苹果实生树个体发育过程中氧化还原平衡态变化及调控的研究

The physiological dynamics and its regulation during vegetative phase change, floral transition and rejuvenation as well are of much significance for understanding the ontogenetic mechanism and the techniques for propagation of elite cultivars in perennial woody plants. MiR156 has been proven to be both necessary and sufficient for the juvenile phase, and vegetative phase change is initiated by a signal(s) produced by leaf primordia, which acts by repressing the transcription of specific members of the miR156 gene family. So far, these signals and the related pathway are not discovered. Redox homeostasis and redox regulation are closely involved in growth, differentiation and development in both animals and plants, however, whether and how redox regulation is interlinked with ontogenesis in perennial woody plants are not unambiguously clear. From our previous experiments by using 2-D fluorescence difference gel electrophoresis (2-D DIGE) and suppressed subtractive hybridization (SSH), we found significant differences in leaf H2O2 contents, in abundance and the activities of many leaf redox related enzymes and in the expression levels of redox associated genes between ontogenetic phases. In the present experiments, we further confirm the dynamics of redox homeostasis during ontogenesis in apple seedling via the guidance of miR156 and we identify the associated interdependent or independent pathways of glutathione-ascorbate peroxide metabolism. Then, the differentially expressed genes will be sub-cellular localized by using tobacco cell suspension immuno-fluorescent labeling. Finally, the relationship between miR156 and redox regulation will be clarified by application of exogenous generators and inhibitors of both peroxides and their scavengers, and also by transgenic test through apple and tobacco. The result will be important for understanding the mechanism of ontogenesis in higher plants.

研究多年生木本植物阶段转变及返童过程中生理与调控的变化，对认识个体发育的机制，指导育种和良种繁育有意义。近年已明确miR156是童期的充分必要条件，且miR156的上游调控信号来源于叶原基，但这种（些）上游调控信号及调控机理尚未被揭示。氧化还原平衡态及调控是生物发育过程的重要调控途径，而氧化还原与多年生木本植物个体发育是否存在或存在何种联系仍不清楚。申请人用2-DE DIGE和SSH分析发现了苹果实生树个体发育的不同阶段叶H2O2、氧化还原相关酶含量、酶活性及相关基因表达量均存在显著差异。本项目以miR156作标记，经检测苹果实生树个体发育不同阶段氧化还原活性物质、酶活性及基因表达量的动态变化，以确证氧化还原平衡态变化的途径，通过免疫荧光标记对差异表达基因进行亚细胞定位。再通过转基因、外施氧化还原促进或抑制剂，明确氧化还原调控与miR156的关系。对认识植物个体发育及调控机制有意义。

miR156是童期的充分必要条件，且miR156的上游调控信号来源于叶原基，但这种（些）上游调控信号及调控机理尚未被揭示。氧化还原平衡态及调控是生物发育过程的重要调控途径，而氧化还原与多年生木本植物个体发育是否存在或存在何种联系仍不清楚。本项目进一步证实了苹果实生树个体发育过程中H2O2水平逐渐升高，GSH含量及GSH/GSSG比值逐渐降低。成龄期H2O2主要积累于叶绿体。成龄期叶片MdRboh基因家族主要成员的表达量显著降低（MdRbohK1除外），使质外体氧化还原环境偏向还原，可溶性GGT酶活性受到抑制而降低，使组织水平GSH含量下降。苹果实生树个体发育过程中随着GSH水平降低，MdMIR156a5和MdMIRa12表达显著降低，导致miR156水平显著降低。结果对进一步认识高等植物个体发育有意义。