月季细胞周期相关蛋白RhAPC3b调控花朵大小的机制解析

Rose (Rosa sp.) is the most important cut flower worldwide. Flower size, as one of the important commodity features, directly determines the economic value of rose. Therefore, unraveling the mechanism for controlling flower size is very crucial for the rose cultivation. Previously, through RNA-seq and gene function analysis, we found the anaphase-promoting complex/cyclosome (APC/C) subunit RhAPC3b, which is related to cell cycle, may be involved in regulating flower size. It has been reported that the APC/C complex determines cell endoreduplication, and affects cell size and differentiation. However, its function in flower development remains unknown. In this project, we will generate transgenic rose plants by overexpressing RhAPC3b or knocking out RhAPC3b through CRISPR-based gene editing to confirm its function in regulating flower size. We will screen candidate upstream regulators of RhAPC3b by Yeast One Hybrid, and validate the interactions between upstream regulators and RhAPC3b promoter via ChIP-PCR, EMSA and dual-luciferase reporter assay. We will analyze gene function of upstream regulators by generating VIGS-based gene silencing plants and stable transgenic plants in rose. This research project will hopefully provide new data for revealing the role of cell cycle in controlling flower size, and also be beneficial for molecular breeding of rose plants.

月季是世界范围内第一大切花，花朵大小是月季重要的商品特征，直接决定了月季的经济价值。因此，解析控制月季花朵大小的分子机制，对于月季品种培育具有重要意义。申请者前期通过转录组和基因功能分析，发现细胞周期中的后期促进复合体（APC/C）亚基RhAPC3b可能参与调节花朵大小。APC/C复合体决定了细胞的内源复制进而可影响细胞大小和分化，但其在花发育中的功能尚无报道。本项目以RhAPC3b为切入点，拟通过对月季稳定过表达RhAPC3b植株和CRISPR基因编辑植株进行表型分析，确认RhAPC3b在调控花朵大小中的功能；通过酵母单杂交筛选文库筛选其上游调节因子，并利用ChIP-PCR、EMSA和双荧光素酶技术进行结合验证；VIGS基因沉默和稳定转基因技术验证上游调节因子的基因功能。本项目的实施将有望为揭示细胞周期调控在花朵大小决定中的作用提供新的数据，同时也可为月季品质改良分子育种提供基因储备。

月季是世界范围内第一大切花，花朵大小是月季重要的商品特征，直接决定了月季的经济价值。因此，解析控制月季花朵大小的分子机制，对于月季品种培育具有重要意义。细胞周期中的后期促进复合体（APC/C）是一类调控植物发育和细胞周期的多亚基复合体。申请者前期通过转录组和基因功能分析，发现细胞周期中的后期促进复合体（APC/C）亚基RhAPC3b可能参与调节花朵大小。本项目以RhAPC3b为切入点，通过拟南芥异源表达、月季病毒诱导沉默进行表型分析，确认RhAPC3b通过影响维管束发育调控花朵大小。且RhAPC3b的启动子活性表明，RhAPC3b在维管束中也有较高的表达。通过酵母单杂交筛选文库筛选其上游调节因子，并利用ChIP-PCR、EMSA和双荧光素酶技术进行结合验证，鉴定到一个HD-ZIP I家族转录因子，RhPMP1。病毒诱导的基因沉默和稳定转基因结果表明RhPMP1通过调控维管束发育调节花朵大小。本项目的实施为揭示细胞周期调控在花朵大小决定中的作用提供新的数据，同时也可为月季品质改良分子育种提供基因储备。