拟南芥内质网蛋白翻译调控基因SOL1在干旱胁迫适应中的作用机制研究

Intracellular perturbations such as cellular Ca2+ concentration or redox homeostasis caused by a variety of stressors, e.g. viral infection, energy deprivation, NH4+ toxicity, heat stress, drought or salt stress, induce the unbalance of protein folding, leading to the accumulation of unfolded proteins and endoplasmic reticulum (ER) stress. In mammalian cells, there are three ER receptors including activating transcription factor 6 (ATF6), inositol requiring kinase 1 (IRE1) and double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK), which sense ER stress signals and activate the downstream response trying to reestablish the balance of protein folding. In Arabidopsis, bZIP17 and bZIP28, similar to ATF6, and bZIP60 as the target of IRE1, were reported in ER stress signals. However, the PERK pathway sensing ER stress to regulate protein translation was neither reported in plants. An Arabidopsis mutant lew1 (leaf wilting 1), isolated by the applicant, activates earlier ER signals to reestablish the balance of protein folding than wild type, due to unfolded proteins caused by a certain defect of protein glycosylation, so that mutants lew1 are more resistant to drought stress than wild type. The applicant mutagenized the drought-resistant mutants lew1 and isolated a recovered mutant sol1 (suppressor of lew1 1). The mutation site of sol1 has been found, and primary evidences show that SOL1 mediates protein translation in ER stress signaling, suggesting that plants slow down protein translation to reduce the demand of protein folding in drought stress conditions, similar to PERK pathway in animals.The application aims to uncover the mechanism of SOL1 in controlling protein translation and the adaptive regulation of protein translation in drought stress, using mutants lew1, lew1sol1 and sol1 as the materials. By means of physilogical analysis, biochemical assays, profile analysis of gene expression and protein technology, the adaptive mechanism of SOL1 in ER signaling to regulate protein translation in response to drought stress will be disclosed, as a new pathway of drought stress and ER stress signaling in plants and the rationale to increase the resistant ability to drought stress in crops by genetic engineering and traditional breeding.

各种胁迫因子引起内质网(Endoplasmic Reticulum, ER)蛋白折叠失衡，引发ER胁迫。动物三种ER受体ATF6、IRE1及PERK感应ER胁迫，激活下游防御机制。拟南芥中仅发现有类似ATF6及IRE1途径，类似PERK的蛋白翻译调控在植物界尚未报道。申请人之前筛选到拟南芥突变体lew1，由于蛋白糖基化受损而产生较多错误折叠的蛋白，更早地激活了ER信号防御机制，比野生型更抗干旱。EMS诱变lew1，申请人筛选到抗旱表型恢复的突变体sol1，已找到突变位点，初步表明SOL1在类似PERK介导的蛋白翻译调控中起作用。本项目以突变体lew1、lew1sol1及sol1为材料，通过生理生化、转录组分析及蛋白技术等，阐明SOL1在ER蛋白翻译调控中的作用机理，揭示ER蛋白翻译调控在植物抗旱中的作用，为植物抗旱研究及ER信号途径开辟新路径，同时为改良作物的抗旱性及抗旱育种提供理论基础。

酵母中，逆境胁迫下GCN1与GCN2互作，激活GCN2，使蛋白翻译起始因子eIF2α发生磷酸化，而eIF2α的磷酸化使蛋白翻译水平下降，帮助酵母适应逆境胁迫。通过EMS诱变技术我们筛选到得到一个对冷胁迫敏感的突变体atgcn1-1，利用图位克隆的方法克隆了拟南芥AtGCN1，证实其与拟南芥GCN2互作，在SA、UV、冷胁迫及氨基酸饥饿时磷酸化eIF2α，而且eIF2α磷酸化是植物适应冷胁迫所必需的。atgcn1-1 及atgcn1-2 突变体中eIF2α不能发生磷酸化，且突变体对冷胁迫敏感。对核糖体RNA进行分布分析发现，与野生型相比，突变体atgcn1-1的蛋白翻译水平降低了。提取总RNA及核糖体RNA进行测序，结果表明eIF2α磷酸化可选择性地调控基因的转录及翻译。进一步分析表明，植物中eIF2α发生磷酸化后对上游没有uORF或含有强起始密码子（G/ANNATGG）的mRNA有一定的翻译优先性。另外，更重要的是，虽然突变体atgcn1-1的蛋白翻译水平与野生型相比降低了，但是叶绿体及线粒体中蛋白的翻译水平却升高了。以上实验结果表明，AtGCN1所介导的冷胁迫下蛋白翻译水平的抑制是植物适应冷胁迫所必需的，而且eIF2α磷酸化影响植物的开花时间、生长发育、种子形成及特定基因的转录及翻译。