茶树不同外植体再分化潜力差异的表观遗传机制研究

Transgenic technology is an important approach for identifying the function of new gene and improving germplasm of the tea plant. Redifferentiation frequency of the tissues is quite low in vitro culture of tea plant, which is the main bottleneck of application of transgenic technology. However, the molecule mechanism on the redifferetiation obstacle of tissues from the tea plant is still unclear. In this study, the epigenetic differences of the genomic DNA methylations, histone modifications, sRNA components and gene expression patterns will be extensively compared in the dedifferentiated and redifferentiated materials as well as in the initial tea explants with different regeneration potentials. The effects of the DNA methylation and histone modification inhibitors on the change of epigenetic factors and the redifferentiation potentials of the explants will also be tested thoroughly. The variation patterns of the epigenetic factors in the explants during the dedifferentiation and redifferentiation process will be uncovered.The relationship between redifferentiation potential of the different explants and their epigenetic factors will be established and elucidated finally. These findings will help to reveal the regulatory mechanisms in molecule level during the dedifferentiation and redifferentiation process of tea explants, and will also provide a theoretical basis for improving the regeneration and transformation system of tea plant.

转基因技术是茶树基因功能鉴定、种质改良的重要手段，离体组织再分化困难是茶树转基因的主要技术瓶颈。但茶树离体组织再分化困难的内在分子机制目前仍不明确。本项目采用表观遗传学分析手段，比较不同再分化潜力茶树外植体及其脱分化和再分化衍生材料的基因组DNA甲基化、组蛋白修饰、sRNA组成及基因表达等表观遗传因子差异，分析DNA甲基化和组蛋白修饰抑制剂处理对外植体再分化潜力及表观遗传因子的影响，明确茶树外植体在脱分化和再分化过程中表观遗传因子的变化规律，探明不同外植体再分化潜力与DNA甲基化、组蛋白修饰等表观遗传因子之间的关联关系。为阐明茶树外植体脱分化和再分化分子调控机制创造条件，同时也可为建立和完善高效茶树再生和转化体系提供重要理论依据。

离体组织再分化困难是影响茶树转基因效率的关键瓶颈因素之一，本项目研究了不同茶树外植体脱分化再分化过程中的DNA甲基化、组蛋白修饰和基因表达模式变化，以期探明不同外植体再分化能力差异的分子机制，结果显示，茶树茎段和叶片脱分化形成愈伤时DNA甲基化程度均下降，但降幅及半/全甲基化比例变化差异明显，再分化成根后甲基化程度差异小；在MSAP差异序列中的胞嘧啶普遍存在甲基化修饰，且多数甲基化状态在组培过程保持不变，而可变甲基化占比较高的AAC/TTC2-3等特异位点可能参与茶树脱分化和再分化调节。茎段与叶片H3修饰水平差异明显，但不同外植体愈伤分化的根中H3修饰差异较小；H3K79me2、H3K36me2、H3K14ac和H3K56ac等修饰与DNA甲基化水平显著相关；miR156a等sRNA在脱分化和再分化过程中表达强度差异显著，进而影响SBP1等靶基因活性。激素响应和信号转导、核糖体、光合作用、次生代谢以及甾醇类和蜡质合成等相关代谢途径基因的差异表达是外植体脱分化和再分化的直接影响因素；愈伤形成与叶绿体退化以及乙烯和细胞分裂素/生长素代谢诱发的细胞增殖密切相关，根的分化则与离子转运、极性生长、质体重建等有关；而芽分化与miR156a(-5p)、miR166a(-3p)、miR167d、miR393c-3p和miR396e-3p低表达及其相应靶基因高表达有关。在含4mg/L 6-BA+0.1mg/L IBA+0.5mg/L GA3的MS培养基中可将愈伤转化成体胚，并在含2mg/L 6-BA+0.1mg/L IBA的MS培养基中再生出芽，而去乙酰化抑制剂TSA处理也可提高愈伤直接出芽频率。可见，原始外植体DNA甲基化水平和位点特异性、组蛋白H3K79me2等位点修饰状态、以及激素响应等功能基因和miR156a等sRNA表达模式与不同组织脱分化和再分化能力差异有密切联系。相关研究为全面了解茶树脱分化和再分化过程中表观遗传核心调控机制奠定了基础，也为茶树再生体系进一步完善创造了条件。