EAGER: Application of transcriptomics to investigate organism-environment relationships in marine zooplankton

智力优势：该项目将发展转录组学方法来研究基因调控作为环境周期的功能和对实验操作的响应。目前，建立海洋浮游动物生理状态，特别是海洋物种生理状态的工具很少。基于定量转录组的分子方法可以作为强大的工具来获得在该领域收集的个体和群体的生理特征。结合实验室实验，转录组分析将提供一种新的方法来理解海洋生物与环境的相互作用。该项目将重点研究一种典型的浮游甲壳类动物Calanus finmarchicus，以开发分子工具。C. finmarchicus是一种类calanoid桡足动物，在北大西洋非常丰富，种群从缅因湾和拉布拉多海一直延伸到北海。焦磷酸测序和微阵列技术将用于开发一种诊断工具，以确定C. finmarchicus的生理状态。生理状态测量的目的是确定种群中的个体是否正在生长，是否正在合成或分解储存脂质，是否代谢活跃和/或经历环境压力。该项目的具体目标包括：代表年周期不同阶段（春末-初夏、初秋、滞育个体）的前成虫（桡足动物V期[CV]）转录组的高通量测序分析序列数据，发现季节性调节基因，开发生态相关微阵列。该芯片的探针将包括季节性调节基因、参与环境应激反应的基因和控制基因。对从缅因湾收集并储存在液氮中的现有样本以及实验操纵的动物进行微阵列的初步测试。更广泛的影响：1)将对研究生和本科生进行这些技术的培训。至少有两名海洋科学/生物海洋学研究生将参与该项目，并在实验台上接受转录组学和生物信息学方面的培训。本科生将通过NSF REU-site资助在Mount Desert Island生物实验室参与该项目。本科培训将是跨学科的，包括生物海洋学和分子生物学技术。2)本研究获得的结果和序列将存入数据库并向公众开放。序列将使用聚类工具进行分析，并使用基因本体软件进行注释，然后将序列提交到国家生物技术信息中心（NCBI）数据库。总的来说，桡足类的基因序列数据仍然很少，而计划提交的报告将使鱿鱼类桡足类的可用信息增加十倍或更多。

Intellectual Merit: This project will develop transcriptomics approaches to investigate gene regulation as a function of environmental cycles and in response to experimental manipulation. Currently, there are few tools to establish physiological state of marine zooplankton, in particular for oceanic species. Molecular approaches based on quantifying the transcriptome could serve as powerful tools to obtain a physiological profile for individuals and groups of individuals collected in the field. In combination with laboratory experiments, transcriptome analysis will provide a new approach to understanding organism-environment interactions in the pelagic zone.The PI will focus on a model planktonic crustacean, Calanus finmarchicus, to develop the molecular tools. C. finmarchicus, a calanoid copepod, is highly abundant in the North Atlantic, with populations extending from the Gulf of Maine and Labrador Sea to the North Sea. Pyrosequencing and microarray technologies will be used to develop a diagnostic tool to determine physiological state in C. finmarchicus. The goal of having a measurement of physiological state is to determine if individuals in the population are growing, are synthesizing or catabolizing storage lipids, and are metabolically active and/or experiencing environmental stress. Specific objectives of this project include:1. High throughput sequencing of C. finmarchicus transcriptome from pre-adult (copepodid stage V [CV]) individuals representing distinct phases of the annual cycle (late spring-early summer, early fall, diapausing individuals).2. Analysis of the sequence data for discovery of seasonally regulated genes for the development of an ecologically relevant microarray. Probes for this microarray will include seasonally regulated genes, genes involved in the environmental stress response and control genes.3. Preliminary testing of microarray on existing samples collected from the Gulf of Maine and stored in liquid nitrogen, as well as on experimentally manipulated animals.Broader Impacts: 1) Training of graduate and undergraduate students in these techniques will occur. At least two graduate students in Marine Sciences/Biological Oceanography will participate in the project and be trained in transcriptomics at the bench and in bioinformatics. Undergraduate students will participate in the project at the Mount Desert Island Biological Laboratory through the NSF REU-site funding. Undergraduate training will be interdisciplinary and include both biological oceanography and molecular biology techniques. 2) Results and sequences obtained in this study will be contributed to databanks and made available to the general public. Sequences will be analyzed using clustering tools and annotated using gene ontology software, followed by sequence submission to the National Center for Biotechnology Information (NCBI) database. Gene sequence data for copepods in general are still sparse, and the planned submission will increase information available for calanoid copepods by ten-fold or more.