臺灣博碩士論文加值系統

本研究以菩提藻(Grateloupia filicina)葉狀體(thallus)為材料，探討其在晝夜週期、長期暗處理和鹽分逆境下，多元胺與紅藻澱粉代謝間的相關。光照期和黑暗期各為12小時的晝夜週期試驗中，菩提藻葉狀體在光照初期的紅藻澱粉含量無變化，可溶性糖和putrescine(Put)的含量與兩種紅藻澱粉降解酵素(α-glucosidase(EC 3.2.1.20)和phosphorylase(EC 2.4.1.1))的活性皆下降，蛋白質大幅上升。進入黑暗期後，紅藻澱粉與蛋白質含量下降，可溶性糖略微降低，Put含量與兩種紅藻澱粉降解酵素的活性都增加，Put含量在第20小時達高峰值。長期暗處理試驗中，紅藻澱粉和蛋白質的含量與phosphorylase的活性皆呈現下降趨勢，可溶性糖含量在第四天後才開始下降，Put含量和α-glucosidase的活性在暗處理前三天略微上升，其後迅速下降。

鹽分逆境的試驗採用15 ‰為低鹽逆境，45 ‰為高鹽逆境，控制組鹽度為30 ‰，並外加Put與其合成抑制劑(D-arginine (D-Arg)和methylornithine(MO))。經此處理24小時後，再移至無外加Put及其合成抑制劑的培養液中培育兩星期，結果顯示45 ‰的鹽度會抑制菩提藻葉狀體的生長，提升可溶性糖與Proline的含量；鹽度15 ‰的相對生長速率和各內含物的變化都與控制組相似。Put合成抑制劑D-Arg可顯著抑制Put含量並抑制相對生長速率，MO則無處理效應。在45 ‰鹽度下外加Put可使相對生長速率高於控制組。此外，45 ‰鹽度下紅藻澱粉降解酵素之活性與可溶性糖含量亦受D-Arg抑制，但Proline的含量增加。有鑑於24小時短期處理效應的顯著性可能會隨時間而降低，進一步於30 ‰和45 ‰兩鹽度下外加Put與其合成抑制劑。於24小時後立即分析其生理變化，結果除了phosphorylase的活性無處理效應外，Put、紅藻澱粉、可溶性糖、Proline的含量與α-glucosidase活性之變化均比兩週後才分析之效應明顯。

綜合上述結果，自晝夜週期和長期暗處理試驗可發現Put含量變化與紅藻澱粉降解酵素活性變化有一致性，在鹽分逆境和外加Put及其合成抑制劑的試驗中，可進一步證實Put可調節紅藻澱粉降解酵素的活性以促進紅藻澱粉的分解。

Thallus of Grateloupia filicina was used for investigating the correlation of polyamines and metabolism of floridean starch under a photoperiod, a long-term darkness and two salt stress(15 ‰, 45 ‰). In photoperiod experiment which was under intermittent illumination of 12 hr light and 12 hr darkness, floridean starch level was found unchanged at the beginning of the light period. Soluble sugar, putrescine (Put) content and activity of two floridean starch degrading enzyme (α-glucosidase (EC 3.2.1.20) and phosphorylase (EC 2.4.1.1)) evidently decreased simultaneous, and protein content obviously increased. During the following dark period, there was significantly declined in floridean starch and protein content, and slightly decreased in soluble sugar. On the contrary, Put content and activity of two floridean starch degrading enzyme remarkably increased, and Put level was accumulated to a maximum value(123.76±4.46 nmol/Fw g) at the 20th hr of photoperiod.The experiment of long-term darkness indicated floridean starch and protein content, phosphorylase activity showed a declined pattern, soluble sugar decreased at the 4th day, Put content and α-glucosidase activity slightly raised during the first 3 days of darkness, and then rapidly declined.

In the experiment of salt stress used 15 ‰ seawater as hyposaline stress, 45 ‰ seawater as hypersaline stress and 30 ‰ seawater as control. Thallus was treated with Put and it’s synthetic inhibitors (D-arginine (D-Arg) and methylornithine(MO)) for 24 hr, then removed to cultural medium without Put and it’s synthetic inhibitors. The results indicated growth rate reduced, soluble sugar and protein increased at 45 ‰ salinity. The change of specific growth rate and ingredients at 15 ‰ were similar to control. Put synthetic inhibitor, D-Arg, could significantly decrease Put content and specific growth rate. There wae no effect in MO. At 45 ‰ salinity, the specific growth rate of thallus treated with Put was highter than at 30 ‰ salinity. In addition, activity of floridean starch degrading enzyme and soluble sugar were inhibited by D-Arg, and increaseing proline level at 45 ‰. Due to 24 hr short-term significant treatment effect may decline with time,and an additional Put and it’s synthetic inhibitor were added to the medium of 30 ‰ and 45 ‰ salinity, and immediately, analysis it’s physiological change after 24 hr. The results indicated phosphorylase activity showed no significantly different, and α-glucosidase activity, Put, floridean starch, soluble sugar and protein content were more significant than 24 hr short-term treatments.

As a whole, after photoperiod and long-term darkness treatments, change in Put content was consistent with the activity of floridean starch degrading enzyme. In the experiment by adding Put and synthetic inhibitors under salt stress were proved that Put may regulate activity of floridean starch degrading enzyme, and to stimulate the floridean starch degradation.


Key words:
Grateloupia filicina、salt stress、floridean starch、floridean starch degrading enzyme、proline、polyamine、polyamine synthetic inhibitors

縮寫表……………………………………………………………………1
英文摘要…………………………………………………………………2
中文摘要…………………………………………………………………4
壹、前言…………………………………………………………………6
貳、前人研究……………………………………………………………8
一、菩提藻的生活史與培養技術…………………………………8
二、紅藻澱粉的代謝途徑之相關研究……………………………9
三、多元胺的代謝與功能…………………………………………14
(一)多元胺的代謝………………………………………….15
(二)多元胺的功能………………………………………….16
四、鹽分逆境對海生藻類生長的影響……………………………19
參、材料與方法…………………………………………………………25
一、菩提藻絲狀體(filament)的來源與培養條件………………25
二、菩提藻葉狀體(thallus)的誘導……………………………….25
三、試驗方法………………………………………………………26
1.菩提藻在晝夜週期下之生理變化的觀察……………….26
2.菩提藻在暗處理下之生理變化的觀察………………….26
3.菩提藻在鹽分逆境下外加put及其合成抑制劑後的生理變化…………………………………………………………26
(1)鹽分逆境對菩提藻葉狀體的相對生長速率之影響….26
(2)外加put與其合成抑制劑對菩提藻葉狀體的相對生長速率及Put含量之影響……………………………27
(3)鹽逆境下外加put和其合成抑制劑對菩提藻葉狀體的澱粉代謝之影響…………………………………. 28
四、分析方法………………………………………………………29
1.可溶性糖與蛋白質含量的分析…………………………. .29
2.紅藻澱粉含量的分析……………………………………. .29
3.紅藻澱粉降解酵素活性的分析…………………………. .30
4.多元胺含量的分析………………………………………. .31
5.脯胺酸含量的分析………………………………………. .32
五、統計方式………………………………………………………33
肆、結果…………………………………………………………………34
一、菩提藻在晝夜週期下之生理變化的觀察……………………34
二、菩提藻在暗處理下之生理變化的觀察………………………39
三、菩提藻在鹽分逆境下外加put及其合成抑制劑後的生理變化……………………………………………………………..43
伍、討論…………………………………………………………………63
一、菩提藻在光照週期下的生理變化……………………………63
二、菩提藻在暗處理下的生理變化………………………………65
三、菩提藻在鹽分逆境下外加put及其合成抑制劑後的生理變化……………………………………………………………. .67
陸、結語…………………………………………………………………76
柒、參考文獻……………………………………………………………77
捌、附錄…………………………………………………………………87
附錄一、菩提藻的生活史…………………………………………87
附錄二、 A.紅藻澱粉的代謝途徑………………………………88
B.NDP-sugars pool概念圖……………………………89
附錄三、 A.多元胺的合成途徑………………………………….90
B.多元胺的降解途徑………………………………….91
附錄四、菩提藻培養液之組成成份………………………………92
附錄五、紅藻(Caloglossa leprieurii)的抗鹽機制……………….. 93

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