拟南芥生长素组成型反应突变体的鉴定及其突变体car1－1根尖分生组织维持缺陷的机理研究

Phytohormone auxin controls every aspect of plant individual development, including the patterning of root meristem. Previous studies confirmed that auxin plays key roles in the maintenance of root stem cell niche and the differentiation of their daughter cells in Arabidopsis. Although the model of auxin signaling pathway regulated by F-box protein TIR1/AFBs as auxin receptors and mediated by SCF ubiquitin ligase is well established.To explore new components in auxin regulation complex, a new screening method for harvest more constitutive auxin response (cars) mutants was continued to impliment in this application: Firstly, the seeds of Col-0 wild type plants with auxin response marker DR5:GFP will be mutagenized by EMS, and the T2 mutants with constitutive or hypersensitive response to auxin will be picked out using fluorescent dissect microscope.Secondly, their mutation genes will be mapped and cloned after phenotyping in detail. In this study, we will also plan to investigate the molecular causes of the deficiency of root stem cell niche maintenance in the mutant car1-1 ,one of which was screened previously.

生长素不仅控制着植物个体发育的各个方面，同时也精确地调控着植物根尖分生组织特异性的形态建成，特别是根尖干细胞池的维持以及子细胞的定向分化。目前我们已经建立了以F-BOX蛋白TIR1/AFBs为受体的，SCF蛋白复合体介导的生长素信号转导调控途径。本研究中为了鉴定新的生长素信号转导调控途径，同时找寻SCF介导的蛋白降解中的可能存在的未知修饰途径，我们新发展了一个以生长素反应特异荧光蛋白（DR5:GFP）为主，结合其他生长素反应标记为辅的，荧光显微镜下筛选突变体的方法，继续饱和特异性地筛选生长素反应超敏感或组成型反应的突变体cars系列（constitutive auxin response Mutants). 同时在本项目中，我们将对已筛选到的一个转录相关的突变体car1-1，进行根尖分生组织维持缺陷的分子机理研究。

本课题我们通过对带有生长素反应荧光标记DR5::GFP的COL野生型拟南芥种子，进行EMS诱导突变，获得了12个生长素反应组成型突变的株系，精细定位了4个突变体，突变体表型恢复实验验证了其中2个，card1-1 和card2-1。初步探究了CARD2蛋白的组织与细胞器定位，利用酵母和YFP分裂互补实验证实CARD2蛋白可以与核孔复合物蛋白的相互作用，突变体可以抑制mRNA的穿核运输。详细研究了CARD1蛋白的CTD结构域对于植物应激转录活性的精细调控的重要性，转录组测序结果证实了植物不同应激反应的信号转导途径，在突变体中得到富集；突变体个体发育的整体缺陷来源于植物茎尖和根尖的干细胞区维持不稳定，干细胞荧光标记显示突变体干细胞谱系分区界限不清，由此我们提出CARD1的CTD全长对于植物基因转录的时空和水平的精确控制是必需的，而基因转录的时空和水平的精确控制又是植物根尖和茎尖干细胞群维持分生区活性所必需的。由此根据本课题的研究结果，我们提出可能存在至少两种信号调控机制影响植物生长素信号的转导。