PRFB FY 2021: Characterizing the symbiosome membrane in two divergent cnidarian lineages to investigate the cellular mechanisms and evolution of coral-algal symbiosis

该行动资助了美国国家科学基金会2022财年生物学博士后研究奖学金，研究基因组，环境和表型之间相互作用的生命规则的综合研究。该奖学金支持将以创新方式对生活规则领域作出贡献的研究员的研究和培训。该研究员将使用尖端的基因编辑和分子技术来研究控制珊瑚和与珊瑚一起生活的藻类之间共生关系的基本机制，这是珊瑚健康的一个关键方面。气候变化威胁到这种共生关系的稳定性，因此可能影响珊瑚的生存。因此，更好地理解这种关系可能有助于制定珊瑚生存的解决方案。该研究员将通过在马里兰州巴尔的摩的推广活动中展示他们的工作，为当地K-12学生开发一门使用海葵的动手课程，以及为俄勒冈州立大学无脊椎动物生物学实验室设计一门基于研究的课程，来支持科学的多样性和包容性。在刺胞-藻类光共生中，藻类共生体居住在吞噬体膜内，称为共生体。蛋白质定位于共生体膜以维持共生，其中一些蛋白质在独立进化光共生的宿主中是共享的。该研究员将描述两个独立获得光共生的不同刺胞动物谱系的共生体膜蛋白质组：海葵Exaiptasia diaphana和珊瑚Acropora millepora。为了表征共生体膜蛋白质组，首先，将从每个宿主物种中分离藻类细胞，并用抗体标记和流式细胞术富集共生体阳性藻类细胞。然后，用生物化学方法纯化共生体膜蛋白，并用串联质谱法进行鉴定。然后，他将用CRISPR/Cas9敲除两种宿主体内的每个基因，测试共生体膜蛋白的功能。将敲除幼虫接种藻类共生体，并测量共生体密度。据预测，敲除对共生至关重要的基因会对共生密度产生强烈的影响，而那些不那么重要的基因则会产生较弱的影响。最后，该研究员将在俄勒冈州立大学开展以课程为基础的本科研究经验，学生将在共生早期阶段通过一个时间过程来表征E. diaphana幼虫共生体膜蛋白的基因表达。这些基因的表达预计会根据它们的功能在时间上发生变化。该研究员将由其赞助商在生物信息学，蛋白质组学，CRISPR/cas9敲除技术和课程开发方面进行培训。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This action funds an NSF Postdoctoral Research Fellowship in Biology for FY 2022, Integrative Research Investigating the Rules of Life Governing Interactions Between Genomes, Environment and Phenotypes. The fellowship supports research and training of the fellow that will contribute to the area of Rules of Life in innovative ways. The fellow will use cutting-edge gene-editing and molecular techniques to study the fundamental mechanisms governing the symbiosis between corals and algae that live with the coral, which is a key aspect of coral health. Climate change threatens the stability of this symbiosis and therefore can impact coral survival. Better understanding this relationship therefore may help with the development of solutions for coral survival. The fellow will support diversity and inclusion in the sciences by presenting their work at outreach events in Baltimore, MD, developing a hands-on curriculum for local K-12 students using a sea anemone, and by designing a research-based course for Oregon State University’s Invertebrate Biology lab.In cnidarian-algal photosymbiosis, the algal symbiont resides within a phagosomal membrane, called the symbiosome. Proteins localize to the symbiosome membrane to maintain symbiosis, and several of these proteins are shared among hosts that evolved photosymbiosis independently. The fellow will characterize the symbiosome membrane proteome in two divergent cnidarian lineages that have independently gained photosymbiosis: the sea anemone Exaiptasia diaphana and the coral Acropora millepora. To characterize the symbiosome membrane proteome, first, algal cells will be isolated from each host species and symbiosome-positive algal cells will be enriched with antibody labeling and flow cytometry. Then, symbiosome membrane proteins will be purified with biochemistry and identified using tandem mass spectrometry. The fellow will then test the function of symbiosome membrane proteins by knocking out each gene with CRISPR/Cas9 in the two host species. Knockout larvae will be inoculated with algal symbionts and symbiont density will be measured. Knockouts of genes critical for symbiosis are predicted to have a strong effect on symbiont density, while those that are less vital will have weaker effects. Finally, the fellow will develop a course-based undergraduate research experience at Oregon State University where students will characterize the gene expression of symbiosome membrane proteins in E. diaphana larvae through a time course during the early stages of symbiosis. Expression of these genes are expected to vary temporally depending on their function. The fellow will be trained by his sponsors in bioinformatics, proteomics, CRISPR/cas9 knockout techniques, and curriculum development.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.