磷酸化修饰的bZIP转录因子调控香蕉果实成熟的机理解析

Fruit ripening is modulated by various internal and external factors, including plant hormones, transcription factors, temperature, light, humidity and nutritional substances, among which transcription factors along with their regulatory networks play an essential role in the process. The bZIP (basic leucine zipper) transcription factors are reported to function in plant diverse developmental processes, responses to biotic/abiotic stresses, as well as plant primary and secondary metabolites biosynthesis. However, the precise roles of bZIP transcription factors in fruit ripening remain largely unknown. In this project, the banana fruit are used and two bZIP transcription factors from banana which were named MabZIP21 and MabZIP47 are found to be up-regulated and phosphorylated in the ripening stage. Thus the aims of our project are to characterize the target genes of MabZIP21 and MabZIP47 by using DNA-protein interaction strategies, and to find out their interacting proteins such as protein kinases and transcription factors through protein-protein interaction approaches. Moreover, the effects of MabZIP21 and MabZIP47 that are phosphorylated on the regulation of their target genes are investigated by using the transient expression methods. Finally, their biological functions in fruit ripening are also validated via the transgenic transformation in tomato fruit. The results of our project might pave the way to an understanding of the molecular mechanism of phosphorylated transcription factors in controlling fruit ripening, and provide a theoretical basis for improving the quality of fruits and vegetables during storage and transportation.

果实成熟受内在和外在诸多因素调控，包括植物激素、转录因子、温度、光照、水分和营养物质等，其中，转录因子介导的分子调控网络在果实成熟过程起着重要作用。bZIP（basic leucine zipper）转录因子参与植物发育过程、响应生物非生物胁迫，以及植物初生/次生代谢物质合成等，但bZIP调控果实成熟的具体机制仍不明确。本项目拟以香蕉果实为试材，在获得果实成熟上调和受磷酸化修饰的MabZIP21和MabZIP47基础上，采用DNA-蛋白质互作技术研究它们调控的下游靶基因，通过蛋白质-蛋白质互作方法鉴定与它们互作的蛋白激酶和转录因子，并运用瞬时表达法分析MabZIP21和MabZIP47磷酸化修饰后对下游靶基因调控的影响，最后利用农杆菌侵染法转化番茄分别鉴定它们的生物学功能。研究结果为深入揭示磷酸化修饰的转录因子调控果实成熟的分子机制奠定基础，也为改善果蔬贮运品质提供理论依据。

转录因子介导的转录调控机制在果实成熟过程中发挥重要作用，但有关转录因子的翻译后修饰如磷酸化是如何调控果实成熟仍很大程度未知。本课题通过转录组、DAP-Seq、凝胶阻滞迁移、染色体免疫共沉淀、蛋白质互作、体外磷酸化检测等手段，深入研究bZIP转录因子及其磷酸化修饰调控香蕉果实成熟的分子机制。研究结果加深了人们对呼吸跃变型果实成熟调控机理的认识，同时从多角度完善果实成熟的分子调控网络。主要研究结果如下：.①探明了低温抑制MabZIP4/5活性是低温贮藏导致香蕉香气含量下降的主要原因。MabZIP4直接结合BanAAT启动子，而MabZIP5直接结合MaMT1、MaACY1、MaAGT1和BanAAT启动子；低温贮藏抑制了MabZIP4/5活性进而下调香气合成相关基因的转录。.②发现了MabZIP93的磷酸化修饰调控香蕉果实软化的分子机制。MaMPK2磷酸化果实成熟上调的转录因子MabZIP93，磷酸化修饰后的MabZIP93能显著提高靶基因MaPL2、MaPE1、MaXTH23和MaXGT1的转录，从而促进香蕉果实成熟软化。.③揭示了磷酸化修饰MabZIP74调节香蕉果实乙烯合成的作用机理。转录抑制子MabZIP74抑制乙烯合成基因MaACO1/4的转录，随着果实成熟进程，MaMAPK11-3促进MabZIP74磷酸化，磷酸化后的MabZIP74解除了其对MaACO1/4的抑制效应，从而促进果实乙烯合成。.④解析了磷酸化修饰MabZIP21参与香蕉果实成熟的调控模式。蛋白质磷酸化组学显示MabZIP21蛋白在果实成熟显示出较高的磷酸化水平。MabZIP21与促分裂原活化蛋白激酶MaMPK6-3相互作用，体外磷酸化实验揭示了MabZIP21被MaMPK6-3磷酸化，并鉴定出T318和S436是重要的磷酸化位点。体内和体外蛋白质磷酸化增强了MabZIP21介导的转录激活能力。