GENETICS AND FUNCTION OF THE ARABIDOPSIS CIRCADIAN CLOC

昼夜节律影响人类的许多生理功能，并影响药物治疗的疗效、副作用和毒性。打乱日常节律可能会产生显著的有害影响，导致慢性失眠等疾病。对生物钟的更好理解将有助于进一步优化许多药物治疗和改进睡眠障碍的治疗方法。在几个模式生物中的研究导致了一些与昼夜节律有关的基因的识别。然而，在高等植物中还没有明确的时钟基因被克隆出来。我的研究计划的目的是：1)在模式植物拟南芥中寻找新的时钟突变体，2)确定几个参与植物昼夜节律调节的Myb类转录因子的功能。由于时钟成分的过度表达扰乱了生物钟的计时，我将使用‘激活标签’构造来驱动基因过度表达来插入突变植物。在拟南芥的昼夜节律突变体中，开花时间被打乱，使我们能够首先筛选比野生型植物更早或更晚开花的植物(一种比直接寻找昼夜节律突变体更容易的测试)。然后，通过检测时钟控制的荧光素酶报告基因的表达模式，将识别出昼夜节律改变的植物。在消除光感受器信号通路改变的植物后，将使用激活标签克隆昼夜节律突变。这些基因将作为转基因引入植物中，以确认它们在过度表达时扰乱时钟的能力。还将检测与植物时钟功能有关的七个Myb样转录因子家族的功能活性。虽然哺乳动物Myb蛋白使用两个Myb结构域与DNA结合，但这些植物蛋白都包含一个DNA结合域，这表明它们以同源或异源二聚体的形式与DNA结合。这些蛋白质自身和彼此结合的能力将通过酵母双杂交分析来评估，它们一致的DNA结合特异性将通过基于PCR的随机寡聚物结合试验来确定。这些Myb蛋白反式激活可能的靶基因的能力也将被评估。

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.