Collaborative Research: Development of Molecular and Biochemical Markers for Nutrient Stress and Toxin Production in Pseudo-Nitzchia

这个项目的两个相互交织的目标是确定伪菱形藻在产毒条件下表达的一系列基因，以及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。一系列生理学实验将允许对基因表达研究产生的分子探针进行评估。反过来，这些分子探针将被用来询问自然种群，并帮助确定田间假硝化细菌的生理状态。最终目标是找到与田间毒素生产相关的特定基因转录本或基因表达模式。将检验以下假设：H1：在伪菱形藻中存在表达模式与毒素产生高度相关的基因或一套基因。H2：蒙特利湾毒素产生的一个主要触发因素是硅酸盐的限制，因此某些海洋条件允许水华的发展。硅酸盐限制可能会使细胞对微量金属(如铜)的压力敏感，毒素(软骨藻酸)可以起到金属离子缓冲剂的作用。批量和连续培养将受到硅酸盐、铜和铁的压力。将对生长、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)进行评估。在不同的应激源下，获取细胞，用于构建消减文库。根据这些文库开发的基因阵列将为现场测试提供分子探针。通过生理学实验产生的信息，将有助于识别与毒素产生有关的基因，但不是一般新陈代谢。实验室工作将与有限的现场计划相结合，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有助于初步描述介导环境诱因(如硅酸盐和金属)产生毒素的细胞途径。

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.