Shanghai Center for Plant Stress Biology, CAS Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China.
Center for Research in Agricultural Genomics (CRAG), Barcelona, Spain.
Methods Mol Biol. 2022;2382:115-127. doi: 10.1007/978-1-0716-1744-1_7.
The availability of a fast and controlled mitotic model system that could simplify the generation of genetic material and reduce the experimental time from months to days would largely benefit research in plant cell division. In this protocol, we propose the use of pavement cells of Nicotiana benthamiana leaves to study cell division, which is artificially induced by Agrobacterium-mediated transient overexpression of the transcription factor E2Fb. The cell division-inducing overexpression of E2Fb can be combined with the expression of fluorescent protein-tagged proteins of interest or with dyes, which could be visualized throughout the cell cycle under the microscope. This simple and affordable method enables the study of cell cycle regulation and cell division in plants, from genome replication to cell wall formation, in a fast and controlled manner, and can be used for functional studies when coupled with chemical inhibitors or reverse genetic approaches.
一个快速且可控的有丝分裂模型系统的可用性将极大地有益于植物细胞分裂的研究，因为它可以简化遗传物质的生成，并将实验时间从数月缩短至数天。在本方案中，我们建议使用黄花烟叶片的 pavement 细胞来研究细胞分裂，该分裂是通过农杆菌介导的转录因子 E2Fb 的瞬时过表达来人为诱导的。E2Fb 的诱导性细胞分裂过表达可以与感兴趣的荧光蛋白标记蛋白的表达或与染料结合使用，这些染料可以在显微镜下整个细胞周期中进行可视化。这种简单且经济实惠的方法能够以快速且可控的方式研究植物中的细胞周期调控和细胞分裂，从基因组复制到细胞壁形成，并且可以与化学抑制剂或反向遗传学方法结合使用，用于功能研究。
