Cellular and transcriptional regulation of the seafood toxin domoic acid

项目总结/摘要 海洋有害藻华（HABs）对人类健康构成重大环境威胁，沿海 生态系统和海洋食物供应。由于气候变化，有害藻华预计将继续增加， 严重性和频率，影响沿海社区的数百万人。有史以来最大的赤潮，包括 主要是有毒的假菱形硅藻，横跨北美西海岸，从阿拉斯加到巴哈 2015年期间的半岛。这种水华产生的高水平的神经毒素软骨藻酸（DA）导致了主要的 太平洋西北部的渔业关闭，以保护人类健康。虽然水华监测和毒素检测 是相当复杂的，相对来说，我们对海洋有害藻华产生毒素的基本原理知之甚少 物种了解编码赤潮毒素生物合成的基因的调控和表达， 通过引入遗传成分来帮助预测水华的潜在毒性，从而改进水华监测。 这个建议的目标是描述DA生物合成的细胞和转录调控 利用实验室模拟系统，培养的拟菱形藻属（Pseudo-nitzschia spp.）分离物和环境样本。我 最近的工作揭示了编码拟菱形藻中DA生物合成途径的成簇基因。我 现在我计划将我的研究扩展到硅藻系统中DA生物合成（dab）基因的研究。通过描述 DA生物合成发生在细胞中，我们可以将DA生物合成置于整个细胞生理学的背景下。 此外，在培养物和环境样本中追踪dab基因表达可以帮助我们将 基因转录到水华毒性。我假设，一个完整的表征细胞和转录 毒素生物合成的调节将使我们能够在转录水平上监测拟菱形藻HAB的毒性。 我们的研究结果也将有助于社会了解海洋条件，诱导DA生产的盛开。 为了实现这些目标，我将使用该模型探索Dab酶的亚细胞定位 硅藻Phaeodactylum tricornutum作为异源宿主，使用微生物学特化的分子技术。下一个是 我将探讨dab基因在假菱形藻属菌株中的表达。在各种DA诱导下生长 培养条件，探讨环境输入的毒性。最后，我将应用我们的研究结果，从培养 通过从每周过滤器样本生成元转录组学数据集，对更大的环境进行实验 在2015年北美伪菱形藻水华之前、期间和之后在蒙特雷湾采集的。 这个建议是为了补充我以前对DA生物合成的研究，使我能够继续研究。 进一步的学术培训机会，以及我的博士学位。摩尔教授和艾伦教授我 为了涵盖所有要素，我们选择了具有多样性和互补性科学专长的顾问 的拟议研究。

Project Summary/Abstract Oceanic harmful algal blooms (HABs) pose a major environmental threat to human health, coastal ecosystems, and marine food supplies. Due to climate change, HABs are anticipated to continue increasing in severity and frequency, impacting millions in coastal communities. The largest HAB ever recorded, comprised primarily of toxic Pseudo-nitzschia diatoms, spanned the North American west coast from Alaska to the Baja peninsula during 2015. High levels of the neurotoxin domoic acid (DA) produced by this bloom resulted in major fishery closures in the Pacific Northwest to protect human health. Although bloom monitoring and toxin detection is quite sophisticated, relatively little is known about the underlying basis for toxin production among marine HAB species. Understanding the regulation and expression of the genes encoding HAB toxin biosynthesis could improve bloom monitoring by introducing a genetic component to help predict a bloom’s potential for toxicity. The goal of this proposal is to characterize the cellular and transcriptional regulation of DA biosynthesis by using laboratory model systems, cultured Pseudo-nitzschia spp. isolates, and environmental samples. My recent work has uncovered the clustered genes that encode the DA biosynthetic pathway in Pseudo-nitzschia. I now plan to extend my research to study DA biosynthesis (dab) genes in diatom systems. By describing where DA biosynthesis takes place in the cell, we can place DA biosynthesis in the context of overall cellular physiology. In addition, tracking dab gene expression both in culture and in environmental samples can help us correlate gene transcription to bloom toxicity. I hypothesize that a full characterization of the cellular and transcriptional regulation of toxin biosynthesis will enable us to monitor Pseudo-nitzschia HAB toxicity at the transcript level. Our findings will also help the community understand the oceanic conditions that induce DA production in blooms. To achieve these goals, I will explore the subcellular localization of the Dab enzymes using the model diatom Phaeodactylum tricornutum as a heterologous host using diatom-specialized molecular techniques. Next, I will explore dab gene expression in isolates of Pseudo-nitzschia spp. grown under a variety of DA-inducing culture conditions to explore environmental inputs to toxicity. Finally, I will apply our findings from the culturing experiments to the larger environment by generating metatranscriptomics datasets from weekly filter samples collected in Monterrey Bay before, during, and after the 2015 North American Pseudo-nitzschia bloom. This proposal is designed to supplement my prior research on DA biosynthesis, allowing me to pursue further academic training opportunities together with my Ph.D. co-advisors, Professors Moore and Allen. My advisors have been chosen for their diverse and complementary scientific expertise in order to cover all elements of the proposed research.