黄花苜蓿E3泛素连接酶Mf630参与内质网胁迫响应的分子机制

Endoplasmic reticulum (ER) stress response is important for maintaining cell homeostasis. It has been largely studied in yeast and mammals; however, there is little knowledge in plants and the relation genes are homologs of yeast or mammals. Previous work has been reported that ER stress is triggered by abiotic stress like drought and salinity, thus, it is important to study the molecular mechanisms underlying ER stress response and therefore identify genes which are responsible to stress response, providing insight into molecular breeding in crops. In this study, we identified a gene from Medicago falcata, Mf630 with unknown function, which encoded a RING finger protein and was induced by salt, drought and ER stress. We further found that Mf630 displayed E3 ubiquitin ligase activity and localized in ER membrane of plant cell. Moreover, we found that Mf630 interacted with Sec61γ, a subunit of Sec61 complex and Mf630 had no effect on degradation of Sec61γ in ER. Based on these works, we will further combine the physiology, genetic, biochemistry and molecular biology approaches to study Mf630 physical function, interaction with Sec61γ and identifying other components which may involve in unfolded proteins degradation system, so that we will get a deep understanding about biological interaction between Mf630 and Sec61γ and uncover the precise molecular mechanism of Mf630 in regulating ER stress response.

内质网胁迫响应对维持细胞内稳态十分重要，目前研究主要集中在酵母和哺乳动物，植物中较少且为对酵母或动物同源基因的研究。盐, 旱等环境胁迫能导致植物内质网胁迫，研究植物内质网胁迫响应机制对抗逆育种和新品种创制有重要意义。本课题组从黄花苜蓿中克隆到一个受盐和干旱诱导的含RING finger的基因Mf630。 前期结果表明Mf630有体外多聚泛素化活性，定位于内质网膜, 响应内质网胁迫，与内质网蛋白转运复合体Sec61的γ亚基相互作用，但不影响Sec61γ降解。在此基础上, 拟深入研究Mf630参与内质网胁迫的分子机制，主要研究内容包括以生理、遗传，生物化学和分子生物学手段，确定胁迫诱导Mf630的生理学功能，分析 Mf630与Sec61复合体的相互作用，发掘参与胁迫产生变性蛋白降解系统的组分，明确Mf630与 Sec61γ作用的生物学意义，阐明Mf630参与内质网胁迫的分子机制。

内质网胁迫响应是维持细胞内稳态的重要反应。正常条件下，内质网分子伴侣帮助蛋白质正确折叠；胁迫条件下，错误折叠蛋白质通过内质网相关降解途径（endoplasmic reticulum associated degradation，ERAD）降解，二者之间存在精确平衡，如果该平衡被打破，则会产生内质网胁迫。植物在面临高盐、干旱、高温等非生物胁迫或者生物胁迫下，均能导致内质网胁迫。本研究在分析黄花苜蓿非生物胁迫响应表达谱时，发现了一个受盐和干旱显著诱导表达的 RING finger 基因，当时克隆并命名为 Mf630。BLAST 比对分析表明 Mf630 是一个新基因，在植物中没有相关同源基因的研究报道。按照发表文章杂志编辑的建议，我们后来用MfSTMIR (M. falcata salt tunicamycin-induced RING finger protein)替代Mf630。我们的研究结果首次发现了植物参与ERAD的E3泛素连接酶MfSTMIR，体外生化实验证实MfSTMIR能够促进ERAD通用底物CPY*的降解。我们进一步构建了35S:MfSTMIR过表达转基因株系、MtSTMIR RNAi植物和Mtstmir-1突变体，通过遗传学证据证实了在盐胁迫和内质网胁迫下MtSTMIR能够激活ERAD相关的标记基因的诱导表达，体内实验证实了它是ERAD通路的活性组分。本研究提出了MfSTMIR工作模型：MfSTMIR与内质网胁迫关键转运通道蛋白Sec61的γ亚基互作，和泛素结合蛋白UBC32协同参与由Sec61转运出的错误折叠蛋白的降解。本研究阐明了MfSTMIR参与内质网胁迫的分子机制，明确了MfSTMIR 和 Sec61γ作用的生物学意义，丰富了对植物内质网胁迫响应的认识，为植物抗逆育种提供了新的理论基础。本项目作为第一标注发表2篇高水平SCI论文，包括：1篇Plant Journal和1篇Plant Cell。培养博士生4名，硕士生1名。