 5030, Moncton, New Brunswick E1C 9B6, Canada
5 Biology Department, Clark University, 950 Main Street, Worcester, MA 01610, USA
6 Present address: Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, MA 02139, USA
7 Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada
8 Department of Microbiology and Immunology, Vail Building Room 208, Dartmouth Medical School, Hanover, NH 03755, USA
BMC Molecular Biology 2013, 14:25 doi:10.1186/1471-2199-14-25Published: 1 November 2013
Pseudo-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.
Through 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.
Our 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.