Genomic and Transcriptomic Comparison of Iron and Light Physiology in Coastal and Oceanic Diatoms

智力优势：硅藻是海洋中最重要的初级生产者之一。沿海物种可以对海洋表层营养物质的动态输入做出反应，并形成大规模的生物量繁殖。海洋中大部分浮游植物的生长受到一种或另一种营养物质的限制，在这些环境中生存的物种必须能够在这种低营养条件下发挥作用。海洋硅藻生长对铁的需要量较低，可能通过改变需铁代谢途径的组成而进化到在低铁环境中生存。铁和光响应途径密切相关，因为光合作用需要大量铁，并且可能限制生长和生物碳泵的效率。生理和野外研究表明，许多硅藻易受铁/光共限，但我们缺乏关于共限的生化基础以及不同硅藻种之间的差异的信息。该项目将使用基因组学工具的组合来研究生态上重要的海硅藻属的沿海和海洋硅藻如何对不同的铁和光条件做出反应。研究人员将比较海洋硅藻T. oceanica的基因组序列，该序列最近由PI与Illumina公司合作测序，与已发表的硅藻基因组进行比较，以确定海洋和沿海硅藻在铁和光代谢方面的潜在差异和相似性。他们将使用标准化的表达序列标签文库（EST）来表征T. oceanica（一种海洋菌株weissflogii）和T. rotula（生长在限制铁和充满铁的条件下以及低光和生长饱和光水平下的海岸硅藻）的转录组。他们将利用数字基因表达（DGE）来量化这些实验中转录组全响应的基因表达水平，并利用EST数据来绘制DGE标签。DGE和EST实验的数据将用于比较硅藻代谢对不同光和铁浓度的反应，并确定天然硅藻种群中以下限制的靶基因。这些基因的表达将在时间过程实验中进行监测，通过额外的铁和光照水平操作，使用定量PCR （qPCR）识别指示不同生理状态的基因标记。研究人员还将设计针对选定数量的蛋白质的抗体来监测蛋白质表达，他们将使用qPCR和抗体来跟踪从富铁和限铁环境之间的巡航过渡中收集的实地样品中的硅藻群落的反应，这是其他资助研究工作的一部分。这项工作将进一步加深我们对硅藻如何适应不同环境以及它们生态成功的遗传基础的理解。这项工作的结果将帮助我们预测硅藻如何应对气候变化导致的分层增加所导致的光照变化，并帮助预测来自不同栖息地的物种是否会有相似或不同的反应。更广泛的影响：该项目将为生态上重要的物种建立基因组基础设施。PI Jenkins教授基因组序列分析课程，在提议的工作中开发和测试的分析工具将纳入本课程。本课程学生将参与海洋巨藻基因组的注释。该项目将资助一名研究生和一名新的女性博士后研究员。将制作向高中学生宣传的视频。研究人员和学生将录制有关浮游植物的重要性、铁和光如何控制海洋光合作用、基因组学工具如何用于理解生态问题的视频片段。面向高中受众的内容将被上传到他们的SciVee网站（http://www.scivee.tv/user/bjenkins,http://www.scivee.tv/user/dreuxchappell）上，SciVee是一个由NSF和其他机构支持的网站。这项工作对社会有益，因为它将帮助我们了解在海洋中调节碳循环的生物是如何应对气候变化和营养动态变化的。这项工作的结果将使我们更好地了解硅藻的代谢，并进一步了解它们的生态学

Intellectual Merit: Diatoms are among the most important primary producers in the ocean. Coastal species can respond to dynamic inputs of nutrients into the surface ocean and form large biomass blooms. Phytoplankton growth in much of the ocean is limited by one nutrient or another, and species that persist in these environments must be able to function under these low nutrient conditions. Oceanic diatom species have lower Fe requirements for growth and may have evolved to subsist in low Fe environments by changing the composition of Fe-demanding metabolic pathways. Iron and light responsive pathways are intimately linked because of the large Fe requirement of photosynthesis and the potential for both to limit growth and the efficiency of the biological carbon pump. Physiological and field studies have shown that many diatoms are susceptible to Fe/light co-limitation, but we lack information on the biochemical basis for co-limitation and how this differs between diatom species. This project will use a combination of genomics tools to investigate how coastal and oceanic diatoms in the ecologically important Thalassiosira genus respond to differing conditions of Fe and light. The investigators will compare the genome sequence of the oceanic diatom T. oceanica, which has recently been sequenced by the PI in collaboration with Illumina, Inc., to published diatom genomes to identify potential differences and similarities in the Fe and light metabolism in oceanic and coastal diatoms. They will use normalized libraries of Expressed Sequence Tags (EST) to characterize the transcriptome of T. oceanica, an oceanic strain of T. weissflogii, and the coastal diatom T. rotula grown in a matrix of Fe-limiting and replete conditions and at low and growth-saturating light levels. And they will quantify gene expression levels for the transcriptome-wide response in these experiments using digital gene expression (DGE), and use the EST data to map the DGE tags. Data from the DGE and EST experiments will be used to compare how diatom metabolism responds to variable light and Fe concentrations and to identify target genes for following limitation in natural diatom populations. Expression of these genes will be monitored in time-course experiments with additional manipulations of Fe and light levels to identify gene markers indicative of different physiological states using quantitative PCR (qPCR). The investigators will also design antibodies to a select number of proteins to monitor protein expression, and they will use qPCR and antibodies to follow responses in diatom communities in field samples collected from a cruise transitioning between Fe-replete and Fe-limiting environments as part of other funded research efforts. This work will further our understanding of how diatoms are adapted to different environments and what the genetic basis for their ecological success may be. Results from this work will help us predict how diatoms may respond to changing light regimes as a result of increased stratification due to climate change and help predict if species from different habitats will have similar or varied responses.Broader Impacts: The project will build genomic infrastructure for ecologically important species. PI Jenkins teaches a course in genome sequence analysis and the analysis tools developed and tested during the proposed work will be incorporated into this course. Students in the course will participate in the annotation of the T. oceanica genome. The project will support a graduate student and a new female postdoctoral investigator. Video for outreach to high school students will be developed. The investigators and students will record video episodes on the importance of phytoplankton, how iron and light control ocean photosynthesis, and how genomics tools can be used to understand ecological questions. Content aimed at a high school audience will be uploaded to their sites on SciVee (http://www.scivee.tv/user/bjenkins,http://www.scivee.tv/user/dreuxchappell), a site supported by NSF and others. The work benefits society because it will help us understand how organisms that mediate carbon cycling in the ocean respond to scenarios of changing climate and changing nutrient dynamics. Results from the proposed work will provide us with a much better view of diatom metabolism and further our understanding of their ecology