泛素连接酶E3及其底物蛋白在番茄果实成熟中的功能研究

Ripening is an important attribute of fruit, directly associated with fruit quality and storage life. It is important to elucidate the mechanisms regulating fruit ripening both in theoretical basis and practical implications. Ubiquitin mediated protein degradation plays important roles in many cellular processes, including plant development and disease resistance, however, its function in fruit ripening remain poorly understood. In our previous study, we have demonstrated that the ubiquitin conjugating enzyme E2 participated in the regulation of fruit ripening, however, the regulatory mechanism remains unclear. In ubiquitin proteosome pathway, the ubiquitin ligase E3 is able to interact with E2 and contribute to the specificity of ubiquitination. This project is planed to carry out an extensive search of the tomato genome to identify all potential E3 ligase and select those associated with fruit ripening for functional analysis. To characterize the ripening-related E3s, we describe the expression profile of E3s during fruit ripening using the RNA-seq combined with quantitative RT-PCR technique, determine the substrate proteins of these E3 ligases by quantitative proteomics, and furthermore, perform the methods of virus-induced gene silencing and protein interaction to explore the important roles of these E3s and their substrate proteins in fruit ripening. Based on these work, we can provide theory basis for engineering technology to control fruit ripening and senescence.

成熟是果实的重要生理特性，直接影响果实的采后品质和贮藏时间，研究果实成熟调控机制具有重要的理论意义和应用价值。泛素介导的蛋白质降解途径与植物发育、抗病反应等多种生命过程密切相关，但该途径在果实成熟中的作用少有报道。我们前期研究结果表明泛素结合酶E2参与了果实成熟调控，但其具体的作用机制仍不清楚。泛素/蛋白酶体途径为级联放大系统，其中与泛素结合酶E2互作的泛素连接酶E3在底物特异性识别等方面发挥着重要作用。本项目拟从全基因组水平寻找参与果实成熟的泛素连接酶E3并深入解析它们的功能。通过基因表达分析鉴定与成熟相关的E3蛋白，利用定量蛋白质组学探寻E3蛋白的作用底物，并综合运用基因沉默、蛋白质相互作用等技术明确相关蛋白在果实成熟过程中的作用。这些研究结果将为进一步利用基因工程技术来控制果实成熟衰老提供理论依据。