牡丹B类ARR-WUS互作响应细胞分裂素调控愈伤组织形成再生芽研究

牡丹集观赏、食用和药用于一身，市场潜力巨大。但无性繁殖效率低、高效再生体系缺乏严重阻碍了牡丹产业和基础研究的发展。完善再生体系是高效扩繁和实现遗传转化的关键所在。申请人前期研究发现细胞分裂素能够诱导牡丹愈伤形成再生芽，但不同牡丹基因型和外植体再生芽诱导率差异巨大。本研究基于已建立的‘秋发1号’叶片再生芽诱导体系，利用组织学观察确定愈伤诱导再生芽的关键时期，采用RNA-seq等初步筛选得到了2个关键B类ARR和WUS候选基因。通过转染拟南芥突变体验证其生物学功能；利用原位杂交技术研究ARR和WUS的组织表达模式，确定其响应时期和作用部位；运用双荧光素酶报告基因检测试验和EMSA技术研究ARR-WUS的互作模式，并明确细胞分裂素对其互作效应的影响。项目的实施有望阐明牡丹B类ARR-WUS互作调控愈伤组织形成再生芽的机制，为增强牡丹特定品种、外植体的再生诱导率以及基因工程育种奠定理论基础。

Tree peony can be used for ornamental, edible and medicinal purposes with a huge market value. However, low asexual reproduction efficiency and the lack of efficient regeneration system have seriously hindered the development of tree peony industry and basic research. Improving regeneration system is vital for efficient propagation and genetic transformation. We found that cytokinin can induce tree peony shoot regeneration from callus, but the regeneration efficiency of different peony genotypes and explants varied greatly. In this study, the mechanism of cytokinin-regulated shoot regeneration will be studied based on the established shoot regeneration system from leaves of ‘Qiufa 1’. Key induction periods of shoot regeneration from callus were determined by structural features. Two key genes of B-type ARR and WUS were screened based on RNA-seq and so on. We will verify the function of the key genes using genetic transformation technique. The tissue expression patterns of ARR and WUS during shoot regeneration from callus will be studied by in situ hybridization, determining its response period and action site. Dual-luciferase reporter gene assay and EMSA techniques will be used to investigate the interaction model of ARR-WUS, and determine the effect of cytokinin on their interaction. The implementation of this project is expected to elucidate the mechanism of shoot regeneration from callus by type B ARR-WUS interaction in tree peony, and lay a theoretical foundation for enhancing the regeneration efficiency for specific variety and explant, and genetically engineered breeding.