Collaborative Research: Expression profiling and functional genomics of a pennate diatom: Mechanisms of iron acquisition, stress acclimation, and recovery

合作研究：羽状硅藻的表达谱和功能基因组学：铁获取、应激适应和恢复的机制

• 批准号: 0727997
• 负责人: Andrew Allen
• 金额: $ 60万
• 依托单位: J. Craig Venter Institute, Inc.
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2011-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0727997&HistoricalAwards=false
• 关键词: Collaborative; Research; Expression; profiling; functional

Iron (Fe) availability plays an increasingly well known role regulating the fate of upwelled nitrate and determining the size structure and community composition of phytoplankton assemblages in the ocean. All Fe enrichment experiments conducted to date have reported increases in the biomass and photosynthetic capacity of diatoms. Mounting evidence from field experiments, detailed physiological investigation, and genomic sequence data suggest fundamental differences in Fe bioavailability and uptake mechanisms, storage capacity, and stress recovery between pennate and centric diatoms. Pennate diatoms often dominate the phytoplankton assemblage after mesoscale Fe addition experiments because, in part, they are able to maintain cell viability during long periods of chronic Fe stress. The underlying molecular bases for these adaptations are virtually unknown. Preliminary primary metabolite data of Fe-limited P. tricornutum suggest that metabolic reconfigurations are necessary to meet increased demand for Fe-stress metabolites such as those involved in defense from reactive oxygen species (ROS) and intracellular metal chelation. Cellular nitrogen (N) status, and the accumulation of glutamate in particular, appears likely to play a primary role in recovery from Fe stress. This project capitalizes on the extremely well annotated Phaeodactylum tricornutum genome sequence to characterize global patterns of gene expression in response to shifts into and out of Fe and N stress and over the course of the diel cycle. The primary goal is to determine the molecular and physiological processes that constrain and define different phases and levels of Fe-stress acclimation. Oceanic physiological regimes have recently been defined according to different combinations of Fe and N availability and physiological indicators of the resident phytoplankton. This research will provide molecular-level insights into defense, acclimation, and regulatory mechanisms and pathways that govern survival strategies in situations of oceanographically-relevant stress and thus are of major ecological and biogeochemical consequence. Preliminary EST and partial genome microarray data, for example, indicate that chaperones and proteases play a significant role in monitoring cellular health and balancing the difference between investment in defense or activation of programmed cell death (PCD). The proposed research will provide insights into the regulation of this fascinating and delicate balance. Such basic cellular processes play an important biogeochemical role in controlling bloom dynamics and regulating particle flux. Analysis of global gene expression will be compared with state of the art monitoring of intracellular metal levels and primary metabolite profiles using ICP-MS and gas chromatograph-mass spectroscopy (GC-MS) to determine the factors that determine cell survivability. The combination of global gene expression profiling and analysis of intracellular metal and metabolite pools will supply, for the first time, a holistic picture of the global cellular response of a marine pennate diatom to Fe-stress. P. tricornutum transcriptome profiles resulting from exposure to Fe - hydroxamate siderophores and heme-bound Fe (two classes of Fe binding ligands that are believed to comprise two major components of Fe in seawater) will be evaluated to understand the network of genes involved in recognizing and assimilating these compounds. An advanced reverse-genetics system for manipulating levels gene expression in P. tricornutum will be used to evaluate the specific role of particular genes and pathways in facilitating Fe stress acclimation.Broader Impacts: This research integrates important current themes in biogeochemistry, microbial ecology, marine sciences, and genome biology and will provide insight into factors that control the distribution and nutrient biogeochemistry of diatoms. By partnering with Affymetrix, through their Microbiology Program, a diatom microarray resource will be made available for the first time for open purchase and use. As part of the proposed research, a high school teacher from one of the local school systems with large underrepresented student populations will be recruited to work on a related topic. Upon completion of his/her paid internship, the teacher will design a classroom activity for use the following school year. As a further point of dissemination, the activity will be incorporated into a curriculum installment focused on marine and phytoplankton genomics for an existing mobile laboratory program called DISCOVER GENOMICS!, which interacts with middle school students in the Washington, D.C. Metropolitan area.

铁（Fe）的可利用性在调节海洋中涌升硝酸盐的命运和决定浮游植物组合的大小结构和群落组成方面起着越来越众所周知的作用。迄今为止进行的所有铁富集实验都报告了硅藻生物量和光合能力的增加。越来越多的证据，从现场实验，详细的生理调查，和基因组序列数据表明，铁的生物利用度和吸收机制，存储能力，和压力恢复羽状和中心硅藻之间的根本差异。羽状硅藻往往占主导地位的浮游植物组合后，中尺度的Fe添加实验，因为在某种程度上，他们能够保持细胞活力，在长期的慢性铁胁迫。这些适应的潜在分子基础实际上是未知的。铁限制三角褐指藻的初步初级代谢物数据表明，代谢重构是必要的，以满足铁胁迫代谢物的需求增加，如参与防御活性氧（ROS）和细胞内金属螯合。细胞氮（N）的状态，特别是谷氨酸的积累，似乎可能发挥主要作用，从铁胁迫的恢复。该项目利用非常好的注释三角褐指藻基因组序列来表征全球模式的基因表达，以响应转入和转出铁和氮胁迫，并在昼夜循环的过程。主要目标是确定分子和生理过程，限制和定义不同阶段和水平的铁胁迫驯化。根据浮游植物铁氮有效性和生理指标的不同组合，海洋生理机制最近被定义。这项研究将提供分子水平的见解防御，驯化和监管机制和途径，管理在海洋学相关的压力的情况下生存策略，因此是主要的生态和地球化学后果。例如，初步的EST和部分基因组微阵列数据表明，分子伴侣和蛋白酶在监测细胞健康和平衡防御投资或程序性细胞死亡（PCD）激活之间的差异方面发挥着重要作用。这项拟议中的研究将为这种迷人而微妙的平衡的调节提供见解。这种基本的细胞过程在控制水华动态和调节颗粒通量方面发挥着重要的生态地球化学作用。将总体基因表达分析与使用ICP-MS和气相色谱-质谱（GC-MS）监测细胞内金属水平和初级代谢产物谱的最新技术水平进行比较，以确定决定细胞存活率的因素。全球基因表达谱和细胞内金属和代谢物池的分析相结合，将提供，第一次，一个海洋羽状硅藻的Fe-应力的全球细胞反应的整体图片。将评估由暴露于Fe -异羟肟酸铁载体和血红素结合的Fe（两类Fe结合配体，据信其包含海水中Fe的两种主要组分）产生的三角褐指藻转录组谱，以了解参与识别和同化这些化合物的基因网络。一个先进的反向遗传学系统操纵水平的基因表达的三角褐指藻将被用来评估特定的基因和途径的具体作用，促进铁胁迫acquisition.Broader影响：这项研究整合了重要的当前主题在地球化学，微生物生态学，海洋科学和基因组生物学，并将提供深入了解的因素，控制分布和营养盐地球化学的硅藻。通过与Affybet合作，通过他们的微生物学计划，硅藻微阵列资源将首次开放购买和使用。作为拟议研究的一部分，将征聘一名来自当地学校系统的高中教师从事相关专题的研究。在他/她的带薪实习结束后，教师将设计一个课堂活动，用于下一学年。作为进一步的传播点，该活动将被纳入一个名为“发现基因组学！”的现有移动的实验室项目的海洋和浮游植物基因组学课程，该组织与华盛顿，华盛顿特区大都会区的中学生互动。