塔瑪亞歷山大藻之細胞內生物螢光和光動螢光韻律之研究

Abstract

一直以來，渦鞭毛藻的生物螢光都是研究者感興趣的研究課題，然而經過多年的研究，其螢光特性和生物及化學機制仍然尚未解開。 渦鞭毛藻的生物螢光已被證實是由一種結構上類似葉綠素a的螢光素,在酵素催化下發生氧化還原反應或者受紫外線激發時，能夠放出波長470nm的藍綠色螢光。透過螢光顯微鏡及共軛焦顯微鏡，我們發現塔瑪亞歷山大藻之螢光素在暗周期時會聚集成小球狀（螢光胞器）且分布於細胞表面，而此螢光胞器在光周期時卻不存在；不帶有生物螢光之微小亞歷山大藻無論在光或暗周期都未發現螢光胞器；此外，我們也發現塔瑪亞歷山大藻中的葉綠體在光周期時會分布在細胞表面，而暗周期時會往細胞中心聚集，這種現像僅在Pyrocystis中存在。 由前人研究得知，渦鞭毛藻之生物螢光只表現於暗周期且必須由物理性的刺激所激發，因此我們設計了一個可透過搖動器震盪藻液，並以單眼相機同步測量生物螢光的裝置。其他渦鞭毛藻的生物螢光通常被生理時鐘所調控，然而在塔瑪亞歷山大藻的生物螢光周期中，我們發現其生物螢光只固定發生在光暗周期變化中的暗周期，且非常容易受到光線的抑制及調控。由本研究所觀察到的現像，我們認為塔瑪亞歷山大藻的生物螢光存在著螢光素的生物時鐘表現，然而伴隨著其與螢光酵素的生物螢光表現卻受到光線的干擾而抑制，這是相同渦鞭毛藻生物螢光的種類中，尚未被引述的現像。雖然其生物螢光表現受到光抑制，我們也發現其螢光胞器內的螢光酵素依然會在連續4小時強光的照射下被破壞殆盡，而光線之移除可以馬上啟動另一次的生物螢光周期。 另外，我們也發現塔瑪亞歷山大藻生物螢光會隨著不同光照長短的光暗周期（8:16光暗周期，12：12光暗周期和16：8光暗周期）進行不超過±2小時的調整。總結來說，雖然本研究室仍未對渦鞭毛藻生物螢光韻律之研究有更深入的探討，但目前這些對塔瑪亞歷山大藻的生物螢光週期的研究，有助於我们更進一步了解渦鞭毛藻的生物時鐘和其特性。

Bioluminescence of dinoflagellates is one of the interesting topics which have attracted the attention of researchers, but its characters and mechanisms are yet to be determined. Bioluminescence of A. tamarense was observed only in the dark period upon physical stimulation by shaking, but not in the light period when the cultures were maintained and acclimated to a daily 24 hours cycle of alternative light and dark period. These observations were being operated by using our newly designed instrument. Photoluminescence of the in vivo luciferin was detected under a fluorescent and a laser scanning confocal microscope that showed the luciferin-containing scintillons being located on the peripheral of cell cytoplasm and appeared only in dark period. It was also evidenced by the absence of such photoluminescence in both light and dark period of non-bioluminescent Alexandrium minutum. At the meantime, the chlorophyll a containing chloroplasts were found to be located at the cell peripheral during light period and in the center during dark period, which only being reported in Pyrocystis species so far. Although bioluminescence in other dinoflagellates was being described as circadian-regulated, our observation on A. tamarense bioluminescence showed more light-regulated. Shorten of the dark period also shorten the period of bioluminescence. However, unlike the light-regulated bioluminescence expression in A. tamarense, the aggregation of luciferin observed by its photoluminescence were found unaffected by light and followed the circadian rhythm for the first two cycles and diminished under continuous dim light. This expression was also different from other dinoflagellates which their circadian rhythms can sustain for up to twenty cycles or more. Besides, the circadian rhythmic photoluminescence of luciferin aggregation could be broken down by continuous four hours of strong illumination, while removal of light could induce another cycle of bioluminescence as in our observation in A. tamarense. Furthermore, this intracellular rhythm can be acclimated, but within a limited capacity, to different photoperiod (LD 8:16, LD 12:12 and LD 16:8) and remained a 12 ± 2 hours of bioluminescence duration. Although our study in A. tamarense bioluminescence characteristics was just the initiatory to this field, further investigations of those findings can not only lead to a deeper understanding of the circadian control in bioluminescence of this species, but also the circadian oscillation principles common to all organisms.