光線誘導甘藷懸浮細胞中澱粉粒結合性澱粉合成酵素基因概日韻律表現之探討

Light-Induced Circadian Expression of Starch Granule-Bound Starch Synthase Gene in Cell Suspension Cultures of Sweet Potato

本研究之主要目的在探討甘藷懸浮細胞是否適合做為探討概日韻律調控機制之有效工具。我們以澱粉粒結合性澱粉合成酵素（granule-bound starch synthase: GBSS）基因做為此研究之標誌基因。GBSS基因在葉部的表現呈現概日韻律，但在黑暗環境連續培養的懸浮細胞中則表現恆定。因光線為啟動生物時鐘的因子之一，故將黑暗培養之懸浮細胞轉移至十六小時光照、八小時黑暗的生長箱中培養七天再進行北方雜合分析，其結果顯示經光線誘導後GBSS基因的表現與在葉部所觀察到的概日韻律相似。除了光照因子外，我們亦探討其他環境因子（例如：滲透逆境）對GBSS基因表現的影響。將細胞培養在高滲透壓的培養基中會使GBSS基因的表現增強。但在此高滲透壓的環境下，GBSS基因受光線誘導的韻律性表現則會受到抑制。在此研究中不僅指出光線可做為一環境訊息啟動生物時鐘，亦證明懸浮細胞可做為研究概日韻律的一便利系統。

Attempts were made to evaluate whether suspension cell cultures of sweet potato could be used as a tool for studying gene expression controlled by circadian clocks. Starch granule-bound starch synthase (GBSS) gene, which exhibited a circadian rhythm in the leaf tissue but expressed constantly in dark-adapted cell cultures, was used as a marker gene for the study. Because light is considered as a major signal to set circadian clocks, dark-adapted cells were entrained by 16 h light/8 h dark photoperiod for 7 days before determination for the expression of GBSS gene. Results of RNA blot analysis showed that fluctuations of GBSS mRNA during a diurnal cycle could be induced in cell cultures and the pattern of rhythm was similar to that of the leaf tissue. In addition to light effect, other environmental factors such as osmotic stress were also evaluated. Expression of GBSS gene was enhanced by 600 mM sorbitol in the dark-adapted cells; however, the pattern of circadian rhythm established under 16 L/8 D photoperiod was repressed by the high osmotic effect. These results not only indicate that the circadian clock could be set by light but also provide evidence that suspension cells might be used for studying the regulation of circadian rhythm.