Genetic and Molecular Interactors in Circadian Clock Function

摘要生物钟已经在一定程度上应对了协调整个生物体的过程与环境中经常可预测的变化的挑战。这种内部计时系统既提供了对即将到来的变化的预期，也提供了对环境快速变化的缓冲。在植物中，环境和发展之间的密切协调到目前为止是不言而喻的。开花时间和休眠开始只是协调植物环境和发育时间的重要性的两个例子，而生物钟在其中起着至关重要的作用。最近的克隆进展表明，尽管植物和动物拥有相同的基本昼夜节律特性，但这两个王国的昼夜节律系统的分子组成明显不同。这项工作的长期目标是从分子上更好地理解植物昼夜节律的作用机制。最近，影响昼夜节律和幅度的基因产物的鉴定迅速增加，但这些因素之间的关系尚不清楚。这项工作旨在通过结合分子和遗传策略，从拟南芥F-box蛋白ZEITLUPE(ZTL)向外工作，了解这些关系。这个建议的目的是：1.长周期ZTL-1表型增强子的鉴定和定位克隆。对突变表型的抑制子或增强子进行基因筛选已成为一种常见且成功的策略，用于确定遗传层次中的主要参与者，并确定先前被认为是独立的通路之间的意外相互作用。已鉴定出7个增强ZTL-1长周期表型的突变体。除了主要的昼夜节律表型外，每个突变体的特征是下胚轴长度和开花时间的变化。每个基因都被物理作图以确定所涉及的基因座的数量，然后基于图谱的克隆将被用来识别负责的基因。心律失常ZTL过表达表型抑制子的鉴定和位置克隆。ZTL的高结构性过表达会导致心律失常。很可能通过寻找能够下调ZTL介导的活性并在持续存在高ZTL水平(反式突变)的情况下恢复节律性的基因，可能会发现一类独特的相互作用因子。已鉴定出两个独立分离的反式突变，它们赋予ZTL高表达系野生型(24.5h)或略短(22.5h)的周期。每个候选基因都在进一步表征和定位以确定等位基因，基于图谱的克隆正在进行中以确定涉及的基因(S)。将确定在植物提取物中与ZTL相互作用的蛋白质。这些蛋白质可以帮助我们深入了解ZTL的作用机制。这项研究的更广泛的科学益处包括对生物钟的更清晰的理解，以及潜在的农业后果。昼夜节律系统与开花途径的相互作用意味着有可能增加对开花时间的控制。一些农业物种的纬度范围受到日长的限制，因此操纵时钟组件可能有助于扩大一些植物的生长范围。从培训的角度来看，实验室的研究环境是任何系列讲座都无法替代的动手学习体验。本项目将雇用本科生、研究生和博士后研究员。这些人的研究经验将拓宽他们的科学专长，并为他们未来的PI、博士后和教师的发展做出贡献。

SummaryThe challenge of coordinating whole-organism processes with often-predictable changes in the environment has been met to some extent by the circadian clock. This internal timing system provides for both the anticipation of coming changes and as a buffer against rapid variation in the environment. In plants, close coordination between the environment and development is by now axiomatic. Flowering time and dormancy onset are just two examples of the importance of coordinating environmental and developmental timing in plants, and the circadian clock plays a crucial role in this. Recent cloning advances have shown that the molecular components of plant and animal circadian systems are distinctly different, though the two kingdoms share the same basic circadian properties.The long-term goal of this work is a better molecular understanding of the mechanism of action of the plant circadian clock. There has been a recent rapid increase in the identification of gene products that affect circadian period and amplitude, but the relationship between these factors remains unclear. This work aims at understanding these relationships by working outwards from one component, the Arabidopsis F-box protein ZEITLUPE (ZTL), using a combination of molecular and genetic strategies. The objectives of this proposal are: 1. Characterization and positional cloning of the enhancers of the long period ztl-1 phenotype. Genetic screens for suppressors or enhancers of mutant phenotypes have become a common and successful strategy for identifying the principle players in a genetic hierarchy and for identifying unexpected interactions between pathways previously thought to be independent. Seven mutants that enhance the long period phenotype of ztl-1 have been identified. In addition to the primary circadian phenotype, each mutant is being characterized for changes in hypocotyl length and flowering time. Each is being physically mapped to determine the number of loci involved, and map-based cloning will then be used to identify the responsible genes.2. Characterization and positional cloning of suppressors of the arrhythmic ZTL overexpression phenotype. High constitutive overexpression of ZTL causes arrhythmicity. It is likely that a unique class of interactors may be found by searching for genes that can down regulate ZTL-mediated activity and restore rhythmicity in the continued presence of high ZTL levels (trans mutations). Two independently-isolated trans mutations that confer wild type (24.5 h) or slightly shorter (22.5 h) periodicity to a high ZTL overexpressing line have been identified. Each candidate is being further characterized and mapped to determine allelism, and map-based cloning is ongoing to identify the gene(s) involved.3. Proteins that interact with ZTL in plant extracts will be identified. These proteins should provide insight into the mechanism of action of ZTL. The broader scientific benefits of this research include a clearer understanding of the circadian clock, with potential agricultural consequences. The interaction of the circadian system with flowering pathways implies a potential for increased control over the timing of floral initiation. The latitudinal range of some agricultural species is limited by daylength, so manipulation of clock components could contribute to increasing the growing range of some plants. From the perspective of training, the laboratory research environment is a hands-on learning experience that no lecture series can replace. The present project will employ undergraduates, graduate students and post-doctoral fellows. The research experiences of these people will broaden their scientific expertise and contribute to their development as future PI's, post-docs and teachers.