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

Katie Rose Boissonneault,David E Housman,Stephen S Bates,Jerry Pelletier,Deborah L Robertson,Deborah A Hogan,Sean Milton,Brooks M Henningsen

doi:10.1186/1471-2199-14-25

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.

Highlights

Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid
Pseudo-nitzschia growth and toxin production for microarray studies Samples for microarray analysis were obtained from three biological experiments using Ps-n strain CL-125
Previous studies indicated that Si is the limiting nutrient for Ps-n cells grown in batch cultures with medium f/2 [9,10,14]; we presume that the cells in these experiments were Si-limited during stationary phase

Summary

Introduction

Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. 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. The marine diatom Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) produces the neurotoxin domoic acid (DA), which causes amnesic shellfish poisoning (ASP) [1,2,3,4]. Production of DA by Ps-n, and at least 14 other members of the genus Pseudo-nitzschia, has been verified in oceanic regions throughout the world, primarily in coastal and upwelling zones [7,8]. The biosynthetic pathways leading to DA production and the genes that govern these pathways remain unresolved [12,13]

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