SGER: Proof of concept on using spliced leader to study dinoflagellate gene expression in the ocean

智力优势：甲藻在海洋中的丰度和固碳潜力仅次于硅藻，对造礁珊瑚的生长至关重要，是有害藻类大量繁殖的最重要因素。尽管进行了广泛的实验室和野外实验，但对甲藻的生长以及与自然海洋生态系统中的环境和其他生物的相互作用的了解仍然非常有限，因为人工实验(实验室和田间)的研究结果往往无法解释自然界中甲藻的动态。能够在其他共存生物中原位测量天然甲藻组合的基因表达，将增加对甲藻相关海洋过程的了解，而不需要进行实验操作。最近，研究人员发现了一个甲藻特异的反式剪接前导序列(DinoSL)。这些发现有力地表明，DinoSL可用于从多种生物贡献的混合RNA样本中分离甲藻mRNA，这将有助于甲藻相关海洋学和生态学过程的研究。然而，这一概念尚未得到系统评估的证实，人们怀疑这项技术是否1)专用于甲藻，2)通用于甲藻核基因组中的所有mRNA。对DinoSL技术适用性的证明将为甲藻海洋学研究提供新的工具，减轻甲藻研究的技术障碍，以考察固碳潜力、放牧、压力、对气候变化的反应、与其他浮游植物的竞争、生长以及对自然环境中珊瑚生长的贡献。在这个项目中，研究人员将使用DinoSL作为甲藻培养物的选择性引物，构建并测序甲藻培养物、纯种和与其他浮游植物混合的cDNA文库，并验证只有和所有甲藻基因将被分离。广泛影响：如果该项目成功，该项目将为甲藻研究带来一个创新的工具，对未来的海洋学研究具有深远的意义。此外，研究人员计划使用微小共生菌作为模式生物，这是一种对珊瑚礁生长至关重要的内生共生菌。如果提议的概念得到证实，基于DinoSL的技术将立即适用于该物种和其他甲藻物种的研究。此外，该项目将为微小旱生沙门氏菌产生大量的数据集，这将对未来的研究有用。此外，被证实的概念可能会成为研究人员正在与神秘水族馆合作的水族馆展览的补充。这次展览将包括介绍有害的藻华，甲藻如何对其做出贡献，甲藻毒素如何影响生态系统和公众健康，以及甲藻基因组如何调节这些活动。此外，在这项研究中获得的信息可能会被添加到关于甲藻的生命百科全书(EoL)在线页面。

Intellectual merit: Dinoflagellates are second only to diatoms in abundance and the potential of carbon fixation in the ocean, are essential for growth of reef-building corals, and are the most important contributors to harmful algal blooms. Despite extensive laboratory and field experiments, knowledge of dinoflagellate growth and interaction with the environment and other organisms in natural marine ecosystems is still very limited because research results from manipulated experiments (laboratory and field) often do not explain dinoflagellate dynamics in nature. The ability to measure in situ gene expression for a natural dinoflagellate assemblage amidst other co-existing organisms would increase understanding of dinoflagellate-associated oceanographic processes without experimental manipulation. Recently, the investigator has identified a dinoflagellate-specific trans-spliced leader sequence (DinoSL). These findings strongly suggest that DinoSL can be used to isolate dinoflagellate mRNA from a mixed RNA sample contributed by many organisms, which would facilitate studies of dinoflagellate-associated oceanographic and ecological processes. However, the concept has not been proven with systematic evaluation, and there is skepticism about whether this technique will be 1) specific for dinoflagellates and 2) universal for all mRNAs from the dinoflagellate nuclear genome. Proof of the suitability of the DinoSL-based technique will provide a new tool for dinoflagellate oceanographic research, alleviating technical obstacles for dinoflagellate studies to examine the potential of carbon fixation, grazing, stress, response to climate change, competition with other phytoplankton, growth, and contribution to coral growth in the natural environment. In this project, the investigator will construct and sequence cDNA libraries using DinoSL as the selective primer for dinoflagellate cultures, pure and mixed with other phytoplankton, and verify that only and all dinoflagellate genes will be isolated.Broader impact: If successful, this project will bring about an innovative tool for dinoflagellate research with far-reaching implications for future oceanographic studies. Furthermore, the investigator plans to use Symbiodinium microadriaticum, an endosymbiont essential for reef growth, as a model organism. If the proposed concept is proved, the DinoSL-based technique will be immediately applicable to research on this and other dinoflagellate species. Furthermore, this project will generate a substantial data set for the S. microadriaticum that will be useful for future research.. Furthermore, the proved concept may become an addition to the aquarium exhibit the investigators are working on with Mystic Aquarium. This exhibit will include an introduction to harmful algal blooms, how dinoflagellates contribute to them, how dinoflagellate toxins impact the ecosystem and public health, and how dinoflagellate genomes regulate these activities. In addition, information obtained in this study may be added to the Encyclopedia of Life (EoL) online pages on dinoflagellates.