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

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。人们对任何特定海水样本中的浮游植物物种组成知之甚少，更不用说这些物种的生长速度了。观察到的浮游植物的生长速度和对环境条件的反应范围广泛，对物种演替和生物种群的效率具有明显的影响。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特定物种的rRNA靶向探针；2)一种新出现的分子系统发育探针，称为肽核酸(PNAS)，高度敏感，相对容易设计和使用；由于大多数浮游植物的分裂周期都是以日明暗周期为阶段的，因此可以通过监测DNA细胞周期在一段时间内的分布来确定种群的内在增长率。如果有关生物体的流式细胞术特征(色素荧光/光散射特征)足够独特(如原核生物微浮游杆菌原氯球菌)，自然样品中的DNA分布(用荧光染色测量)可以提供物种水平的生长速度。直接对真核浮游植物细胞周期进行流式细胞术分析是不现实的，因为给定样本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含量或细胞分裂时间不同。因此，研究人员将使用荧光标记的PNA rRNA探针选择性地分析特定物种的细胞。目标物种将从沿海和开阔海域的海底采样点存在的浮游植物中选择。他们将从通过流式细胞仪细胞分选或粒度分级纯化的浮游植物中提取DNA，并使用PCR扩增真核rDNA。结合终端限制性片段长度多态性(T-RFLP)分析和从选定的位点产生的18S克隆文库的测序，将被用于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定rRNA探针。调查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度信息)。除了特定的增长率，这些分析将为真核浮游植物在海洋中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。

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.