| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2003: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2002: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
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.
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