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研究生: 莊政霖
Cheng-Lin Chuang
論文名稱: 劍尖槍鎖管發光器官內共生發光細菌之鑑定及特性分析研究
Identification and characterization of the symbiotic bioluminescent bacteria from the light organ of swordtip squid [Uroteuthis (Photololigo) edulis]
指導教授: 王玉麒
Wang, Yu-Chie
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2011
畢業學年度: 99
語文別: 中文
論文頁數: 135
中文關鍵詞: 鎖管發光器官發光細菌共生生物發光生長曲線
英文關鍵詞: Loliginidae, light organ, bioluminescent bacteria, symbiosis, bioluminescence, growth curve
論文種類: 學術論文
相關次數: 點閱:137下載:6
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  • 前人研究 (Fukasawa & Dunlap, 1986) 指出共生於劍尖槍鎖管 [Uroteuthis (Photololigo) edulis] 發光器官內的發光細菌為Photobacterium leiognathi,然而本研究有新的發現。本研究從臺灣北部海域釣取劍尖槍鎖管,並就其發光器官內的共生細菌進行基因、生理和生化特性的探討。「16S rDNA」和「luxF」兩種基因的序列比對和特徵分析的結果顯示,25株分離自五尾劍尖槍鎖管的發光細菌中,有24株為Photobacterium mandapamensis,另外1株為P. leiognathi。因此,劍尖槍鎖管發光器官內的共生細菌應大多數為P. mandapamensis,僅少數發光器官內有P. mandapamensis和P. leiognathi同時共生 (cosymbiosis) 的情形。本研究分離的24株P. mandapamensis中,有7株的發光基因操縱組 (lux operon) 呈現部分雙套 (merodiploid) 之情形,這些菌株也對生活環境的「溫度」和「鹽度」因子有差異性的表現,而發光強度上更出現有1~60倍的差異。根據這些基因變異及生理、生化特性的差別,本研究推測P. mandapamensis與劍尖槍鎖管建立共生關係的初始,是由多個已具變異特性的菌株由海水中進入發光器官內拓殖,而非僅由單一菌株進入拓殖的情形。此外,本研究也發現除了基因型的差異之外,某些生理和生化特性的差異也可被應用於區分親緣關係接近的P. mandapamensis和P. leiognathi二菌種,包括P. mandapamensis可於含7% NaCl的培養液中生長,但P. leiognathi則否;P. mandapamensis的離胺酸去羧酶 (lysine decarboxylase) 測試呈陰性反應,而P. leiognathi則呈陽性反應;P. mandapamensis具有同化蘋果酸 (malate) 的能力,而P. leiognathi則否。而本研究所建立之發光細菌的簡易、準確生長測量方法,更可成為其他發光細菌研究的重要研究方法參考。

    The previous study (Fukasawa & Dunlap, 1986) identified the bioluminescent bacteria that symbiosed in the light organ of swordtip squids [Uroteuthis (Photololigo) edulis] is Photobacterium leiognathi. However, the current study proposes a contradictory identification. In our study, 25 strains of symbiotic bacteria were isolated from the light organs of five swordtip squids collected off the coast of north Taiwan. Among these 25 isolates, 24 were identified to be Photobacterium mandapamensis, while the rest one being P. leiognathi, based upon the results of comparative analyses of the 16S rDNA and the luxF gene. Therefore, the predominant bacterial symbiont of swordtip squid should be P. mandapamensis, and only in rare cases that both P. mandapamensis and P. leiognathi might be cosymbiosed within the same light organ. Since we have observed among the 24 P. mandapamensis isolates some featured differences of genetic, physiological, and biochemical characteristics, it is hypothesized that polyclonal bacteria may have entered the light organ from the sea water during its early stage of development. Moreover, the current studies unraveled some physiological and biochemical features that can be employed to distinguish the two close-related bacterial species of P. mandapamensis and P. leiognathi . The current studies have also developed a simple and accurate photometric method for measuring the growth of luminescent bacteria.

    摘要 1 Abstract 2 壹、緒論 3 一、生物發光 (bioluminescence) 3 二、生物發光的機制 4 三、生物發光的可能功用 6 (1)、避免被捕食 (predator avoidance) 6 (2)、吸引獵物 (prey attraction) 7 (3)、傳遞訊息 (intraspecific communication)、繁殖 (reproduction) 8 四、發光細菌 (bioluminescent bacteria) 8 五、發光細菌的發光基因操縱組 [lux operon, luxCDAB(F)EG] 11 六、常見海洋發光生物的研究模式系統 (model system) 12 七、臺灣附近海域的鎖管科 (Loliginidae) 動物 18 八、劍尖槍鎖管 [Uroteuthis (Photololigo) edulis] 21 九、研究目的 22 貳、研究材料與方法 23 一、實驗動物的採集與鑑定 23 (1)、夜釣鎖管 23 (2)、鎖管物種的鑑定 23 二、鎖管之共生發光細菌的分離、純化與保存 23 (1)、發光細菌的分離與純化 23 (2)、發光細菌之菌種保存 24 (3)、標準菌株 24 三、16S ribosomal RNA (16S rRNA) 的基因 (16S rDNA) 分析 25 (1)、聚合酶連鎖反應 (polymerase chain reaction, PCR) 25 (2)、限制酵素 (restriction endonucleases) 切割反應 26 (3)、DNA電泳分析 27 (4)、定序分析 28 四、luxF基因的分析 28 (1)、PCR反應 28 (2)、定序分析 30 五、劍尖槍鎖管共生發光細菌的生長曲線 (growth curve) 測定 30 (1)、OD700與細菌密度 [colony forming unit (CFU)/ml] 的關係 30 (2)、溫度對細菌生長曲線的影響 32 (3)、鹽度對細菌生長曲線的影響 32 (4)、兩種劍尖槍鎖管共生發光細菌的生長相容性測試 33 (5)、統計分析 34 六、生化特性測試 34 七、發光特性分析 35 (1)、發光波長範圍測定 35 (2)、發光強度測定 35 八、劍尖槍鎖管共生發光細菌的屬性分析及形態觀察 36 (1)、革蘭氏陰、陽性菌的鑑定 36 (2)、穿透式電子顯微鏡 (transmission electron microscopy, TEM) 觀察 36 參、結果 38 一、實驗動物的採集與鑑定 38 二、劍尖槍鎖管之共生發光細菌的分離 43 三、共生發光細菌的16S rDNA分析 47 (1)、限制酵素切割圖譜 47 (2)、基因定序分析 51 四、luxF基因的分析 58 (1)、PCR檢測 58 (2)、PCR產物定序分析 66 五、共生發光細菌的生長特性分析 74 (1)、OD700與細菌密度 [colony forming unit (CFU)/ml] 的關係 74 (2)、溫度對共生發光細菌生長的影響 78 (3)、鹽度對共生發光細菌生長的影響 83 六、共生發光細菌的生化特性分析 90 七、共生發光細菌的發光特性分析 92 (1)、發光波長範圍測定 92 (2)、發光強度測定 95 八、共生發光細菌的顯微觀察結果 98 九、不同共生發光細菌的相容性測試 102 肆、討論 104 一、劍尖槍鎖管發光器官內共生發光細菌的鑑定 105 二、部分劍尖槍鎖管的發光器官內可能同時共生 (cosymbiosis) 著P. mandapamensis以及P. leiognathi兩菌種 110 三、P. mandapamensis的發光基因操縱組呈現部分雙套 (merodiploidy) 之情形 112 四、劍尖槍鎖管發光器官內共生的P. mandapamensis,其基因型與表現型特徵具有多樣性 113 五、劍尖槍鎖管發光器官內共生的發光細菌,其發光強度、生長情況具有差異性 114 六、以OD讀值來估算發光細菌之數量,必須先考量菌株的發光波長特性及菌株間散射光線的能力是否相同 115 七、結論 116 參考文獻 118 附錄 125 附錄一、NBSA培養基及NBS培養液的製備 125 附錄二、luxF基因檢測產物序列比對 (BLAST) 結果 126 附錄三、中英對照表 129

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