两组类受体激酶调控拟南芥根生长发育的分子机理

发布时间：2018-06-11 19:15

本文选题：类受体蛋白激酶 + 拟南芥　；参考：《兰州大学》2017年博士论文

【摘要】：在长期适应陆生环境的过程中,植物进化出根这一重要的营养器官,来执行固定、吸收养分和水分、合成以及运输等生理功能。根尖中的干细胞微环境,包括静止中心细胞和周围的干细胞,对于根的正常生长发育非常重要。在根尖干细胞微环境的维持过程中,不同类型细胞间的信号通讯对于细胞的命运具有决定性的作用。静止中心细胞合成并分泌根分生组织生长因子RGFs上调PLT1和PLT2进而促进干细胞分裂,以维持干细胞微环境的活力,但RGFs的受体仍然是一个未解之谜。本实验室一系列的证据表明BAK1不仅作为共受体调控油菜素内酯的信号转导,还参与不依赖于油菜素内酯信号的根发育途径。本实验室利用酵母双杂交系统筛选了161个LRR-RLKs,鉴定到83个与BAK1有相互作用,本研究采用遗传学的方法分析了两组LRR-RLKs的功能。第一组类受体蛋白激酶含有五个成员,被命名为RGF1 insensitives(RGIs,RGI1到RGI5)。RGIs的单重T-DNA插入突变体被分离到,但没有表现出明显表型。我们构建了一系列的双重、三重、四重和五重缺失突变体,表现出或多或少的根发育缺陷表型,五重缺失突变体表现出分生区缩短的严重根短表型。我们进一步利用完全独立的T-DNA插入突变体构建了第二套五重缺失突变体,与第一套五重缺失突变体表型几乎完全相同。RGIs五重缺失突变体中PLT1和PLT2表达下调到几乎不能检测,用RGI2的启动子异位表达PLT2能够极大地恢复五重缺失突变体的根尖分生区缺陷。RGIs的五重缺失突变体对外源施加的磺酸化的RGF1完全不敏感。体外实验斑点印迹和pull-down分析表明RGF1能够与RGI1的胞外结构域结合。外源施加RGF1能够在体内诱导RGI1和RGI2的磷酸化增强,表明RGIs能够感知并传导RGF1的信号。RGF1能够诱导RGI1的泛素化和降解,还能够诱导BAK1的磷酸化增强。这些结果表明,RGIs作为RGF1的受体,在RGF1-PLT介导的根分生区发育过程中有着不可或缺的作用。第二组类受体蛋白激酶含有三个成员,在根中有一定程度表达。超表达RLK101和RLK219都能导致初生根变短的表型。三重缺失突变体表现出萌发加快和根生长加快的表型,还导致了若干ARR表达上调。总的来说,我们对第一组类受体蛋白激酶RGIs进行了深入的研究,综合遗传、生理、生化、细胞等方法证明了RGIs作为RGF1的受体参与拟南芥根尖干细胞微环境自调控的过程。我们还对第二组类受体蛋白激酶做了初步的研究,证明了其负调控拟南芥根生长发育的功能。
[Abstract]:In the process of adapting to terrestrial environment for a long time, the plant evolved root as an important vegetative organ to perform physiological functions such as fixation, absorption of nutrients and water, synthesis and transportation. The stem cell microenvironment in the root tip, including stationary center cells and surrounding stem cells, is important for the normal growth and development of the root. In the process of maintaining the microenvironment of root tip stem cells, the signal communication among different types of cells plays a decisive role in the fate of cells. The synthesis and secretion of root meristem growth factor (RGFs) up-regulated PLT1 and PLT2 in order to maintain the viability of stem cell microenvironment, but the receptor of RGFs remains a mystery. A series of evidences in our laboratory indicate that BAK1 not only regulates the signal transduction of rapesinolide as a co-receptor, but also participates in the root development pathway which is independent of the Brassinolide signal. A total of 161 LRR-RLKs were screened by yeast two-hybrid system and 83 of them were identified as interacting with BAK1. The function of two groups of LRR-RLKs was analyzed by genetic method. The first group of receptor-like protein kinases containing five members, named RGF1 in sensitives-RGIsRGI1 to RGI5, RGIs, were isolated, but showed no obvious phenotype. We constructed a series of double, triple, quadruple and quintuple deletion mutants, which showed more or less defective phenotypes of root development, and five deletion mutants showed severe short root phenotypes with shortened meristem regions. We further constructed a second set of quintuple deletion mutants using completely independent T-DNA insertion mutants. The expression of PLT1 and PLT2 was down-regulated to almost undetectable in the first set of quintuple deletion mutants. The heterotopic expression of PLT2 in the promoter of RGI2 could significantly restore the apical meristematic defect of the quintuple mutant. The quintuple deletion mutant of RGIs was completely insensitive to exogenous sulfonic acid applied to RGF1. In vitro dot blot and pull-down analysis showed that RGF1 could bind to the extracellular domain of RGI1. Exogenous RGF1 could induce the increase of phosphorylation of RGI1 and RGI2 in vivo, which indicated that RGIs could sense and transmit the signal of RGF1. RGF1 could induce the ubiquification and degradation of RGI1, and enhance the phosphorylation of BAK1. These results suggest that RGIs, as a receptor of RGF1, play an indispensable role in the development of root meristem mediated by RGF1-PLT. The second group of receptor-like protein kinases contains three members and is expressed to a certain extent in the root. Overexpression of RLK101 and RLK219 both resulted in shorter primary rooting phenotypes. Triple deletion mutants showed the phenotype of accelerated germination and rapid root growth, and also resulted in a number of arr expression upregulation. In general, the first group of receptor-like protein kinases (RGIs) have been studied in depth. It has been proved that RGIs are involved in the process of microenvironment self-regulation of Arabidopsis thaliana root tip stem cells as RGF1 receptors by comprehensive genetic, physiological, biochemical and cellular methods. We also made a preliminary study on the second group of receptor-like protein kinases, which demonstrated the function of negative regulation of Arabidopsis root growth and development.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2;Q945

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