Qi Xingyun, Zheng Huanquan
Department of Biology, McGill University, Montreal, QC, Canada.
Methods Mol Biol. 2013;1043:103-12. doi: 10.1007/978-1-62703-532-3_11.
Rab proteins are key regulators of membrane transport in eukaryotes. Recent evidence from different species supports the notion that some Rab proteins are crucial for cytokinesis, a pivotal procedure for successful cell division. As a family of monomeric small GTPases of the Ras superfamily, the function of Rab proteins is modulated by guanine nucleotide binding and hydrolysis. To investigate the function of Rab proteins, creating dominant negative or constitutively active mutant forms of a Rab protein is a widely used approach. To study cytokinesis in plant cells, using fluorescent dye to highlight the cell shape and the nuclei, and to monitor the formation of the newly formed cell plate in mitotic cells, is easy and useful. In this chapter, we describe detailed methods for (1) generating transgenic plants expressing dominant negative or constitutively active form of RAB-A1c; (2) fluorescent staining of cell shape, cell wall, and nuclei of mitotic root tip cells; (3) fluorescent staining of newly formed cell plate; and (4) detecting fluorescent signals using Confocal Laser Scanning Microscopy in the genetic model plant species Arabidopsis thaliana.
Rab蛋白是真核生物中膜运输的关键调节因子。来自不同物种的最新证据支持这样一种观点，即某些Rab蛋白对胞质分裂至关重要，而胞质分裂是细胞成功分裂的关键过程。作为Ras超家族的单体小GTP酶家族，Rab蛋白的功能受鸟嘌呤核苷酸结合和水解的调节。为了研究Rab蛋白的功能，创建Rab蛋白的显性负性或组成型活性突变形式是一种广泛使用的方法。为了研究植物细胞中的胞质分裂，使用荧光染料突出细胞形状和细胞核，并监测有丝分裂细胞中新形成的细胞板的形成，既简单又有用。在本章中，我们描述了详细的方法，包括：（1）生成表达RAB - A1c显性负性或组成型活性形式的转基因植物；（2）对有丝分裂根尖细胞的细胞形状、细胞壁和细胞核进行荧光染色；（3）对新形成的细胞板进行荧光染色；（4）在遗传模式植物拟南芥中使用共聚焦激光扫描显微镜检测荧光信号。
