微管蛋白荧光探针的制备及其在蚕豆叶片保卫细胞中的组装  

Preparation of Rhodamine-labelled Tubulin and Its Reassembly in Guard Cells of Leaves (Vicia faba L.)

作　　者：于荣[1] 袁明[2] 王学臣[2] 

机构地区：[1]首都师范大学生物系,北京100037 [2]中国农业大学生物学院植物生理生化国家重点实验室,北京100094

出　　处：《农业生物技术学报》2004年第1期48-51,共4页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划项目(No.G19990117);北京市科技新星计划(B类)

摘　　要：从新鲜猪脑中提取微管蛋白(两个循环的解聚甘聚合超速离心)，经DEAE-Sephadex A50离子层析纯化及罗丹明荧光标记，得到浓度为10．8mg/mL和染料蛋白比0．6(Dye／Protein=0．6)的猪脑微管蛋白荧光探针。将其显微注射于蚕豆(vicia faba L.)叶片下表皮的保卫细胞中，激光共聚焦扫描显微镜(confocal laser scanning microscope，CLSM）下观察，发现该荧光标记微管蛋白可以顺利地组装到保卫细胞的微管骨架上，5～10min即可清楚地观察到整合于保卫细胞中的微管网络。在开放气孔保卫细胞中，周质微管(cortical microtubule)沿细胞的腹壁向背壁辐射状排布；而关闭气孔保卫细胞中周质微管则解聚为零碎小片段。以上实验结果为在活体条件下，进一步精细研究气孔运动过程中保卫细胞微管骨架的动态变化提供了可靠的实验基础。From fresh pig brain, tubulin was extracted (two cycles of polymerizat ion and depolymerization), purified (chromatography of DEAE-Sephadex A50) and la beled with 5(6)-TAMRA, SE. We prepared 10.8 mg/mL rhodamine-labelled tubulin pro be (Dye/Protein=0.6), then microinjected into living guard cells of leaves (Vici a faba L.). 5~10 min later, microtubules network of guard cells could be clearly observed under confocal laser scanning microscope (CLSM). In opening guard cell s, the cortical MTs radiated from ventral wall to dorsal wall and in closed guar d cells the radial MTs were completely depolymerized into random and diffuse fra gments. This results showed that pig brain tubulin could incorporate into guard cells, microtubules easily, and it is possible to examine the dynamic behavior o f microtubules directly during stomatal movement in vivo .

关 键 词：微管蛋白荧光探针 显微注射 蚕豆 保卫细胞 组装 

分 类 号：Q943.2[生物学—植物学] S643.6[农业科学—蔬菜学]