Collaborative Research: Development of Molecular and Biochemical Signatures for the Detection of Toxin Production in Pseudo-nitzchia spp. Under Nutrient Stress

该项目的两个相互交织的目标是确定假菱形藻在毒素产生条件下表达的基因组，并更好地了解环境条件和导致毒素产生的生理反应之间的联系。一组生理学实验将允许评估从基因表达研究产生的分子探针。反过来，分子探针将被用来询问自然种群，并帮助确定该领域的伪菱形藻的生理状态。最终的目标是找到一个特定的基因转录本或基因表达的模式，与毒素的生产领域。以下假设将被测试：H1：有基因或一套基因的表达模式是高度相关的毒素生产在伪菱形藻。H2：蒙特雷湾毒素产生的主要触发因素是硅酸盐限制，因此某些海洋条件允许水华发展。H3：硅酸盐限制可能使细胞对痕量金属（例如铜）应激敏感，并且毒素（软骨藻酸）可以起到金属离子缓冲剂的作用。将评估生长、底物利用和生理参数（可变荧光、营养配额、氨基酸库，包括软骨藻酸）。收获细胞用于在不同应激源下开发cDNA消减文库。从这些文库中开发的基因阵列将为现场测试提供分子探针。 生理学实验产生的信息将有助于鉴定与毒素产生有关的基因，而不是一般代谢。 实验室工作将与有限的实地项目相结合，以评估环境触发因素（如铜、硅酸盐、铁应力）和测试分子探针。 从分子表达和生理测定的结果将允许介导的环境触发器（如硅酸盐和金属）的毒素生产的细胞途径的初步描述。

The two intertwined goals of this project are to determine the suite of genes expressed by Pseudo-nitzschia under toxin-producing conditions, and to acquire a better understanding of the connections between environmental conditions and physiological responses leading to toxin production. A set of physiological experiments will permit evaluation of molecular probes generated from gene expression studies. In turn, the molecular probes will be used tointerrogate natural populations and help determine the physiological status of Pseudo-nitzschia in the field. The ultimate goal is to find a specific gene transcript or a pattern of gene expression that is correlated with toxin production in the field. The following hypotheses will be tested: H1: There are genes or a suite of genes whose expression pattern is highly correlated with toxin production in Pseudo-nitzschia. H2: A primary trigger for toxin production in Monterey Bay is silicate limitation, so that certain oceanographic conditions permit bloom development. H3: Silicate limitation may sensitize cells to trace-metal (e.g. copper) stress and the toxin (domoic acid) can function as a metal ion buffer.Batch and continuous cultures will be stressed with silicate, copper, and iron. Growth, substrate utilization, and physiological parameters (variable fluorescence, nutrient quotas, amino acid pools, including domoic acid) will be assessed. Cells will be harvested for development of cDNA subtraction libraries under different stressors. Gene arrays developed from these libraries will provide molecular probes for field testing. Identification of genes related to toxin production, but not general metabolism, will be facilitated by information generated by the physiology experiments. The laboratory work will be combined with a limited field program for assessment of environmental triggers (e.g. copper, silicate, iron stress) and for testing of the molecular probes. Results from the molecular expression and physiological assays will permit an initial description of the cellular pathways mediating environmental triggers (e.g silicate and metals) for production of toxin.