本研究目的主要是探討大型海藻石蓴 (Ulva lactuca L.) 細胞內acid phosphatase (ACP; EC 3.1.3.2) 與磷饑餓之關係。加磷 (100 mM NaH2PO4) 藻體呈深綠色多皺褶而磷饑餓 (1 mM NaH2PO4) 藻體則為淺綠色少皺褶。相對於加磷藻體，磷饑餓明顯抑制生長，處理前2天生長與加磷藻體相似，第3天後降低而第7天後即無生長。磷饑餓藻體之soluble reactive phosphorus (SRP) 含量在第3天下降而soluble non-reactive phosphorus (SNRP)、total soluble phosphorus (TSP)、polyphosphate及磷 (P) 含量則持續下降。碳:磷 (C:P) 及氮:磷 (N:P) molar ratio在磷饑餓後第3天開始增加至第7天達到最高值。細胞內ACP活性在磷饑餓第3天開始增加，至第14天達加磷藻體之16倍，細胞外alkaline phosphatase (AP; EC 3.1.3.2) 活性在第2天開始增加至第4天達最高點。細胞內ACP活性及細胞外的AP活性與SRP、SNRP、PP及磷含量呈負相關。Native PAGE活性染色顯示磷饑餓誘導10條ACP isoenzymes而IEF活性染色顯示有9條ACP isoenzymes被誘導。醣蛋白分析磷饑餓14天所誘導之ACP isoenzymes (Native PAGE)顯示ACP 1及ACP 2為醣蛋白。添加100 mM NaH2PO4至磷饑餓藻體之磷恢復試驗顯示藻體生長在第5天始恢復，SRP、SNRP、TSP及磷含量及C:P及N:P molar ratio在第1天恢復至加磷藻體之水平，細胞外AP活性第2天開始降低而第8天達最低點，細胞內ACP活性第3天開始降低而第8天達最低點。Phosphate相似物Phi (phosphite) 1 mM 會抑制磷饑餓所誘導的細胞外AP及細胞內ACP活性增高。本研究指出磷饑餓所誘導或促進石蓴ACP活性增高與polyphosphate及有機磷分解以提供生長代謝之磷有關。

The roles of phosphorus (P) starvation on the induction of intracellular acid phosphatase (ACP; EC 3.1.3.2) activity have been studied in a marine macroalga Ulva lactuca L. In comparison to creasy and dark green appearance in P-sufficient thalli (100 mM NaH2PO4), P-starved thalli (1 mM NaH2PO4) showed less crease and light green appearance. On exposure to 1 mM NaH2PO4, the growth rate, the contents of SRP, PP and P, and tissue C:P and N:P molar ratio decreased at day 3 and the contents of SNRP, TSP and polyphosphate decreased immediately. Intracellular ACP activity increased at day 3 after exposure to 1 mM NaH2PO4 and reached 16 folds of P-sufficient thalli at day 14, while extracellular alkaline phosphatase (AP; EC 3.1.3.1) activity increased at day 2 and reached the plateau after 4 days. Activity staining both on Native PAGE and IEF gel showed the induction of 10 and 9 ACP isoenzymes, respectively. Changes in intracellular ACP and extracellular AP activities were negatively correlated with SRP, SNRP, PP and P contents. After transferred to 100 mM NaH2PO4, the growth rate of 10 day-starvated thalli recovered after 5 days, the contents of SRP、SNRP、TSP and P, and the C:P and N:P molar ratio recovered to the level of P-sufficient thalli at day 1. When recovered to 100 mM NaH2PO4, extracellular AP activity of 10 day-starvated thalli decreased at day 2 and reached the minimum after day 8, while intracellular ACP activity decreased at day 3 and reached the minimum after day 8. The analog of Pi, Phi (1 mM) inhibited the intracellular ACP and extracellular AP activities induced by P swtarvation. The results of present investigation show that ACP has a role in the enhancement of P availability in U. lactuca via the enzymatically degradation of polyphosphates and organic P when suffers P deficiency.

目錄

章次 　 頁數
一、緒論．．．．．．．．．．．．．．．．．．．．01
二、材料與方法．．．．．．．．．．．．．．．．．06
(一) 材料栽培及處理．．．．．．．．．．．．．．06
(二) 分析方法．．．．．．．．．．．．．．．．．07
1. 生長速率測定．．．．．．．．．．．．．．．．07
2. 藻體碳含量測定．．．．．．．．．．．．．．．08
3. 藻體氮及磷含量測定．．．．．．．．．．．．．08
4. 藻體SRP、SNRP、TSP、PP及polyphosphate含量測量．．．．．．．．．．．．．．．．．．．．．．12
5. 磷酸分解酵素活性測定．．．．．．．．．．．．13
6. ACP電泳分析．．．．．．．．．．．．.．．．．16
(三) 統計方法．．．．．．．．．．．．．．．．．17
三、結果．．．．．．．．．．．．．．．．．．．19
(一) 外表型態與生長．．．．．．．．．．．．．19
(二) 碳、氮、磷、TSP、SNRP與PP含量分析．．．．．．．．．．．．．．．．．．．．．．19
(三) 細胞外AP活性．．．．．．．．．．．．．．27
(四) 細胞內ACP活性．．．．．．．．．．．．． 27
(五) 細胞內ACP活性、細胞外AP活性及SRP、SNRP、TSP、PP及磷含量之關係．．．．．．．．．．．．．27
(六) 電泳分析．．．．．．．．．．．．．．．．30
(七)　磷恢復試驗之生長．．．．．．．．．．．．38
(八)　磷恢復試驗之碳、氮、磷、SRP、TSP、SNR及PP含量測定．．．．．．．．．．．．．．．．．．．．38
(九)　磷恢復試驗之細胞外AP活性測定．．．．．．38
(十) 磷恢復試驗之細胞內ACP活性測定．．．．．．38
(十一) Phi 對生長、細胞外AP活性及細胞內ACP活性之影響．．．．．．．．．．．．．．．．．．．．．．．38
四、討論與結論．．．．．．．．．．．．．．．．45
五、參考文獻．．．．．．．．．．．．．．．．．51
六、藥品配製．．．．．．．．．．．．．．．．．57

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