Collaborative Research: Combining rRNA Probes and Cell Cycle Analyses to Investigate In Situ Growth Rates of Eukaryotic Phytoplankton

合作研究：结合 rRNA 探针和细胞周期分析来研究真核浮游植物的原位生长速率

• 批准号: 0099078
• 负责人: Virginia Armbrust
• 金额: $ 31.27万
• 依托单位: University of Washington
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0099078&HistoricalAwards=false
• 关键词: Collaborative; Research; Combining; rRNA; Probes

The goal of the project is to assess both species diversity and the distribution of growth rates among eukaryotic phytoplankton populations under natural conditions. Relatively little is known about phytoplankton species composition in any particular sample of seawater, let alone the growth rates of those species. The wide range of growth rates and responses to environmental conditions observed for phytoplankton has obvious implications for species succession and the efficiency of the biological puinp. The investigators will focus on eukaryotic phytoplankton because these cells are responsible for the bulk of production in many regions, including most marine phytoplankton blooms. They will employ a three-tiered approach that combines: 1) high-throughput DNA sequencing and fragment analyses to allow us to determine species diversity and to design species-specific rRNA-targeted probes for ecologically relevant organisms; 2) a newly emerging class of molecular phylogenetic probes known as peptide nucleic acids (PNAs), which are highly sensitive and relatively easy to design and use; and 3) flow cytometric cell cycle analyses of PNA-labeled cells to determine intrinsic growth rates of targeted species.Since the division cycles of most phytoplankton are phased to the daily light:dark cycle, the intrinsic growth rate of a population can be determined by monitoring DNA cell cycle distributions over a diel period. If the flow cytometric signature (pigment fluorescence/light scatter characteristics) of the organism in question is sufficiently distinctive (as with the prokaryotic picoplankter Prochlorococcus), DNA distributions (measured with a fluorescent stain) in natural samples can provide growth rates at the species level. Direct flow cytometric analyses of eukaryotic phytoplankton cell cycling are not practical because multiple species in a given sample typically have similar morphology and flow cytometric signatures, but different amounts of DNA or cell division timing. Therefore, the investigators will selectively analyze cells of particular species by using fluorescentlylabelled PNA rRNA probes. Target species will be chosen from among the phytoplankton present at diel sampling sites in coastal and open ocean waters. They will extract DNA from phytoplankton purified either by flow cytometric cell sorting or size fractionation and amplify eukaryotic rDNA using PCR. A combination of terminal restriction fraction length polymorphism (T-RFLP) analysis and the sequencing of 18S clone libraries generated from selected sites will be used to select species of interest for further study. Specific rRNA probes to selected species will then be designed. The invetigators will use fluorescently-labeled PNA probes, in conjunction with DNA staining and dual-beam flow cytometry, to obtain cell cycle (and thus growth rate information for target species). In addition to speciesTm growth rates, these analyses will provide new perspectives on the distribution and diversity of eukaryotic phytoplankton in the sea and will improve our capabilities to address a variety of oceanographic questions on ecologically relevant scales.

该项目的目标是评估自然条件下真核浮游植物种群的物种多样性和生长率分布。对于任何特定海水样本中的浮游植物物种组成，人们所知相对较少，更不用说这些物种的生长速度了。浮游植物的生长速率和对环境条件的反应范围很广，这对物种演替和生物种群的效率有明显的影响。研究人员将把重点放在真核浮游植物上，因为这些细胞负责许多地区的大部分生产，包括大多数海洋浮游植物水华。他们将采用一种三层方法，结合：1）高通量DNA测序和片段分析，使我们能够确定物种多样性，并为生态相关生物设计物种特异性rRNA靶向探针; 2）一类新出现的分子系统发育探针，称为肽核酸（PNAs），具有高度灵敏度，相对容易设计和使用;（3）用流式细胞仪分析PNA标记细胞的细胞周期，以确定目标种的内禀生长率。由于大多数浮游植物的分裂周期是以昼夜光暗周期为阶段的，因此，通过监测DNA细胞周期在昼夜周期中的分布，可以确定种群的内禀生长率。如果所讨论的生物体的流式细胞术特征（色素荧光/光散射特征）足够独特（如原核微球菌原绿球藻），天然样品中的DNA分布（用荧光染色测量）可以提供物种水平的生长率。真核浮游植物细胞周期的直接流式细胞术分析是不切实际的，因为给定样品中的多个物种通常具有相似的形态和流式细胞术特征，但DNA或细胞分裂时间的量不同。因此，研究人员将通过使用荧光标记的PNA rRNA探针来选择性地分析特定物种的细胞。目标物种将从沿海和公海沃茨昼夜采样点的浮游植物中选择。他们将从浮游植物中提取DNA，通过流式细胞仪细胞分选或大小分级纯化，并使用PCR扩增真核rDNA。将使用末端限制性片段长度多态性（T-RFLP）分析和从选定位点产生的18 S克隆文库测序的组合来选择感兴趣的物种进行进一步研究。然后将设计针对选定物种的特异性rRNA探针。研究人员将使用荧光标记的PNA探针，结合DNA染色和双光束流式细胞术，以获得细胞周期（从而获得目标物种的生长速率信息）。除了物种Tm生长率之外，这些分析还将为海洋中真核浮游植物的分布和多样性提供新的视角，并将提高我们在生态相关规模上解决各种海洋学问题的能力。