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

EDGE FGT：用于定义全球有害藻华产生硅藻、拟菱形藻表型的重要新分子遗传工具。

• 批准号: 2103715
• 负责人: Andrew Allen
• 金额: $ 160万
• 依托单位: J. Craig Venter Institute, Inc.
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2103715&HistoricalAwards=false
• 关键词: EDGE; FGT; Essential; New; Molecular

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.

有害藻华（HABs）威胁着全球生态系统、渔业和人类健康，是由一种硅藻属伪菱形藻（Pseudo-nitzschia）、沿着几种甲藻属和蓝藻引起的。在过去的十年里，各种假菱形藻在澳大利亚、巴西、加州、智利、法国、缅因州湾、印度尼西亚、意大利和突尼斯的沿海沃茨水域产生了神经毒素软骨藻酸（DA）的毁灭性水华。目前，海洋酸化、大气CO2浓度增加、降水和营养物质压力将如何影响生产力以及伪菱形藻有害生物的全球范围，这方面存在很大的不确定性。事实上，长期趋势已经出现，有毒的假菱形藻属。在温暖的海洋温度下，在温暖的年份，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，以及我们最近对迄今未知的DA生物合成途径的发现和表征，使我们处于建立假菱形藻的理想位置，作为新的硅藻模型在这样做的过程中，分子生物学方法将被开发，使基因组到表型研究的监管和生物合成的DA。具体来说，一种方法来转换Pseudo-nitzschia sp驱动的细菌接合将被开发和传播到世界各地的海洋实验室。 本研究将建立一种新的模式硅藻--拟菱形藻，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-表型组联系，全球有害藻华。采取双轨制的方法，我们建议显着升级的功能基因组和分子生物学工具目前使用的硅藻社区。第一部分，我们将构建一种细菌-硅藻接合方案和一个伴随的、可维持的附加体，用于在澳大利亚拟菱形藻和多纹藻（两种全球分散的高产毒硅藻物种）中进行转基因递送。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。轨道二，与强大的遗传操作工具，为拟菱形藻属。我们将开发一种新的硅藻工具包，用于分子和生物化学应用。首先开发的三种工具是a）纳米抗体荧光蛋白（Nb-FP），可以在体内抑制或激活靶蛋白的功能; B）诱导型荧光生物传感器（UnaG），可以检测实验室或海水培养物中的DA; c）邻近标记酶：TurboID和miniTurbo，可以深入了解体内蛋白质-蛋白质相互作用。随着伪菱形藻接合转化方案的发展，我们将在模式生物三角褐指藻中表达和排除每个蛋白质组学工具。该奖项反映了NSF的法定使命，并被认为值得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来支持。