低温胁迫下番茄SUMO E3连接酶SIZ1介导的光破坏防御机制研究

Low temperature stress is one of the main obstacle factors in the production of the thermophilic vegetable crops such as tomato and cucumber in northern China. SUMOylation is an important form of post-translational modification of proteins and plays an important role in the low temperature response of plants. SIZ1, a SUMO E3 ligase, plays a key role in SUMOylation of plant. On the basis of the cloned tomato SUMO E3 ligase gene (SlSIZ1), obtained overexpression and RNAi tomato plants, we are planning to prepare SlSIZ1 antibody and to reveal protein signals corresponding to SlSIZ1 in transgenic plants. Co-IP will be used to investigate interaction between SUMO and VTC1. The APX and some other photosynthesis-related protein’s SUMOylation and interaction by SlSIZ1 will be verified by yeast two-hybrid method. At the same time, we will explore the impact of the process on photosynthesis. Under low temperature stress, effects of tomato SIZ1 on the distribution and transcriptional activity of Whirly protein in chloropast and cell nucleus will be analyzed. The role of SUMOylation by SlSIZ1 in retrograde singnal and in photoprotection mechanism of photosynthesis will be investigated. These researches are important for the cultivation and breeding of chilling-sensitive vegetable plants for chilling tolerance.

低温胁迫是我国北方地区番茄、黄瓜等喜温蔬菜作物保护地生产中的主要障碍因素之一。SUMO化修饰是蛋白质翻译后修饰的一种重要形式，对于植物的低温响应具有重要作用。植物SIZ1是一种SUMO E3连接酶，在SUMO化的过程中起着关键作用。本项目拟在已克隆番茄SUMO E3连接酶基因（SlSIZ1），并获得过表达、RNAi番茄植株的基础上，制备SlSIZ1抗体，Western 杂交检测转基因植株, 通过Co-IP等方法研究VTC1是否被SIZ1介导的SUMO化修饰；利用酵母双杂等方法验证SIZ1与APX及一些光合作用相关蛋白的互作及其SUMO化修饰情况，并揭示该过程对光合作用的影响。研究低温胁迫下番茄SIZ1对Whirly蛋白在叶绿体与细胞核中分布以及转录活性的影响，明确SUMO化修饰在质体信号通路及光合作用的光破坏防御机制中的作用。该研究对于指导喜温蔬菜栽培和抗逆育种具有重要意义。

本课题主要研究了低温胁迫下番茄SIZ1及SIZ2对叶绿体光合作用的影响。①我们获得了Mirco-Tom背景的SIZ1及SIZ2的突变体株系(siz1和siz2)。②我们发现siz1及siz2突变体株系在低温胁迫下较之野生型均不耐低温，BN-PAGE实验分析显示siz1及siz2突变体中光系统蛋白复合体均严重降解。③进一步分析显示在正常生长条件下siz1突变体植株的二氧化碳同化速率、Rubisco含量及活性均低于野生型株系，且低温胁迫能够放大这一效果。而siz2突变体的碳同化速率及Rubisco与野生型无差异。④siz2突变体经低温处理后ROS积累量远高于野生型株系，而且其抗坏血酸含量低于野生型。⑤siz1突变体中，Rubisco小亚基RbsS1在叶绿体中的含量明显减少，其转录水平也低于野生型。进而我们发现SIZ1能够使WHY1 SUMO化，后者是RbcS1的直接上游调控因子。LUC实验显示，SIZ1的过表达能够促进WHY1对RbcS1的激活水平。⑥我们对AsA主要合成基因的转录水平进行了检测，结果发现GGP1在siz1突变体中的表达明显降低。后续我们发现ERF91作为GGP1上游的激活因子能够被SIZ2介导SUMO化，该SUMO化修饰能够促进ERF91对GGP1的激活效果，从而促进AsA的积累，降低低温胁迫产生的氧化伤害。