香蕉泛素连接酶E3介导的泛素化修饰调控果实成熟的机制研究

Banana, as a climacteric fruit, is very susceptible to postharvest losses due to rapid softening. In our previous studies, we have shown that transcription factors (TFs), including NAC, ethylene-responsitive factor (ERF) and bHLH, are involved in the regulation of banana fruit ripening, however, the protein modification of these TFs during fruit ripening is largely unknown. Ubiquitination serves as an important post-translational modification that mediates growth and development, and stress responses in plant. Ubiquitin ligase E3 is the key component of the ubiquitination pathway, while no study about the role and mechanism of protein ubiquitination modification mediated by E3 in the regulation of fruit ripening has been reported. In the present project, in order to reveal the role and mechanism of ubiquitination modification in regulating banana fruit ripening, ripening-related E3 belong to different types in banana fruit are selecetd according to their expression patterns during ripening. Then, ubiquitination modification of important ripening-related TFs, such as NAC, ERF and bHLH mediated by these ripening-related E3 are investigated. Identifying new interaction proteins of these ripening-related E3, and their direct target genes are also analyzed, moreover, the role and the mechanism of E3 in fruit ripening is further confirmed by over-expressing ripening-related E3 in tomato. The results obtained in the project will not only provide new evidence of post-translational modification involving in the regulation of fruit ripening, and expand our understanding about the regulation network of fruit ripening, but also provide essential theoretical support to improve the technology of controlling fruit ripening.

香蕉为乙烯跃变型果实，采后极易成熟腐烂，课题组前期研究发现NAC、乙烯反应因子ERF及bHLH等转录因子在采后香蕉果实成熟过程中具有重要作用，但对这些重要成熟相关转录因子的蛋白质修饰机理知之甚少。泛素化是植物体内一类重要的蛋白质翻译后修饰方式, 在生长发育和抗性反应中发挥重要作用。泛素连接酶E3是泛素化修饰中作用最为关键成员，然而，有关E3介导的泛素化修饰在果实成熟中的作用及机制，尚未见报道。本项目中，在获得与香蕉果实成熟相关的不同类型E3的基础上，分析E3对NAC、ERF及bHLH等重要成熟相关转录因子的泛素化修饰，并筛选、鉴定E3新的互作蛋白及下游靶基因，同时在番茄中超表达与香蕉果实成熟相关的E3基因，以期阐明泛素化修饰在采后果实成熟过程中的作用及机制。研究结果将为果实成熟在翻译后修饰水平上的调控提供新线索，拓展对果实成熟调控网络的认识，同时为改进采后果实成熟控制技术奠定理论基础。

泛素连接酶E3介导的泛素化是植物体内一类重要的蛋白质翻译后修饰方式，在生长发育和抗性反应中发挥重要作用。然而，有关E3介导的泛素化修饰在果实成熟中的作用及机制，尚未见报道。. 基于香蕉果实成熟相关的RNA-seq表达谱和荧光定量PCR，我们筛选获得了8个成熟相关的E3基因；通过酵母双杂、BiFC和免疫共沉淀（CoIP）证实4个RING-finger类型E3，MaRING1、MaRING2、MaRING3和MaXB3分别跟WRKY转录因子MaWRKY31、LBD转录因子MaLBD1、C2H2锌指蛋白MaC2H2-1/2以及NAC转录因子MaNACs存在蛋白互作。我们深入分析了MaXB3和MaNACs互作调控香蕉果实成熟的机制。经凝胶阻滞分析、染色质免疫共沉淀和瞬时表达分析，表明MaNAC1/2可以直接结合乙烯信号负调控因子MaRTH1和MaERF11的启动子并抑制其表达，而MaNAC10/11结合MaACS1和MaACO1的启动子并促进其表达。蛋白质互作和泛素降解实验发现，MaXB3与MaNAC2/10/11、MaACS1和MaACO1互作，并通过26S蛋白酶体途径泛素化降解MaNAC2/10/11、MaACS1和MaACO1，进而削弱MaNAC2/10/11的转录能力和乙烯合成。番茄中超表达MaXB3延缓了果实成熟。更有意思的是，MaNAC1和MaNAC2还可以直接结合MaXB3的启动子并抑制其表达，反馈抑制MaXB3介导的泛素化降解，维持MaNAC2/10/11、MaACS1和MaACO1的蛋白稳定性。这些结果说明E3通过泛素化修饰并降解NAC等转录因子以及乙烯调控因子，影响乙烯信号和合成，进而参与调控香蕉果实成熟。本项目的研究结果丰富和加深了人们对E3介导的泛素化修饰参与果实成熟的机制及其网络的认识，为研发维持采后香蕉果实品质的技术措施提供了科学依据。. 在本项目的资助下，已经在Plant Molecular Biology Reporter、Postharvest Biology Technology、食品科学以及园艺学报等刊物上发表了4篇论文，培养了1名博士研究生，2名硕士研究生提前攻读博士学位。1名项目组成员获聘广东省青年珠江学者。