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一个拟南芥矮化突变体wox1-D的鉴定及相关基因WUSCHEL HOMEOBOX1(WOX1)的功能分析
中文摘要

植物茎端分生组织的两大功能即是其自身的维持与新器官的发生。位于分生组织中心的一群分裂缓慢的细胞是具有全能性的干细胞,分生组织边缘的干细胞不断分化,产生新的器官。我在对拟南芥插入激活突变体库的筛选过程中发现了一株植株矮化的突变体,通过对T-DNA插入位点的鉴定和分析,我发现在该突变体中T-DNA插在一个含有WUSCHEL相关同源异形结构域的转录因子WOX1 (〓USCHEL HOMEOB〓 1)基因的上游。该突变体的分生组织发育异常,茎尖显著小于野生型,并表现出矮化及多枝表型。突变体的叶片较小,呈深绿色,花药发育迟缓导致不育。我用35S增强子构建WOX1的过表达载体转入植物,转基因植物表现出与突变体类似的表型;而用WOX1的RNAi载体去转化突变体,可以使突变体回复到野生型表型。这说明突变体的表型确实是由于WOX1基因的过量表达引起,因此我把该突变体命名为wox1-D。 在wox1-D突变体中,干细胞标志基因CLV3在分生组织中的表达发生下调,并在根与下胚轴的连接处和侧根发生处发生异位表达,这表明WOX1的过量表达影响了CLV3的表达模式,从而导致矮化及多枝表型。此外,wox1-D突变体叶片的细胞明显小于野生型,检测发现在wox1-D中cyclinB1的表达强度明显低于野生型,这暗示WOX1在分生组织中表达模式的变化部分影响了细胞周期的顺利进行,阻滞了侧生器官的发育。 我通过酵母双杂交的方法寻找WOX1可能的相互作用蛋白。通过对拟南芥cDNA文库的筛选我找到了一个可能的靶蛋白SAMDC1,SAMDC1是多胺代谢过程中的一种关键酶。我通过MBP pull-down的方法在体外确认了WOX1与SAMDC1的相互作用。我的结果表明,WOX1参与分生组织的发育调控,这种调控可能是通过调节SAMDC的活性,进而影响多胺代谢,从而调节细胞的分裂活动实现;同时,多胺也有可能反过来调节茎端分生组织的发育。我通过逐步删除WOX1上各结构域的办法检测了WOX1与SAMDC1的结合位点,结果表明WOX1是通过其N端的同源结构域与SAMDC1结合并行使功能的。 关键词:转录因子,分生组织,细胞周期,WUSCHEL相关同源结构域,多胺

英文摘要

Self-perpetuation and organ initiation are the two main tasks separated spatially in plant meristem. Slowly dividing cells in the meristem center act as pluripotent stem cells, and only their derivatives in the meristem periphery specify new organs. Here we report the identification of an Arabidopsis mutant wox1-D, which exhibited subtle defects in the meristem development resulted from increased expression level of the WOX1 (〓USCHEL HOMEOB〓 1) gene. The shoot apex of wox1-D mutant was smaller than that of wild type, while the mutant plants displayed dwarfism and a mildly bushy phenotype. The mutants also produced smaller and darker green leaves and exhibited a failure in anther dehiscence causing male sterility. During the development of wox1-D mutants, expression of the stem cell marker CLV3 is down-regulated in meristem and later reactivated in other regions such as the root-hypocotyl junction and the sites for lateral root initiation. In addition, the organ size and cell size in leaves of wox1-D is smaller than those of wild type, which suggests that the alteration of CLV3 expression pattern in meristem has partially retarded lateral organ development. The fact that the phase-specific expression of cyclinB1 is down-regulated in the wox1-D mutant suggests that cell-cycle arrest is likely to be the main cause of the leaf phenotype in the wox1-D mutant. Therefore, over expression of WOX1 may affect the CLV3 expression profile during meristem development. Using yeast two-hybrid method, a SAMDC protein (SAMDC1) interacting with WOX1 was isolated and their interaction was confirmed via MBP pull-down assay. WOX1 is possibly involved in meristem development via modulating SAMDCs activity and polyamines homeostasis. Our results also suggest that polyamines are likely playing an important role in maintenance of the meristem. To clarify which domain in WOX1 is responsible for the interaction with SAMDC1, we conducted a deletion analysis. The results showed that WOX1 interacted with SAMDC1 through the homeotic domain at its N-terminus. Keywords: transcription factor, meristem, cell cycle, WUSCHEL-related homeobox, polyamines

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