GENETICS AND FUNCTION OF THE ARABIDOPSIS CIRCADIAN CLOC

昼夜节律影响人体的许多生理功能，并影响药物治疗的疗效、副作用和毒性。扰乱日常节律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治疗方法。对几种模式生物的研究已经确定了一些与昼夜节律有关的基因。然而，在高等植物中还没有克隆出明确的时钟基因。我的研究计划的目的是：1)在模式植物拟南芥中发现新的时钟突变体；2)确定几种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。由于时钟成分的过度表达扰乱了昼夜节律，我将使用“激活标签”结构插入诱变植物以驱动基因过度表达。拟南芥昼夜突变体的开花时间被打乱，这使我们能够首先筛选开花时间比野生型植物早或晚的植物（这比直接寻找昼夜突变体更容易）。然后，通过检查生物钟控制的荧光素酶报告基因的表达模式，可以识别昼夜节律改变的植物。在消除具有改变的光感受器信号通路的植物后，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引入植物，以证实它们在过度表达时扰乱生物钟的能力。还将研究与植物时钟功能相关的7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，但这些植物蛋白每个都包含一个DNA结合结构域，这表明它们以同二聚体或异二聚体的方式与DNA结合。这些蛋白结合自身和彼此的能力将通过酵母双杂交分析进行评估，并通过基于pcr的随机寡聚物结合试验确定其一致的dna结合特异性。这些Myb蛋白反激活假定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.