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研究生:劉雅惠
研究生(外文):Ya-Hui Liu
論文名稱:共生宿主對共生藻停止供應營養的反應:以拂塵海葵為例
論文名稱(外文):Symbiotic host response to the cease of nutrient supply from algal symbiont : A case study on Aiptasia pulchella
指導教授:王志騰王志騰引用關係
指導教授(外文):Jih-Terng Wang
口試委員:孟培傑張桂祥王志騰
口試委員(外文):Pei-Jei MengKwee-Siong TewJih-Terng Wang
口試日期:2013-06-14
學位類別:碩士
校院名稱:大仁科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:68
中文關鍵詞:海葵共生藻營養互動蛋白量游離胺基酸
外文關鍵詞:Sea anemonesSymbiodinium spp.Interactive nutritionprotein countfree amino acids
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珊瑚礁生態是一個貧營養鹽的海洋生態系,其中海葵與蟲黃藻(zooxanthellae)的共生是一個重要的生物現象,然而海葵和共生藻(Symbiodinium spp.)的共生關係以及牠們的交互作用機制仍未完全了解。本研究即探討當共生藻無法進行光合作用提供海葵營養物質時,海葵的反應。在光照及完全黑暗狀態下餵食豐年蝦與不餵食的三種處理方式來做比較。結果顯示經過24天,黑暗完全不餵食海葵的重量、蛋白質含量、共生藻密度、光合作用能力 (Fv / Fm) 從第6天開始下降到24天後,分別減少了5 %、80 %、60 %、20 % ( p<0.05),一樣在黑暗中但有餵食宿主的蛋白量僅減少30 % ( p<0.05),並也其共生藻密度與光合作用能力 (Fv / Fm) 與控制組比起來沒有顯著差異(p >0.05)。再者比較處理24天後海葵的生化指標,結果顯示穀氨酸去氫酶(GDH)、總胺基酸以及必需胺基酸有顯著差異(p<0.05);於黑暗中無餵食與有餵食的海葵體內GDH 活性在第12天時開始上升至第24 天,上升的量已超過100 % ;然而在光照並且有餵食的海葵GDH 活性僅有增加17 % ;所以海葵宿主若處於完全黑暗環境中無論是沒有餵食或有餵食,其GDH活性都會比光照有餵食來的高(p < 0.05);海葵在黑暗下有餵食平均會減少59.0 % 總FAA 以及26.4 % EFAA;在完全黑暗不餵食則平均會降低73.8 % 總FAA 以及76.8 % EFAA。另外以人工方式給予碳源來取代光照,結果在完全黑暗狀態下餵食豐年蝦或甘油,經過24天後比較對照組(照光+餵食豐年蝦)與兩處理組之間的重量、蛋白量、共生藻數量、光合作用能力II (Fv /Fm) 、GDH,皆有顯著差異( p < 0.05);兩處理組(餵食豐年蝦及甘油)由第6天開始下降至第24天,其重量、蛋白量、共生藻密度、光合作用能力II (Fv / Fm)與照光時比起來一樣是較低的,則分別減少了重量為30 %、42 %;蛋白量為52 %、65 % ;光合作用能力II (Fv / Fm)為6 %、8 % 以及共生藻密度也減少了53 %、 44%,並且兩處理組的GDH 活性一樣增加,其增加的活性分別都超過100 %(p < 0.05);僅有 MDH無明顯差異( p > 0.05)。由以上結果顯示,在黑暗的環境下共生藻無法行光合作用,不論是餵食豐年蝦或供給碳源,海葵的狀態與照光狀態比起來皆有明顯的差異,所以就算以人工方式額外供給氮源或碳源,只要共生藻無法行光合作用提供營養給宿主,宿主依然無法維持與照光時相似的生理狀態。
Coral reef ecosystem is a nutrient poor marine ecosystem which anemone and zooxanthellae symbiosis is an important biological phenomena. However, mechanisms of symbiosis of anemones and zooxanthellae and their interaction are not yet fully understood. The aim of this research was to explore the anemones response, which when symbiotic algae unable to carry out photosynthesis and provided nutrients to anemones. Three approaches for comparison in the state: (1) light and feeding the Artemia sp.; (2) complete darkness and feeding the Artemia sp.; and (3) complete darkness and not fed. The results showed the anemone’s weight, protein count, alga density, photosynthetic capacity (Fv / Fm) began to decline from the first 6 days to 24 days in darkness and feeding, its weight, protein count, alga density, photosynthetic capacity (Fv / Fm) decreased by 5%, 8%, 60%, 20% ( p < 0.05 ), but the same in the darkness and feeding of host which protein reduction of only 30% ( p < 0.05 ), despite a decrease in the amount, but did not like the no more than feeding anemone to many, and there alga density and photosynthesis capacity (Fv / Fm) compared to the control, there was no significant difference ( p > 0.05 ). Futher comparison anemones biochemical through 24 days indicators showed that glutamate dehydrogenase, total free amino acids and essential amino acids are significantly different ( p < 0.05 ), in the dark with or without feeding anemone’s vivo GDH activity began to rise in the first 12 dasy rising to 24 days volume has exceeded 100%, however, the GDH activity increased by 17% in the general state (light and feeding ), if host is in a completely dark environment, no matter there is no feeding or feeding, the activity will be high than the control ( p < 0.05 ), anemones average decrease of 59% total FAA and 26.4% EFAA in the darkness and feeding; in darkness without feeding will decrease on average 73.8% total FAA and76.8% EFAA. Another way we use of artificial to give carbon to replace lighting. The results are displayed in completely dark state feeding Artemia sp. or glycerol , comparing weight, protein count, alga density, photosynthetic capacity (Fv / Fm) and glutamate dehydrogenase of control (lighting and feeding) and between the two treatment after 24 days which all significant difference ( p < 0.05 ), compare two treatment (fed Artemia sp. and glycerol) and the control (lighting and feeding), two treatment were relatively low began to decrease from the fist 6 days to 24 days, its weight, protein count, alga density, photosynthetic capacity (Fv / Fm), respectively, reducing the weight of 30%, 42%; protein count of 52%, 65%; photosynthetic capacity (Fv / Fm) of 6%, 8% and alga density is reduced by 53%, 44%. Both treatment’s GDH activity is increased, which the activity of both increased by more than 100% ( p < 0.05 ), only malate dehydrogenase was no significant differences ( p > 0.05 ), from the above result, in the case of complete darkness caused by symbiotic algae can not photosynthesize, either not fed Artemia sp. or supply carbon, however the state of anemones compared with light status were have significant differences, so even to use of artificial means supplying additional nitrogen or carbon source, and as long as the symbiotic algae can not to carry out photosynthesis provide nutrition to the host, the host is still unable to maintain the light similar of physiological state.
目錄
中文摘要............................................................................................I
英文摘要......................................................................................... III
謝辭................................................................................................V
目錄...............................................................................................VI
圖次............................................................................................... V
表次...............................................................................................XI
第壹章 前言......................................................................................... 1
第貳章 文獻回顧...................................................................................... 5
2.1 海葵............................................................................................ 5
2.2 共生藻(Symbiodinium)........................................................................... 5
2.3 刺細胞動物與共生藻之間的共生關係................................................................... 6
2.4 宿主和共生體各自扮演的角色......................................................................... 8
2.5 珊瑚礁的重要性................................................................................... 10
2.6 白化(Bleaching)................................................................................ 11
2.7 白化的原因及影響.................................................................................. 11
2.8 白化的生化指標.................................................................................... 13
2.8.1 榖氨酸去氫酶 (glutamate dehydrogenase;GDH)..................................................... 13
2.8.2. 蘋果酸去氫酶 (malate dehydrogenase;MDH) ...................................................... 14
2.8.3 游離胺基酸(free amino acids;FAA).............................................................. 15
第参章 材料與方法....................................................................................... 16
3.1 動物採集與飼養...................................................................................... 16
3.2 動物餵食........................................................................................... 16
3.2.1 豐年蝦孵化........................................................................................ 16
3.2.2 營養混合液配製..................................................................................... 17
3.3 海葵處理的裝置與條件................................................................................. 17
3.3.1 處理裝置.......................................................................................... 17
3.3.2 海葵中共生藻光合作用能力測定....................................................................... 17
3.3.3 海葵共生藻的分離與組織液的製備.......................................................................18
3.4 蘋果酸去氫酶 (MDH)與穀氨酸去氫酶 (GDH)的活性測定....................................................... 18
3.4.1 海葵組織之游離胺基酸 (FAA) 分析..................................................................... 19
3.5 宿主組織均質液中蛋白量的測定.......................................................................... 20
3.6 統計分析............................................................................................ 20
第肆章 結果.............................................................................................. 21
第伍章 討論.............................................................................................. 24
參考文獻................................................................................................. 58

圖目錄
圖1、(A)為共生狀態的海葵(Aiptasia pulchella)、(B)培養於黑暗狀態下, 24 天後的海葵............................. 29
圖2、孵化豐年蝦的裝置...................................................................................... 30
圖3、豐年蝦孵化24 小時後的情形............................................................................. 31
圖4、豐年蝦為向光性的生物,當孵化24 小時後則以遮光的方式使得豐
年蝦往下並朝著有光線的出水口集中............................................................................ 32
圖5、於海水中以豐年蝦餵食海葵(Aiptasia pulchella)的情形..................................................... 33
圖6、海葵麻醉裝置......................................................................................... 34
圖7、標準胺基酸以o-phthaldialdehyde 作管柱前衍生化後之HPLC 分析圖譜.......................................... 35
圖8、於不同處理下海葵(Aiptasia pulchella)宿主重量經過 24 天後的變化情形.......................................36
圖9、不同處理下海葵(Aiptasia pulchella)宿主組織中的蛋白含量經過24天後的變化情形................................ 37
圖10、不同處理下海葵(Aiptasia pulchella)宿主組織中的共生藻光合作用能力經過24 天後的變化情形..................... 38
圖11、不同處理下海葵(Aiptasia pulchella)宿主組織中的共生藻密度經過24 天後的變化情形............................. 39
圖12、不同處理下海葵(Aiptasia pulchella)宿主組織中之GDH 活性經過24 天後的變化情形.............................. 40
圖13、不同處理下海葵(Aiptasia pulchella)宿主組織中之MDH 活性經過24 天後的變化情形.............................. 41
圖14、不同處理下海葵(Aiptasia pulchella)經過 24 天後,宿主組織中總胺基酸含量的變化情形.......................... 42
圖15、不同條件下海葵(Aiptasia pulchella)經過 24 天後,宿主組織中胺基酸組成..................................... 43
圖16、不同條件下海葵(Aiptasia pulchella)經過 24 天後,宿主組織中胺基酸組成之變化................................ 44
圖17、不同環境處理下海葵(Aiptasia pulchella)宿主組織中之Total protein amino acid 經過 18 天後的變化情形......... 45
圖18、不同處理下海葵(Aiptasia pulchella)宿主組織中之Total essential amino acid 經過 24 天後的變化情形........... 46
圖19、兩種不同處理下海葵(Aiptasia pulchella)宿主重量經過24 天後的變化情形....................................... 47
圖20、兩種不同處理下海葵(Aiptasia pulchella)宿主組織中的蛋白含量經過24 天後的變化情形............................. 48
圖21、兩種不同處理下海葵(Aiptasia pulchella)宿主組織中的共生藻密度經過 24 天後的變化情形.......................... 49
圖22、於兩種不同處理下海葵(Aiptasia pulchella)宿主組織中的共生藻光合作用能力經過 24 天後的變化情形................. 50
圖23、兩種不同處理下海葵(Aiptasia pulchella)宿主組織中之GDH 活性經過 24 天後的變化情形............................ 51
圖24、兩種不同處理下海葵(Aiptasia pulchella)宿主組織中之MDH 活性經過 24 天後的變化情形............................ 52

表目錄
表1、Seawater buffer ( SW buffer) (pH 8.2) 的組成............................................................. 53
表2、海葵 (Aiptasia pulchella) 在共生、在完全黑暗有餵食豐年蝦與在完全黑暗沒有餵食的條件下,經過24 天後測試動物組織中之蛋白量、
FAA、MDH、GDH 等生化指標的含量................................................................................. 54
表3、海葵 (Aiptasia pulchella) 在共生、在完全黑暗有餵食豐年蝦與在完全黑暗沒有餵食的條件下,經過24 天後測試宿主之重量、共生藻密
度、光合作用能力等生理指標的含量................................................................................ 55
表4、海葵 (Aiptasia pulchella) 在共生、在完全黑暗有餵食豐年蝦與在完全黑暗有餵食營養液的條件下,經過 24 天後測試宿主之重量、共
生藻密度、光合作用能力等生理指標的含量........................................................................... 56
表5、海葵 (Aiptasia pulchella) 在共生、在共生、在完全黑暗有餵食豐年蝦與在完全黑暗有餵食營養液的條件下,經過24 天後測試動物組織
中之蛋白量、MDH、GDH 等生化指標的含量........................................................................... 57
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