GENETICS AND FUNCTION OF THE ARABIDOPSIS CIRCADIAN CLOC

昼夜节律影响人类的许多生理功能，并影响药物治疗的功效、副作用和毒性。 日常节律的破坏会产生严重的有害影响，导致慢性失眠等疾病。 更好地了解生物钟将允许进一步优化许多药物治疗和改善睡眠障碍的疗法。 对几种模式生物的研究已经鉴定出许多与昼夜节律计时有关的基因。 然而，还没有明确的时钟基因在高等植物中被克隆。我的研究计划的目的是：1）在模式植物拟南芥中鉴定新的时钟突变体，2）确定几个Myb样转录因子的功能，这些转录因子与植物的昼夜节律调节有关。由于过表达的时钟组件扰乱昼夜节律的时间保持，我将插入诱变植物使用“激活标签”的构建驱动基因过表达。开花时间在拟南芥昼夜节律突变体中被破坏，使我们能够首先筛选比野生型植物开花早或晚的植物（比直接寻找昼夜节律突变体更容易的测定）。 然后通过检查时钟控制的荧光素酶报告基因的表达模式来鉴定具有改变的昼夜节律的植物。 在消除具有改变的光感受器信号传导途径的植物后，将使用激活标签克隆昼夜节律突变体。 这些基因将作为转基因导入植物，以证实它们在过度表达时扰乱生物钟的能力。一个家庭的七个Myb样转录因子的植物时钟的功能中牵连的功能活性也将进行检查。 虽然哺乳动物Myb蛋白使用两个Myb结构域结合DNA，但这些植物蛋白各自含有单个DNA结合结构域，表明它们以同源或异源二聚体的形式结合DNA。 通过酵母双杂交分析评估这些蛋白质自身结合和彼此结合的能力，并通过基于PCR的随机寡聚体结合测定确定其共有DNA结合特异性。 还将评估这些Myb蛋白反式激活推定靶基因的能力。

Circadian rhythms influence many physiological functions in humans and affect the efficacy, side effects, and toxicity of drug treatments. Disruption of daily rhythms can have significant deleterious effects, causing disorders such as chronic insomnia. A better understanding of circadian clocks will allow further optimization of many pharmacological treatments and improved therapies for sleep disorders. Studies in several model organisms have led to the identification of a number of genes involved in circadian timekeeping. However, no unequivocal clock genes have yet been cloned in higher plants. The aims of my research proposal are to: 1) identify new clock mutants in the model plant Arabidopsis, and 2) determine the function of several Myb-like transcription factors that have been implicated in circadian regulation in plants. Since overexpression of clock components perturbs circadian timekeeping, I will insertionally mutagenize plants using an 'activation tag' construct to drive gene overexpression. Flowering time is disrupted in Arabidopsis circadian mutants, allowing us to first screen for plants that flower either earlier or later than wild type plants (an easier assay than looking directly for circadian mutants). Plants with altered circadian rhythms will then be identified by examining the expression pattern of a clock-controlled luciferase reporter gene. After eliminating plants with altered photoreceptor-signalling pathways, circadian mutants will be cloned using the activation tag. These genes will be introduced into plants as transgenes to confirm their ability to perturb the clock when overexpressed. The functional activity of a family of seven Myb-like transcription factors implicated in the functioning of the plant clock will also be examined. Although mammalian Myb protein use two Myb domains to bind to DNA, these plant proteins each contain a single DNA binding domain, suggesting that they bind to DNA as homo- or heterodimers. The ability of these proteins to bind to themselves and to each other will be assessed by yeast two-hybrid analysis, and their consensus DNA-binding specificity determined by a PCR-based random oligomer-binding assay. The ability of these Myb proteins to transactivate putative target genes will also be assessed.