拟南芥内质网信号途径旱相关突变体的筛选及基因克隆

The endoplasmic reticulum (ER) is a fastidious membrane system to sense and transmit cell signals, connecting with nuclear membrane. In mammalian cells, there are three ER receptors including double-stranded RNA-activated protein kinase(PKR)-like endoplasmic reticulum kinase (PERK), inositol requiring kinase 1 (IRE1) and activating transcription factor 6 (ATF6). These three ER receptors sense ER stress signals and activate the downstream machanism trying to alleviate the damage of ER stress. In addition to these three ER receptor pathways, there are ER overload response mediated by transcription factor NF-κB and ERAD (ER Associated Degradation) pathway to degrade unfold proteins in ER signaling. In Arabidopsis, only bZIP17 and bZIP28, similar to ATF6, bZIP60, as the target of IRE1, and a small amount of proteins of ERAD pathway were ever reported to mediate ER stress. More importantly, the protective machanism of ER signals in drought stress is largely unknown. The applicant previously isolated an Arabidopsis mutant lew1 (leaf wilting 1). The lew1 mutation caused misfolded proteins due to a certain defect of protein glycosylation, so that mutants lew1 activated ER signals earlier and were more resistant to drought stress than wild type. The application aims to screen recovered mutants and clone drought-related genes in ER signaling through forward and reverse genetic methods, using the drought-resistant mutant lew1 as the materials. By means of physilogical analysis, biochemical assays and protein technology, the adaptive mechanism of ER signaling in drought stress will be uncovered, as the rationale to increase the ability to resist drought stress in crops by genetic engineering and traditional breeding.

内质网 (Endoplasmic Reticulum, ER)能够感受并传递细胞信号。动物ER信号途径除了三种ER受体ATF-6、IRE1及PERK感应并传递逆境信号的途径，还有ER 过载反应和将错误折叠蛋白降解的机制帮助细胞适应逆境。植物中ER信号途径仅发现有类似的ATF6、IRE1及蛋白降解机制，其它途径尚无报道，而且也无研究报道ER信号途径参与植物适应干旱胁迫的响应机制。申请人之前筛选到拟南芥抗旱突变体lew1，由于蛋白糖基化一定程度受损而产生较多错误折叠的蛋白，ER信号途径更早被激活，一些调适性机制提早启动，因此比野生型更抗干旱。本项目以抗旱突变体lew1为材料，利用正向、反向遗传学手段，筛选lew1抗旱表型恢复的突变体，克隆ER信号途径中参与干旱胁迫响应的基因，结合生理生化及蛋白分析，研究ER信号途径在植物适应干旱中的作用机理，为改良作物的抗旱性及抗旱育种提供理论基础。

内质网的主要功能之一是使蛋白发生糖基化以帮助蛋白正确折叠，正确折叠的蛋白才能被运到功能部位行使功能。动物中内质网信号途径研究比较详尽，但植物中研究较少。本项目以蛋白糖基化突变体lew1为材料，利用正向、反向遗传学手段，筛选了6个lew1黑暗及衣霉素敏感表型恢复的突变体株系，同源克隆了内质网信号途径中参与非生物胁迫的基因3个，图位克隆了1个。将同源克隆的基因进行了超表达及CRISPR定点突变，已经得到超表达植株，定点突变材料正在筛选。其他突变体正在进行图位克隆工作。我们的研究表明已知的内质信号途径基因并不能使lew1突变体表型恢复，因此这些lew1黑暗及衣霉素敏感表型恢复的突变体可以帮助我们挖掘到内质网信号途径的新基因，为阐明内质网信号途径在植物非生物胁迫中的作用奠定了基础。