活体烟草BY-2悬浮细胞微丝骨架的标记

微丝骨架是细胞骨架的重要成员,在细胞的多项生理活动中发挥着重要作用.本论文利用荧光标记鬼笔环肽技术和GFP融合蛋白技术,对活体烟草BY-2悬浮细胞中微丝骨架的标记方法进行了探索,结果表明,10nmol/L的Alexa488-Phalloidin为细胞微丝骨架标记的最佳浓度,利用PCR等技术构建pPZP-NtFABD2-GFP植物表达载体后,转化烟草BY-2悬浮细胞,激光共聚焦扫描显微镜观察发现,NtFABD2-GFP融合蛋白能够清晰地显示活体烟草悬浮细胞内的微丝骨架.这些结果为进一步深入研究微丝骨架在活体植物细胞中的功能奠定了基础.

Labeling Methods of Microfilaments in Living Tobacco BY-2 Suspension Cells

Microfilaments,major components of the cytoskeleton,play essential roles in a variety of plant growth and developmental processes.In this paper,labeling methods of microfilaments were investigated in living tobacco BY-2 suspension cells using fluorescent conjugates of phalloidin and GFP fusions to actin-binding domain.The results indicated that 10nmol/L Alexa488-Phalloidin was the optimal concentration for microfilaments localization in tobacco BY-2 cells.Moreover,pPZP-NtFABD2-GFP fusion vector was constructed by PCR amplification and standard cloning techniques and then used for transformed into tobacco BY-2 cells.The localization experiments demonstrated that NtFABD2-GFP fusion protein co-localized with microfilaments stained with rhodamine-phalloidin,suggesting that the fusion protein can specifically label the microfilaments in tobacco BY-2 cells.These results laid a good foundation for further study microfilaments function in living plant cells.