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

植物激素赤霉素对植物细胞培养细胞周期的调控机制研究

• 批准号: 14560056
• 负责人: MITSUHASHI Wataru
• 金额: $ 1.92万
• 依托单位: Yamagata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14560056/
• 关键词: Carrot; Cell cycle; Culture cell; RT-PCR

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.

本研究的目的是（1）分离胡萝卜细胞周期相关基因，（2）建立同步化细胞培养体系，（3）分析同步化细胞中细胞周期相关基因的表达，（4）研究赤霉素生物合成抑制剂对细胞周期进程的影响。使用胡萝卜植物（胡萝卜L.cv.US-Harumakigosun）作为这些实验的植物材料。两种培养的细胞系，EC系或NC系，用于实验。这些细胞系的特点是前者细胞（EC系）能产生体细胞胚，而另一个细胞（NC系）只能增殖而不发育。我们从这些培养细胞中分离了8个细胞周期标志物基因，如细胞周期蛋白D同源物（作为G_1期标志物）、增殖细胞核抗原同源物（作为S期标志物）、细胞周期蛋白B同源物（作为G_2期标志物）和细胞周期蛋白依赖性蛋白激酶抑制剂同源物（作为细胞周期抑制标志物）。用S期起始抑制剂羟基脲处理NC细胞系，建立同步化细胞培养。最后，我们的有丝分裂指数显示约有12-20%的细胞是同步的，并且在该样品中细胞周期同步地经历了两个以上的周期。标记基因表达分析表明，一个细胞周期为24 ~ 30小时，其中G_（2+）+M期约12小时，M+G_1期约10小时，S期约5小时。烯效唑作为赤霉素生物合成抑制剂显著抑制NC细胞增殖。在以后的实验中，我们将阐明赤霉素对细胞增殖的作用。