EDGE FGT: Essential New Molecular Genetic Tools for Defining Phenotype in the Global, Harmful Algal Bloom-producing Diatom, Pseudo-nitzchia spp.

危害全球生态系统、渔业和人类健康的赤潮是由一种硅藻属--伪菱形藻以及几个甲藻属和蓝藻引起的。在过去的十年里，在澳大利亚、巴西、加利福尼亚州、智利、法国、缅因湾、印度尼西亚、意大利和突尼斯的沿海水域，各种伪菱形藻都产生了大量的神经毒素--软骨藻酸(DA)。目前，关于海洋酸化、大气二氧化碳浓度增加、降水和营养胁迫将如何影响生产力和假菱形藻物种赤潮的全球范围，仍存在很大的不确定性。事实上，已经出现了与有毒的伪菱形藻有关的长期趋势。在较温暖的年份普遍存在的较温暖的海洋温度下，水华导致贝类渔业更频繁地受到DA污染。我们在硅藻分子遗传学、当前的硅藻模式生物、海洋生物化学和显微镜方面的集体经验，以及我们最近发现和表征了迄今未知的DA生物合成途径，使我们处于理想的地位，可以建立伪Nitzschia sp.作为新的模式硅藻。在这样做的过程中，将发展分子生物学方法，使对DA的调节和生物合成的基因组到表型组的研究成为可能。具体地说，将开发一种由细菌结合驱动的转化假菱形藻属的方法，并将其传播到世界各地的海洋实验室。我们的研究将建立一种新的硅藻模型--伪菱形藻，并生产和传播最先进的分子生物学方法，以表征驱动软骨藻酸(DA)的基因组-物候组联系，这是有害的藻类在世界范围内大量繁殖。采取双轨方法，我们建议显著升级硅藻群落目前使用的功能基因组和分子生物学工具。Track I，我们将构建一个细菌-硅藻结合方案和一个伴随的、可维护的Episome，用于在两个全球分散的、高度产毒的硅藻物种--拟南菱形藻和多纹藻中传递转基因基因。开发和推广硅藻转化的新方法将增加可用于功能基因组学研究的硅藻的数量和种类。Track II，具有强大的伪菱形藻遗传操作工具。在适当的地方，我们将开发一种新的硅藻工具包，用于分子和生化应用。将开发的前三种工具是：a)纳米体荧光蛋白(NB-FP)，可在体内抑制或激活目标蛋白的功能；b)可诱导的荧光生物传感器(UnaG)，可在实验室或海水培养中检测DA；c)邻近标记酶：TurboID和mini Turbo，可深入了解体内蛋白质之间的相互作用。随着拟菱形藻接合转化协议的开发，我们将表达模式生物三角褐指藻中的每一种蛋白质组工具并对其进行故障排除。该奖项反映了NSF的法定使命，并已通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

Harmful algal blooms (HABs) threatening ecosystems, fisheries and human health worldwide, are driven by one diatom genus, Pseudo-nitzschia, along with several dinoflagellate genera and cyanobacteria. In the last decade, various species of Pseudo-nitzschia have produced devasting blooms of the neurotoxin, domoic acid (DA), in the coastal waters of Australia, Brazil, California, Chile, France, the Gulf of Maine, Indonesia, Italy and Tunisia. Currently there is great uncertainty regarding how ocean acidification, increased concentrations of atmospheric CO2, precipitation and nutrient stress will shape the productivity, and global range of Pseudo-nitzschia species' HABs. Indeed, long term trends have emerged that link toxic Pseudo-nitzschia spp. blooms to more frequent DA contamination of shellfish fisheries in the warmer ocean temperatures that prevail during warmer years. Our collective experience with diatom molecular genetics, current diatom model organisms, marine biochemistry and microscopy and our recent discovery and characterization of the hitherto unknown pathway for DA biosynthesis places us in ideal position to establish, Pseudo-nitzschia sp., as new model diatom. In so doing, molecular biological methods will be developed to enable genome-to-phenome investigations of the regulation and biosynthesis of DA. Specifically, a method to transform Pseudo-nitzschia sp driven by bacterial conjugation will be developed and disseminated to marine labs worldwide. Our research will establish a new model diatom, Pseudo-nitzschia sp., and produce and disseminate state-of-the-art molecular biological methods to characterize the genome-phenome linkages that drive domoic acid (DA), harmful algal blooms worldwide. Taking a two-track approach, we propose to significantly upgrade the functional genomic and molecular biological tools currently used by the diatom community. Track I, We will construct a bacteria-diatom conjugation protocol and a concomitant, maintainable episome for transgenic gene delivery in Pseudo-nitzschia australis and multistriata, two globally dispersed, highly-toxigenic diatom species. Developing and disseminating new methods for diatom transformation will increase the number, and variety of species of diatoms available for functional genomics studies. Track II, With robust genetic manipulation tools for Pseudo-nitzschia spp. in place, we will develop a new diatom toolkit for molecular and biochemical applications. The first three tools to be developed are a) nanobody-fluorescent proteins (Nb-FPs) that can either repress or activate the target protein's function in vivo; b) inducible fluorescent biosensors (UnaG) that can detect DA in laboratory or seawater cultures; c) proximity-labeling enzymes: TurboID and miniTurbo, that provide insights into protein-protein interactions in vivo. As the development of the conjugative transformation protocol for Pseudo-nitzschia spp proceeds, we will express and troubleshoot each of the proteomic tools in the model organism, Phaeodactylum tricornutum.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.