Molecular mechanisms controlled by cytokinin ofM phase progression in division cycle at tobacco BY-2 cells

本研究的目的是确定烟草BY-2细胞分裂周期M期可能受细胞分裂素调控的过程。特别是，我们寻找了细胞分裂素的靶分子事件，主要集中在G2/M期基因转录所需的Ntmyb基因的表达；CDK(细胞周期蛋白依赖的蛋白激酶)的激活和失活；NACK/MAPKKK复合体的形成，这是激活控制胞质分裂的MAP激酶级联所必需的；MAP激酶的激活。我们研究了细胞分裂素的缺失和细胞分裂素加氧酶基因的表达对BY-2细胞M期进程的影响。我们的结果显示，细胞分裂素与不加细胞分裂素孵育的BY-2细胞的M期进展模式没有明显差异。当细胞分裂素氧合酶基因被导入并在转基因拟南芥中表达时，TRACER…的水平然而，更多的G2/M期特异基因的IPT减少。我们还比较了无激素培养和含反式玉米素培养的同步化BY-2细胞的M期进展模式。我们没有观察到在这些实验中获得的两种模式之间的明显差异。当我们与含有或不含细胞分裂素的-2细胞孵育时，我们发现了类似的CDK激活和失活模式。然而，出乎意料的是，我们发现在无激素的培养液中，NACK1蛋白的水平增加了2倍。必须进行进一步的研究，以了解本研究项目获得的意外观测的生理学相关性。虽然我们的结果并没有真正显示细胞分裂素在M期进程中的靶过程，但我们与细胞质分裂相关的研究有助于理解植物细胞胞质分裂调控机制的新方面：(1)CDK在中期之前同时磷酸化NACK1和MAPKKK，导致干扰NACK/MAPKKK复合体的形成，中期之后，这些蛋白被去磷酸化，这诱导了这些蛋白的相互作用，激活了MAP激酶级联，导致细胞质分裂的进展；(2)在拟南芥中，已鉴定出正向或负向控制G2/M期特异转录的MYB因子，并预测了可能参与转录的未知因素。较少

The aim of the present research is to identify processes in M phase of division cycle of tobacco BY-2 cells that might be regulated by cytokinin. In particular, we have sought for target molecular events of cytokinin during phase focusing on expression of Ntmyb genes that are required for transcription of genes during G2/M phase; activation and inactivation of CDK (cyclin-dependent protein kinase); formation &the NACK/MAPKKK complex that is required for activation of the MAP kinase cascade controlling cytokinesis; the activation of the MAP kinase. We have examined effects of depletion of cytokinin and those of expression of genes for cytokinin oxydase, which can degrade this phytohormone, on the M phase progression of BY-2 cells. Our results showed no obvious difference between patterns of M phase progression of BY2 cells incubated with and without cytokinin, respectively. When the cytokinin oxydase genes were introduced and expressed in transgenic Arabidopsis plants, levels of transcr … More ipts of G2/M-phase-specific genes, however, decreased. We also have compared the pattern of the progression of M phase of synchronized BY-2 cells incubated with hormone-free medium to that incubated with trans-zeatin-containing medium. We did not observe clear difference between two patterns obtained in these experiments. We found similar patterns of CDK activation and inactivation when we incubated BY-2 cells with or without cytokinin. Unexpectedly, we, however, found that the level of NACK1 protein increased by 2-fold in the hormone-free medium. Further investigations must be required for understanding of physiological relevance of the unexpected observations obtained by this research project. Although our results did not dearly show target processes of cytokinin during M phase progression, our researches related to cytokinesis have contributed to understanding of new aspects of regulatory mechanisms of cytokinesis in plant cells: (1) CDK phosphorylated both NACK1 and MAPKKK before metaphase, which resulted in interfering the formation of the NACK/MAPKKK complex and after metaphase, these proteins were dephosphorylated, which induced the interaction of these proteins that activated the MAP kinase cascade, leading to the progression of cytokinesis; (2) MYB factors that positively or negatively controlled G2/M-phase-specific transcription in Arabidopsis plants were identified and unidentified factors that may be also involved in the transcription have been predicted. Less