Study on mechanism of cell cycle regulation by a phytohormone gibberellin in plant cell culture

本课题的目的是：(1)从胡萝卜中分离细胞周期依赖性标记基因；(2)建立同步细胞培养；(3)分析同步细胞中细胞周期依赖性标记基因的表达；(4)赤霉素生物合成抑制剂对细胞周期进程的影响。胡萝卜植物(Daucus carota L.cv。这些实验使用US-Harumakigosun)作为植物材料。实验采用EC细胞系和NC细胞系两种培养细胞系。这些细胞系的特点是前者（EC系）能产生体胚，而另一个（NC系）只能增殖而不能发育成细胞。我们从这些培养细胞中分离出8个细胞周期标记基因，如Cyclin D同源物（作为g1期标记）、PCNA同源物（增殖细胞核抗原，作为s期标记）、Cyclin B（作为g2期标记）和ICK同源物（细胞周期蛋白依赖性蛋白激酶抑制剂，作为细胞周期抑制的标记）。用s期起始抑制剂羟基脲处理NC细胞株，建立了同步细胞培养。最后，我们的有丝分裂指数显示，大约有12-20%的细胞是同步的，并且在这个样本中同步经历了2个以上的细胞周期。标记基因表达分析表明，一个细胞周期为24 ~ 30h （G_<2+>+M期约12 h， M+ g_1期约10 h， s期约5 h）。单唑作为赤霉素生物合成抑制剂对NC细胞增殖有明显抑制作用。在我们未来的实验中，我们将通过赤霉素阐明赤霉素在细胞增殖中的作用。

Aims of this project were (1) isolation of cell-cycle dependent marker genes from carrot, (2) establishment of synchronized cell culture, (3)analysis of cell-cycle dependent-marker-gene-expression in the synchronized cells, and (4) effect of gibberellin biosynthetic inhibitor on cell cycle progression. Carrot plants (Daucus carota L.cv.US-Harumakigosun) were used as a plant material for these experiments. Two cultured cell lines, EC line or NC line, were utilized for experiments. Characters of these cell lines were that former cells (EC line) can produce somatic embryo and another one (NC line) is able to only proliferate without cellular development. We isolated 8 genes for cell-cycle markers such as Cyclin D homologue (as G_1-phase marker), PCNA homologue (proliferating cell nuclear antigen, as S-phase marker), Cyclin B(as G_2-phase marker), and ICK homologue (Inhibitor of cyclin dependent protein kinase, as marker for cell cycle inhibition) from these culture cells. Synchronized cell culture was established from NC line by treatment of hydroxylurea that is inhibitor for initiation of S-phase. Finally, our mitotic index showed about 12-20% of total cells were synchronize, and more than 2 cycles of cell cycle were undergone synchronously in this sample. Analysis of marker gene-expression indicated that one cell cycle spends 24〜30hours (G_<2+>+M-phases: about 12 hours, M+G_1-phaases: about 10 hours, and S-phase: about 5 hours). Uniconazole as gibberellin biosynthetic inhibitor significantly inhibited NC cell proliferation. In our future experiments, we will clarify the role of gibberellin for cell proliferation by gibbrerllin.