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

该项目的两个相互交织的目标是确定伪nitzschia在毒素产生条件下表达的基因组合，并更好地了解环境条件与导致毒素产生的生理反应之间的联系。一组生理实验将允许评估从基因表达研究产生的分子探针。反过来，分子探针将被用于询问自然种群，并帮助确定伪尼齐亚在野外的生理状态。最终目标是找到与田间毒素产生相关的特定基因转录物或基因表达模式。以下假设将被检验：H1：假性nitzschia中存在一些基因或一组基因的表达模式与毒素产生高度相关。H2：蒙特利湾产生毒素的主要原因是硅酸盐的限制，因此某些海洋条件允许水华的发展。H3：硅酸盐限制可能使细胞对微量金属（如铜）的压力敏感，而毒素（软骨藻酸）可以作为金属离子缓冲剂。批量和连续培养将用硅酸盐、铜和铁进行强化。将评估生长、底物利用和生理参数（可变荧光、营养配额、氨基酸池，包括软骨藻酸）。在不同的胁迫条件下，收集细胞进行cDNA减法文库的构建。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，而不是一般的代谢。实验室工作将与有限的现场项目相结合，以评估环境触发因素（例如铜、硅酸盐、铁应力）和测试分子探针。分子表达和生理分析的结果将允许对介导环境触发因素（如硅酸盐和金属）产生毒素的细胞途径进行初步描述。

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.