月季RhAG基因在低温导致花朵过度重瓣化中的作用机制解析

Rose is one of the most important cut flowers in the world. In China, production of cut rose flower would have higher profit in the winter than other seasons due to the price. However, low temperatures（<10℃）in the winter would cause formation of excessive petal, resulting in low quality flowers with abnormal shape. Our previous work demonstrated that RhAG plays an important role in petal development under low temperature by In situ hybridazition and VIGS approach and we found some the transcription factor genes with expression differences by comparing the transcriptomic profiles of normal flowers and RhAG-silenced ones. Here, we propose to isolate downstream gene regulated by RhAG. In addition, we try to isolate the potential transcription factors which regulate RhAG by yeast-one-hybrid using the promoter of RhAG. Then we will confirm the function of candidate genes in petal development using VIGS approach. In situ hybridazition will be conducted to show the temporal and spatial expression pattern of candidates, while EMSA, ChIP and transactivation assay will be used to make sure the regulatory relationship of RhAG and the candidates. Base on these works, we try to establish the RhAG-dependent transcriptional regulatory network and the central role of RhAG. The results should shed lights on the molecular mechanism of low-temperature induced excessive and aberrant petal formation in rose, and may provide potential genes for molecular breeding in the future.

月季是全球第一大切花。在我国，月季冬季生产效益最高。然而，低温条件（<10℃）往往引起花瓣过度重瓣化，导致花朵畸形，严重影响切花品质。申请者通过原位杂交和基因沉默等手段，明确了RhAG基因在花朵过度重瓣化中起到了重要作用，并通过对RhAG沉默植株与正常植株的转录组数据比较，发现了多个差异表达的转录因子基因。本项目拟对确受RhAG调控的下游基因展开筛选。同时，利用已克隆的RhAG启动子，开展响应低温，调节RhAG表达的上游转录因子筛选。通过基因沉默方法明确RhAG上游和下游候选基因的功能；基于原位杂交技术分析其时空表达特性；通过EMSA、ChIP和转录激活等技术证实候选基因与RhAG的上下游关系。项目力图从转录层面探讨以RhAG为重要节点的基因调节网络及其在低温调节花朵发育中的作用机制，进而为解决月季冬季生产中花朵畸形这一实践中的瓶颈问题提供理论依据，并为月季品质改良分子育种提供基因储备。

月季是全球第一大切花。我国月季切花70%以上产自云南地区，冬季的短时低温可以引起（<10℃）花瓣过度重瓣化和花朵畸形，严重影响切花品质。申请者之前已明确，花器官发育C-类基因RhAG是引起花朵过度重瓣化的关键因子。基于前述发现，在本项目的支持下，采用酵母单杂交体系对RhAG上游的转录因子进行了筛选，从中发现了一个AP2类转录因子RhANT。通过细胞学和生化方法，对RhANT的生化特性进行了分析。结果发现，RhANT可以与RhAG的启动子直接结合，参与早期花器官的发育。通过互作筛选发现，RhANT蛋白可以与DNA去甲基化酶RhROS1互作。.同时，为理解RhAG调节的下游基因网络，通过比较RhAG沉默株系与对照植株的表达差异，获得了获得RhAG下游基因表达谱，并与花芽中低温响应的基因表达谱进行了整合分析，从中获得了既受RhAG调节又响应低温的基因283个，其中转录因子16个，生长素合成和响应基因3个。对其中的5个基因进行了功能鉴定，发现生长素运输基因RhPILS1参与了花器官的发育。这些发现对于理解低温下切花月季重瓣化的分子机理提供了新的思路，对于月季遗传改良基因工程提供了新的基因资源。