RcAP2和RcAG调控月季‘绿萼’花器官萼片化发育的分子机制研究

Floral organ sepalody is one of the most important phenomena in floral development of ornamental plant. However, litter information is known on its development mechanism. Rosa chinensis cv. Viridiflora is a Chinese old garden rose cultivar, represents an important rose germplasm resource, in which petals, stamens and pistils are converted into sepal-like organs known as sepalody. As being the stable mutant of rose floral development, Viridiflora is the valuable material for researching floral organ development. In our previous study, using grafting and microscopy and PCR experiments, these results strongly show that the flower mutant phenotype of Viridiflora was not caused by phytoplasma infection. Transcriptome comparisons by the mean of RNA-Seq identified some genes involved in floral development in Viridiflora compared to its closely related genotype R. chinensis cv. Old Blush. The expression of RcAP2 and MADS-box genes analyzed using qPCR in different tissues of developing flowers of Viridiflora and Old Blush. The results suggest that the ectopic expression of RcAPETALA2 (RcAP2) in petal, stamen and pistil associated with a down-regulation of the C-class gene RcAGAMOUS (RcAG) correlate with loss of floral identity in Viridiflora. According to the above achievements, the current study will perform promoters cloning of RcAP2 and RcAG, respectively, and analyze its cis-transcription element and Methylation. Then, subcellular position of RcAP2 and RcAG will be performed by combining the methods of fusion protein construction, infection, fluorescence microscope observation. Furthermore, transcriptional activity assay will be employed to verify whether RcAP2 and RcAG possess trans-activation ability alone, and identify interacting proteins of the two genes using yeast two-hybrid system. Finally, RNAi vector of RcAP2 and over-expression vector of RcAG will be constructed, and their functions will be furtherly analyzed by genetic transformation to Viridiflora. These results can not only understand molecular mechanism of RcAP2 and RcAG in floral organ development of R. chinensis, but also provide candidate genes for unique flower shape in ornamental plant.

花器官萼片化是观赏园艺植物发育中的重要现象，但其发育机制一直不清楚。‘绿萼’是中国古老月季最珍贵的种质资源之一，花朵形态表现为花瓣、雄蕊、雌蕊均转变为萼片状，是研究花器官发育的宝贵材料。本项目在前期研究中已确定‘绿萼’独特的表型不是植原体引起；发现 RcAP2在‘绿萼’花瓣、雄蕊和雌蕊中异位上调表达，RcAG在其雌蕊中显著下调表达，表明RcAP2和RcAG在‘绿萼’花器官发育过程中扮演重要角色。在此基础上，本项目首先克隆RcAP2和RcAG启动子，分析转录元件、甲基化情况；其次，利用亚细胞定位、转录激活活性分析及酵母双杂交技术明确RcAP2和RcAG的亚细胞位置、转录活性及互作蛋白；最后，构建RcAP2沉默和RcAG高表达载体及转化验证在‘绿萼’发育过程中的功能。研究结果不仅从理论上解析RcAP2和RcAG调控月季花器官发育的分子机制，同时为该类基因在观赏植物花型遗传改良中的应用奠定基础。

‘绿萼’是中国古老月季最珍贵的种质资源之一，花朵形态表现为花瓣、雄蕊、雌蕊均转变为萼片状，是研究花器官发育的宝贵材料。花器官萼片化是观赏园艺植物发育中的重要现象，但其发育机制一直不清楚。本项目在前期研究发现 RcAP2在‘绿萼’花瓣、雄蕊和雌蕊中异位上调表达，RcAG在其雌蕊中显著下调表达，表明RcAP2和RcAG在‘绿萼’花器官发育过程中扮演重要角色。在此基础上，本项目克隆了RcAG启动子序列，与对照‘月月粉’相对，RcAG的启动子序列CHG和CHH的DNA甲基化水平明显上升，暗示甲基化修饰可能是‘绿萼’萼片化发育过程中扮演重要角色。其次，完成了RcAG和RcAP2的亚细胞定位，发现RcAG 和RcAP2定位在细胞核内，表明RcAP2和RcAG均发挥转录因子的功能。利用VIGS系统结合嫁接技术，完成了RcAP2和RcAG基因的功能分析，发现沉默RcAG基因的‘月月粉’内轮花瓣萼片化；而沉默RcAP2的‘绿萼’发现了有少量雄蕊正常发育。另外，完成了月季‘绿萼’ATAC-seq的测序及分析，发现‘绿萼’的表型变异可能与花发育相关基因的启动子修饰及调控有关。最后，完成了月季‘绿萼’组蛋白27位赖氨酸的三甲基化修饰（H3K27me3）Chip-seq分析，发现组蛋白H3K27me3蛋白在‘绿萼’中的表达显著增强。研究结果不仅从理论上解析了RcAP2和RcAG调控月季花器官发育的分子机制，揭示了表观遗传在花器官发育过程的重要作用，同时为该类基因在观赏植物花型遗传改良中的应用奠定基础。. 项目发表论文5篇，其中3篇SCI收录；培养研究生3人；1人晋升研究员，2人晋升副研究员，项目负责人获得2020年云南省高层次人才培养支持计划“青年拔尖人才”专项。