Investigations into the transcriptome of the toxigenic marine dinoflagellate Alexandrium minutum

摘要

Dinoflagellates of the A. minutum species complex are widely distributed bloom-forming, photosynthetic protists typically producing potent neurotoxins which cause paralytic shellfish poisoning (PSP). Human intoxications usually occur via the ingestion of filter-feeding bivalves, but intoxications after consumption of predatory crabs have also been reported. The same group of toxins is produced by certain freshwater cyanobacteria, in which the gene cluster coding for the PSP toxin-producing enzymes has recently been identified. However, the identification of corresponding genes in dinoflagellates is pending, which might be due to their unusual and little understood genomes.The aim of this thesis is to contribute to understanding gene expression and regulation in dinoflagellates by investigating the transcriptome-level gene expression in A. minutum. The gene expression experiments were based on an expressed sequence tag (EST) library, which was generated from pooled samples originating from different treatments to include the highest possible diversity of cDNA sequences. A set of microarray probes based on this library was used for different gene expression experiments, such as the comparison of toxic and non-toxic strains. Three stains were compared at two different times of the light phase, yielding a group of 192 genes differentially expressed between toxic and non-toxic strains at both timepoints. Another experiment was based on the induction of a fivefold increase in toxin levels by the presence of a copepod grazer. In combination with the data obtained from the strain comparison experiment, this resulted in the identification of two sequences potentially correlated to the ability to produce the toxins.Other treatments were designed to represent extreme values of ecologically significant parameters, such as different salinities or nutrient limitation. While the transcriptomic differences between cultures acclimatised to different salinities remained moderate and indicated a high prevalence of differences in post-transcriptional processes, the differences among growth phases and between nutrient-replete and nutrient-limited batch cultures were considerable. By using combinations of comparisons, several genes consistently up-regulated at the transition between exponential growth and stationary phase were identified, as well as 87 genes consistently associated with N- or P-limitation.In the course of this thesis, I identified several A. minutum genes apparently associated with intracellular toxins, as well as patterns of gene expression indicative of culture growth status and of nutrient limitation. These data substantially add to the emerging field of dinoflagellate transcriptomics. Additionally, they provide a starting point for the potential development of new monitoring tools testing the physiological status or toxin-producing potential of A. minutum populations.