基于生长素响应因子ARF在香蕉后熟进程中的调控机制研究

Banana fruit ripening is a complex, genetically programmed process involving remarkable changes in the color, texture, aroma and flavor of the fruit, which provides the necessary vitamins, amino acids, minerals and other nutrients for human health. Our previous studies revealed that auxin response factor (ARF) might be not only a key transcription factor in auxin signal but also an important link in auxin signal with ethylene signal, which actively involve in the regulation of banana fruit ripening. However, the molecular mechanism is largely unknown, and the downstream target genes of ARF need to be identified. In this project, key ARF associated with banana ripening will be screened using a variety of analysis, such as gene expression profiling, proteomics analysis, western blot and real-time quantitative PCR. Plant expression vector will be constructed, and transformed into tomato using agrobacterium-mediated transformation system. The gene function of ARF will be verified in tomato ripening process. Monoclonal antibody of ARF will be purified and proteins interacted with ARF will be analyzed through a variety of protein interactions technical analysis. Meanwhile, the downstream target genes of ARF will be identified by using ChIP-Seq and yeast one-hybrid technologies. Finally, the pathways of auxin signaling and ethylene signaling will be constructed based on the existing gene expression profiling data and reported data in other references. The crosstalk of these two signaling pathways will be analyzed, which might reveal the regulatory mechanism of ARF in bananas ripening, and improve the model of interaction between auxin and ethylene signaling.

香蕉果实后熟过程中，其品质、风味逐渐形成，为人类健康提供了必要的维生素、氨基酸、矿质营养等。申请人研究发现生长素对香蕉的后熟进程有显著的正调控作用。ARF作为生长素信号中重要的转录因子，可能是生长素信号与乙烯信号相互作用的桥梁。然而，其调控机制目前尚不清楚，靶基因还需进一步实验鉴定。本项目中拟以香蕉果实为材料，利用表达谱、蛋白组、Western blot、定量PCR等手段筛选出香蕉后熟进程中起关键作用的ARF，并构建植物超表达载体，利用农杆菌介导的转化体系转化番茄，验证ARF的基因功能。随后纯化ARF的单克隆抗体，通过多种蛋白互作技术分析与ARF相互作用的蛋白质，同时利用ChIP-Seq和酵母单杂交等手段鉴定ARF调控的下游靶基因。最后依据现有表达谱数据和已报道的文献数据构建生长素信号和乙烯信号的调控路径图，解析ARF在香蕉后熟过程中的调控机制，同时完善生长素信号和乙烯信号相互作用的模式。

香蕉果实后熟是一个复杂的多因素共同调控的生物学过程。期间，果实品质、风味逐渐形成，为人类健康提供了必要的维生素、氨基酸、矿质营养等。然而其调控机制目前尚不清楚，需要进一步挖掘。我们利用表达谱、蛋白质分析香蕉果实自然后熟和乙烯诱导的后熟过程，发现24个生长素信号相关基因、蛋白均显著上调。随后我们利用IAA处理了香蕉果实，发现不仅其后熟过程明显缩短，而且乙烯信号相关基因显著上调。我们筛选并克隆了香蕉ARF17。表达分析发现，ARF17不仅参与了香蕉果实后熟，也参与了香蕉响应低温逆境。亚细胞定位分析发现，ARF17定位于细胞核，具有转录因子活性。在番茄中超表达ARF17后，乙烯信号相关基因显著上调。通过蛋白互作分析发现，ARF17与NHB1、STPP2A、PI4KA1和PP2C12存在明显的相互作用。降解组测序等分析发现，miR160可以靶向ARF17，且二者的表达水平在香蕉后熟和抵御低温逆境过程中呈显著负相关。项目执行期间，发表SCI论文10篇，申请国家发明专利3项，辅助培养硕士研究生1名、博士研究生2名。