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

SGER：使用剪接前导序列研究海洋中甲藻基因表达的概念证明

• 批准号: 0854719
• 负责人: Senjie Lin
• 金额: $ 13.8万
• 依托单位: University of Connecticut
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-11-15 至 2010-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0854719&HistoricalAwards=false
• 关键词: SGER; Proof; concept; using; spliced

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.

智力优点：甲藻在海洋中的丰度和固碳潜力仅次于硅藻，是造礁珊瑚生长所必需的，也是有害藻华的最重要贡献者。尽管进行了大量的实验室和现场实验，但对甲藻生长以及与环境和自然海洋生态系统中其他生物的相互作用的了解仍然非常有限，因为操纵实验（实验室和现场）的研究结果往往不能解释自然界中的甲藻动态。测量原位基因表达的能力，在其他共存的生物体之间的自然甲藻组合将增加了解甲藻相关的海洋过程，而无需实验操作。最近，研究人员已经确定了甲藻特异性反式剪接前导序列（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样品中分离甲藻mRNA，这将有助于研究甲藻相关的海洋学和生态学过程。然而，这一概念尚未得到系统评价的证实，并且有人怀疑这项技术是否1）对甲藻具有特异性，2）对甲藻核基因组的所有mRNA具有通用性。基于DinoSL技术的适用性证明将为甲藻海洋学研究提供一种新工具，减轻甲藻研究的技术障碍，以检查碳固定，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。在该项目中，研究人员将使用DinoSL作为选择性引物构建和测序甲藻培养物的cDNA文库，纯的和与其他浮游植物混合，并验证只有和所有甲藻基因将被分离。更广泛的影响：如果成功，该项目将为甲藻研究带来创新工具，对未来的海洋学研究产生深远的影响。此外，研究人员计划使用共生藻microadriaticum，珊瑚礁生长所必需的内共生体，作为模式生物。如果所提出的概念得到证实，基于DinoSL的技术将立即适用于对这种和其他甲藻物种的研究。此外，该项目将产生大量的数据集的S。这将有助于未来的研究。此外，经过验证的概念可能会成为一个除了水族馆展览的调查人员正在与神秘水族馆。本次展览将包括介绍有害藻华，甲藻如何促进它们，甲藻毒素如何影响生态系统和公共健康，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生命百科全书（EoL）关于腰鞭毛虫的在线页面。