香蕉果实成熟软化过程中淀粉降解的转录调控机制解析

Banana, being a typical climacteric fruit, is highly prone to ripening and softening after harvest. Starch is the primary component in pulp of unripe banana and its content declines dramatically during softening. Starch degradation is a complicated process regulated by several enzymes, but until recently, little information is available about the roles of enzymes involved in starch degradation as well as the mechanism underlying transcriptional regulation of this process. Although transcription factors (TFs) along with their post-translational modifications are known to mediate starch degradation process, some critical issues like whether TFs regulating the starch degradation resulting in ripening and softening of fruit and the corresponding transcriptional regulatory networks are still largely unclear. . In this project, fruit at two different maturity stages were used as experimental materials, members of gene families encoding enzymes involved in starch degradation and their upstream TFs were identified, and then the interacting proteins with these TFs associated with protein modifications are screened and characterized, Moreover, the effect of TFs with post-translational modifications on the regulation of targets involved in starch degradation is also investigated. The results of our project will provide novel information about TFs and their corresponding transcriptional regulatory networks involved in starch degradation, expand our understanding of the relationship between ripening-related TFs along with their post-translational modifications and starch degradation in fruit, and provide a theoretical basis for further elucidating and advancing the regulatory mechanism underlying fruit ripening.

香蕉属于跃变型果实，果实采收后极易成熟软化。未成熟的香蕉果肉主要成分是淀粉，其含量在果实成熟软化过程中急剧下降。淀粉降解是由多种酶共同参与完成的，但目前对于淀粉降解酶在淀粉降解中的作用及其协同调控淀粉降解机制研究较少。转录因子及其蛋白质修饰参与淀粉降解，但转录因子是否可以直接调控果实淀粉降解，从而参与果实成熟软化，其具体的转录调控网络又是什么等关键问题，目前并不清楚。.为此，本项目以两种饱满度不同的香蕉果实为材料，获得参与淀粉降解的主要酶基因家族成员及调控这些成员的转录因子，筛选、鉴定与转录因子修饰有关的互作蛋白，并进一步分析转录因子发生修饰后对其调控淀粉降解关键酶基因的影响。研究结果可以明确参与香蕉果肉淀粉降解相关的转录因子，揭示这些成熟相关转录因子及其蛋白质修饰与果实淀粉降解的关系，对于深入阐明果实成熟调控机制，拓展果实成熟调控网络具有重要的理论意义。

香蕉果实采收后伴随着乙烯释放，淀粉迅速降解并转化为可溶性糖，但是香蕉果实采后成熟进程中的淀粉降解机制目前并未明确。本项目中，我们研究了38个淀粉降解相关酶基因在果实成熟过程中的表达特征，探讨了MaGWD1的调控因子MaAP2a-1，MabHLH6和MYB3等在调控香蕉果实淀粉降解的作用及其机制，获得了以下结果： .1．采后香蕉果实伴随着乙烯释放及果实软化，淀粉含量下降，可溶性糖含量升高；淀粉粒表面由光滑变成平行的凹槽。并且，参与香蕉果实淀粉降解的38个基因中的27个，包括MaGWD1和MaPWD1等在果实成熟中被明显诱导，并且与淀粉转化为可溶性糖的过程一致。而MaGWD2和MaAMY2A等7个基因随果实成熟，表达水平下降。.2 iTRAQ蛋白组学实验分析结果显示，有18个淀粉降解相关酶基因结合到淀粉粒表面，其中包括MaGWD1和MaPWD1等10个蛋白响应果实成熟，并且表达量上升。.3．成熟乙烯能诱导MaGWD1启动子活性，推测MaGWD1在乙烯启动果实淀粉降解中起到重要作用，获得了结合MaGWD1启动子的MabHLH6，MaAP2a-1和MaMYB3；MaAP2a-1及MaMYB3在成熟过程中，转录水平和蛋白水平都受乙烯下调表达，而MabHLH6正好相反。. 4．MaAP2a-1及MaMYB3是转录抑制子，MabHLH6是转录激活子。EMSA和Chip-qPCR以及瞬时表达实验发现，MaAP2a-1能通过结合MaGWD1和MaPWD1等15个淀粉降解基因的启动子元件，抑制这些基因的转录；MaMYB3能明显抑制MaGWD1和MaSEX4等10个淀粉降解相关酶基因的启动子活性；而MabHLH6能激活MaGWD1和MaLSF2等11个淀粉降解酶基因的启动子活性。.5 获得了超表达MaMYB3的番茄转基因果实，并且超表达MaMYB3的番茄转基因果实在成熟进程中淀粉含量降解缓慢。