矮牵牛PhGRL1互作的E3泛素连接酶在乙烯信号和花衰老中的功能分析

Ethylene regulates flower senescence in many ornamental plants. GR/RTE1 is the important component of ethylene signaling pathway. Our previous study showed that PhGRL1 is involved in flower senescence in petunia and one lysine ubiquitination site in PhGRL1 is identified by ubiquitylomes analysis. In addition, some E3 ubiquitin ligases interacting with PhGRL1 were identified by yeast 2-hybrid assay. Protein ubiquitination is an important post-translational modification that plays important roles in protein degradation and plant senescence. We speculate that these E3 ubiquitin ligases could be involved in ethylene signaling transduction and flower senescence by degradation of PhGRL1 in petunia. In this study, the function of these E3 ubiquitin ligases will be identified. ①The interaction of PhGRL1 with E3 ubiquitin ligases will further be confirmed by Y2H, BiFC and Co-IP assays. ② Temporal and spatial expression of these E3 ubiquitin ligase genes and the effects of ethylene on expression of them will be analyzed and the assay of subcellular localization of E3 ubiquitin ligases will be performed using fluorescence techniques. ③ The Crisp/Cas9- and overexpression- vectors of E3 ubiquitin ligase genes will be constructed and the transgenic petunia plants will be acquired. ④ To identify the function of E3 ubiquitin ligase genes, the vase lives and ethylene production of transgenic flowers, ethylene triple response, and the mRNA levels of ethylene signaling component genes will be tested. To know whether PhGRL1 protein is degraded by ubiquitination, the abundance of PhGRL1 protein will be examined in transgenic petunia. The results of this study would be important for the regulation of flower senescence and further clarifying ethylene signaling transduction mechanism.

乙烯是许多观赏植物花衰老的主要因子。GR/RTE1是乙烯信号重要组分，本课题组前期研究表明矮牵牛PhGR-Like 1(PhGRL1)参与了花衰老，泛素化组学分析发现PhGRL1被泛素化修饰，通过酵母双杂交获得与其互作的多个E3泛素连接酶（E3s），而泛素化修饰会导致蛋白质降解且与衰老关系密切，因此我们推测所获的E3s可能参与了乙烯信号转导与花衰老。本项目拟鉴定所获得的E3s在花衰老和乙烯信号转导中的功能。①BiFC和Co-IP等技术进一步鉴定二者的互作；②E3s亚细胞定位，时空表达分析及其对乙烯的应答；③构建E3s基因的沉默和超表达载体，导入矮牵牛。④分析转基因植株表型变化，观察其瓶插期以及幼苗乙烯三重反应表型，测定乙烯释放量等，分析转基因植株PhGRL1蛋白的丰度变化，鉴定E3s在花衰老和乙烯信号转导中的作用。该研究结果将对花衰老调控以及进一步阐明乙烯信号转导机制具有重要意义。

乙烯是许多观赏植物花衰老的主要因子。GR (Green Ripe)是乙烯信号重要组分，本课题组前期研究表明矮牵牛PhGR-Like 1(PhGRL1)参与了花衰老，泛素化组学分析发现PhGRL1被泛素化修饰。本项目用PhGRL1为诱饵，与矮牵牛酵母文库杂交，发现PhGRL1可以与两个E3泛素连接酶PhXB31a和PhUBC28A互作，另还可与PCD相关蛋白PhDHS互作。用BiFC进一步证实PhGRL1可以与PhXB31a互作。PhXB31a沉默植株的叶片发育不正常，花朵瓶插寿命延长1.9天，乙烯释放量减少，PhGRL1蛋白丰度增加，显示PhXB31a可能是通过降解PhGRL1延缓瓶插寿命。PhDHS沉默植株叶色变黄，花朵瓶插寿命延长2.8天，乙烯释放量减少。该结果暗示PhDHS和PhGRL1可能存在一定的关联。而PhUBC28A沉默植株未表现出可见表型变化。用PhXB31a为诱饵与矮牵牛酵母文库杂交，发现PhXB31a可以与PhGRL1和柠檬酸裂解酶亚基PhACLB1互作。PhACLB1和PhACLB2沉默叶片变小，加速了花瓣衰老，乙烯释放量增加。另我们还分析了乙烯处理条件下花瓣蛋白磷酸化修饰和泛素化修饰的关系，发现乙烯处理导致整体磷酸化水平下降，而导致整体泛素化水平上升，有12.1%的蛋白同时发生泛素化和磷酸化修饰。另外我们还探索了乙烯对矮牵牛花瓣蛋白m1A修饰的影响。发表SCI论文8篇，其中一区1篇，二区5篇。培养博士研究生2名，硕士研究生9名。