GFP-mTn融合蛋白对蓝猪耳生活细胞微丝骨架的标记

用农杆菌介导法将嵌合基因GFP-mTn(mTn是微丝结合蛋白Talin的微丝结合域,可以显示活体细胞中微丝的结构)导入蓝猪耳.经激光共聚焦显微镜观察了转基因植株的各种不同组织中融合蛋白的表达和分布情况.在叶片的表皮细胞、保卫细胞、根部的皮层细胞中有融合蛋白的不同程度表达.但仅在保卫细胞中微丝标记状况良好,显示基因表达的组织特异性.经光诱导处于开放态的气孔的保卫细胞微丝呈网状结构,在细胞内无规则分布;经黑暗诱导处于关闭态的气孔保卫细胞中微丝束沿保卫细胞纵轴排列,呈卷曲状分布,并观察到螺旋和环状的微丝结构.在转基因植株的其他部位,例如茎表皮细胞、根毛细胞和花粉粒中,未检测到目的基因的表达.本研究获得的转基因植株为研究气孔运动过程中微丝动态变化提供了有用的材料.

VISUALIZATION OF ACTIN CYTOSKELETON IN LIVING CELLS OF TORENIA FOURINERI USING GFP-mTn FUSION PROTEIN

By transformation mediated by Agrobacterium tumefacien, we successfully transferred the chimeric gene of GFP-mTn (mTn is the binding domain of microfilament binding protein talin from mouse, which can show the microfilament in living cell) into Torenia fournieri. Using confocal laser scanning microscopy (CLSM), the distribution of fusion protein in different kinds of tissues and cell in transgenic Torenia fournieri was observed. GFP fluorescence was found in leaf epidermal cell, stomatal guard cell and root epidermal cell. Actin filaments can be visualized clearly only in guard cells. In the guard cells of open stomata under light, actin filaments arrange reticularly and randomly in cortical cytoplasm. In the guard cells of closed stomata under darkness, actin filaments arrange curly along the longitude of guard cell, and some helix and ring structures were found. GFP fluorescence was not found in other cell types, including stem epidermal cell, root hair cell and reproductive organs. The transgenic Torenia fournieri we got provides a suitable material to study dynamics of actin filament in stomatal guard cell.