雌雄青杨对高锰胁迫光合作用差异响应的叶绿体蛋白质组分析

Male and female Poplus cathayana cuttings responded differently in physiological and biochemical level to high-manganese stress in our previous study. Especially, males had significant higher photosynthesis ability than females did. However, the molecular mechanism of this different photosynthesis responses to high manganese is unclear. In this research, we are going to apply high concentrations of manganese artificially to dioecious tree, P. cathayana, the research material. After that, the chloroplast and chloroplastic proteome was extracted by Percoll density gradient centrifugation and Tris-phenol method respectively. Proteins will separate by 2D-PAGE and the defferential expressed and sex-specific proteins will be picked up for amino acid sequence analysis by ESI-Q-TOF MS/MS method. By searching in proteome database on line, the cellular localization and functional network of these proteins will be clear. Selectively, the differently expressed proteins and Rubisco small and large subunits will be chosed to Western blot hybridization experiments. Combineing with physiological and biochemical analysis and chloroplast ultrastructural observation, the molecular mechanism of different responses in photosynthesis and adaptation strategies to high manganese stress of male and female P. cathayana will be uncover. The results could not only provide informations for functional verifications of different expressed proteins and related genes clone further, but also offer theoretical support for the formulation of plans and measures in tree to fight heavy metal stress.

我们前期研究发现青杨雌雄植株对高锰胁迫存在生理生化差异响应，尤其是高锰胁迫下雄株光合作用显著高于雌株。但雌雄青杨对高锰胁迫光合作用差异性响应的分子机理是什么，目前尚不清楚。本研究拟以雌雄青杨为研究对象，人工模拟高浓度锰胁迫，运用Percoll密度梯度离心技术分离叶绿体，Tris-酚法进行叶绿体蛋白质提取，2D-PAGE双向电泳后选取高锰胁迫应答和性别特异性响应蛋白点进行ESI-Q-TOF MS/MS质谱分析其氨基酸序列，通过数据库搜索，阐明这些蛋白点的细胞定位和功能网络，选择性的对差异蛋白和 Rubisco大小亚基进行 Western blot杂交实验。结合生理生化分析和叶绿体超微结构观察，我们拟揭示雌雄青杨遭受高锰胁迫时性别间光合作用差异响应的分子机制及采取的不同适应策略。研究结果不仅能为下一步差异蛋白功能验证和相关基因克隆提供线索，也可为制定林木抗重金属对策和措施提供理论支撑。

雌雄异株植物因生殖投入的不同而对逆境的抗性存在差异。但人工培育兄妹系欧美杨雌雄之间是否存在高锰胁迫抗性差异以及差异的分子机理，目前尚不清楚。本研究以雌雄欧美杨为研究对象，人工模拟高锰胁迫，运用Percoll 密度梯度离心技术分离叶绿体，Tris-酚法进行叶绿体蛋白质提取，采用iTRAQ技术对叶绿体蛋白质组进行研究。结果发现，欧美杨雌雄植株在温和高锰胁迫环境下的差异蛋白共有量较低，高浓度高锰胁迫的共有量较高；但是欧美杨在温和高锰胁迫下的生理差异较小，高浓度高锰胁迫的生理差异较大。我们预测可能低浓度锰胁迫下的差异表达蛋白不足以在生理生化和形态结构上表现出来；而高浓度锰胁迫的强烈毒性使得蛋白出现大量变性、断裂、失活等现象，表现为较高的共有调控蛋白量。但高浓度高锰胁迫处理前期的胁迫过程才是导致欧美杨雌雄生理生化差异反应的关键过程。不过，这一推测过程有待进一步用时间梯度实验来验证。研究结果不仅能为下一步差异蛋白功能验证和相关基因克隆提供线索，也可为制定林木抗重金属对策和措施提供理论支撑。