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研究生:李明貞
研究生(外文):Ming-Cheng Lee
論文名稱:文蛤(Meretrixlusoria)無氧能量代謝的研究
論文名稱(外文):The study on the anaerobic energy metabolism in hard clam (Meretrix lusoria)
指導教授:李安進
指導教授(外文):An-Chin Lee
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:水產生物學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:99
中文關鍵詞:文蛤丙胺酸琥珀酸丙酸無氧代謝酵素血淋巴酸-鹼平衡
外文關鍵詞:hard clamalaninesuccinatepropionateanaerobic metabolism enzymeshaemolymph acid-base balance
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摘 要

每逢季節交替,溫鹽度變化太大或是環境不適時,文蛤Meretrix lusoria會採取閉殼的方式,由於文蛤為底棲性生物,不易由其行為表現來推測其健康狀態,所以本研究探討無氧環境對文蛤血淋巴酸-鹼平衡的影響,對斧足、血淋巴和無氧培育水中無氧代謝物(琥珀酸、丙胺酸和丙酸)含量的影響以及對斧足中丙酮酸激酶(PK)、丙胺酸氨基轉移酶(AlaT)、磷酸烯醇丙酮酸羧化酶(PEPCK)、延胡索酸還原酶(FR)和合成丙酸酵素活性的影響,希望可以作為文蛤活力的指標。
文蛤暴露於23和30℃空氣中1分鐘,其血淋巴中pO2的含量約下降50﹪,而文蛤暴露於15℃空氣中2分鐘,其血淋巴中pO2也顯著地下降75﹪。文蛤在28℃無氧的環境下2小時,血淋巴中pO2下降60﹪,當無氧處理延長為71小時,其血淋巴pH值顯著地下降,然而pCO2則上升。文蛤在含有597 ppm鉀離子的環境下3小時,其血淋巴pO2下降23﹪。而文蛤在含有100 ppm KCN和缺乏鎂離子的環境下10小時,其血淋巴pO2也分別下降39﹪和42﹪。
文蛤在無氧的環境下,其斧足、血淋巴和無氧培育水中主要的無氧代謝物分別為丙胺酸、琥珀酸以及琥珀酸和丙酸。在斧足中的丙胺酸以及血淋巴中的琥珀酸和丙酸隨著無氧時間的延長而顯著地增加,然而無氧培育水中琥珀酸的含量在無氧的後期逐漸地下降。文蛤在30℃無氧的環境下,僅能存活48小時,然而添加5 ppm的氯黴素可以延長文蛤在無氧環境下存活120小時以上,同時發現氯黴素對文蛤無氧代謝物累積量的影響不顯著。文蛤在缺乏鎂離子的環境下,其斧足中丙胺酸和琥珀酸以及血淋巴中丙胺酸、琥珀酸和丙酸的含量隨著培育時間的延長而顯著地增加,然而在斧足中並沒有測到丙酸的含量。
文蛤在15℃無氧的環境下144小時,其斧足中AlaT和PEPCK的活性沒有顯著的變化,然而PK、FR和合成丙酸酵素的活性則分別先增加至控制組的145﹪、124﹪和300﹪,而後分別下降為控制組的119﹪、117﹪和120﹪。文蛤在30℃無氧的環境下48小時,其斧足中AlaT的活性隨著無氧時間的延長而下降,然而PEPCK和FR的活性則增加,而PK和合成丙酸酵素的活性則分別先增加至控制組的152﹪和300﹪,而後下降為控制組的134﹪和200﹪。
由上述結果得知,測定文蛤斧足和血淋巴中琥珀酸的增加量可作為文蛤活力的指標,除此之外,在夏季時,PEPCK和FR的活性亦可作為文蛤活力的指標。

關鍵詞:文蛤、丙胺酸、琥珀酸、丙酸、無氧代謝酵素、血淋巴酸-鹼平衡
Abstract

Hard clam are closing their shells when changes of season, temperature, salinity and environmental. Hard clam are bottom organisms. This study approaches the effect of anaerobic treatment on the acid-base balance in haemolymph of hard clams, the accumulation of anaerobic metabolites in foot and haemolyph of hard clams as well as in incubated water, and activities of pyruvate kinase (PK), analnine aminotransferase (AlaT), phosphoenopyruvate carboxykinase (PEPCK), fumarate reductase (FR) and propionate-formed enzymes in foot of hard clams.
The pO2 of haemolymph in hard clams exposed to air for 1 minute at 23 and 30℃ is decreased by a half. Furthermore, the remaining of pO2 of haemolymph in hard clams after air exposure for 2 min at 15℃ is 25% of that of control group. Haemolymph pO2 decreases 60% for hard clams incubated in anaerobic condition for 2 hr at 28℃. By extending anaerobic incubation time to 71 hr, haemolymph pH decreases, while pCO2 increases. A 23% decrease in pO2 of haemolymph of hard clams incubated in artificial seawater with 597 ppm K+ for 3 hr is found. Haemolymph pO2 decreases 39% and 42% for hard clams incubated in artificial seawater with 100 ppm of KCN and that lacking Mg2+ for 10 hr, respectively.
In the anaerobic conditions, alanine, succinate as well as succinate and propionate are main anaerobic metabolites in foot, haemolymph, and incubated water, respectively. Alanine in foot, and succinate and propionate in heamolymph and incubation water are accumulated with anaerobic incubation time increased. However, the amount of succinate in incubated water decreases in the late period of anaerobic treatment. Hard clams only survive for 48 hr at 30℃ under anoxia. However they can survive for more than 120 hr by the addition of 5 ppm chloramphenicol at 30℃ under anoxia. The effect of chloramphenicol on the accumulation of anaerobic metabolites is not significant. Alanine and succinate in foot, and alanine, succinate and propionate in haemolymph are accumulated with anoxia time increased, when hard clams are incubated in artificial seawater lacking Mg2+. However, propionate in foot is not detectable.
Changes in the activities of AlaT and PEPCK in foot of hard clams incubated in anaerobic conditions for 144 hr are not significant. However, activities of PK, FR and propionate-formed enzymes in foot first increase to 145%, 124% and 300% of the control group and then thereafter, decrease to 119%, 117% and 120%, respectively. Activities of AlaT, and PEPCK and FR in foot of hard clams incubated in anaerobic condition for 48 hr at 30℃ decrease and increase with anaerobic incubation time increased, respectively. However, the activities of PK and propionate-formed enzymes first increase to 152% and 300% of the control group and then decrease to 134%and 200%, respectively.
The result of the present study was detectable increase amount of succinate in foot and haemolymph of hard clam is indicator of activity. In summer, the activities of PEPCK and FR is indicator of hard clam activity.

Keywords: hard clam, alanine, succinate, propionate, anaerobic metabolism enzymes, haemolymph acid-base balance
目錄
中文摘要 i
英文摘要 iii
第一章 前言
一、本研究的緣由和動機 1
二、二枚貝的簡介 2
三、二枚貝的生長環境 4
四、二枚貝的呼吸作用 7
五、二枚貝的能量代謝 8
第二章 材料與方法
一、 材料 14
(一)實驗動物 15
(二)實驗用耗材、酵素及化學藥品 15
二、方法
(一) 暴露於空氣中、無氧環境或化學物對文蛤血淋
巴酸-鹼平衡和溶氧量的影響 16
(二) 溫度對文蛤在無氧環境下半致死時間的影響 17
(三) 溫度對文蛤無氧代謝物(丙胺酸、琥珀酸和丙
酸)和丙酮酸的影響 18
(四) 人工海水中缺乏鎂離子對文蛤無氧代謝物(丙
胺酸、琥珀酸和丙酸)和丙酮酸的影響 21
(五)溫度對文蛤參與無氧代謝酵素活性的影響 22
第三章 結果
一、暴露於空氣中、無氧環境或化學物對文蛤血淋巴酸-
鹼平衡和溶氧量的影響 29
二、溫度對文蛤在無氧環境下半致死時間的影響 31
三、溫度對文蛤無氧代謝物(丙胺酸、琥珀酸和丙酸)和
丙酮酸的影響 32
四、人工海水中缺乏鎂離子對文蛤無氧代謝物(丙胺酸、
琥珀酸和丙酸)和丙酮酸的影響 36
五、溫度對文蛤參與無氧代謝酵素活性的影響 37
第四章 討論
一、暴露於空氣中、無氧環境或化學物對文蛤血淋巴酸-
鹼平衡和溶氧量的影響 39
二、溫度對文蛤在無氧環境下半致死時間的影響 42
三、溫度對文蛤無氧代謝物(丙胺酸、琥珀酸和丙酸)
和丙酮酸之影響 43
四、人工海水中缺乏鎂離子對文蛤無氧代謝物(丙胺酸、
琥珀酸和丙酸)和丙酮酸的影響 50
五、溫度對文蛤參與無氧代謝酵素活性的影響 50
第五章 結論 55
參考文獻 56
表 76
圖 84
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