具脫氮能力的假弧菌屬細菌(Alphaproteobacteria; Rhodobacterales; Rhodobac teraceae)廣泛存在於海洋環境中(特別是海綿)。假弧菌屬菌於2004年，在台灣墾丁國家公園南灣海水中首次被分離與描述。這些兼性厭氧的海洋細菌常在海洋無脊椎動物中被發現，如海鞘、被囊動物、藻類、管蟲、珊瑚與海綿，事實上，假弧菌屬細菌被認為與宿主共生關係中扮演重要角色，此外，多個研究指出此屬菌株具廣效的抗菌活性，可為發現新穎活性天然物的來源。在本研究中，由9種海綿樣本以培養的方法篩選假弧菌屬菌株，海綿樣本採集自國立海洋生物博物館(NMMBA)水族實驗中心與南灣附近海域。從三種不同選擇性培養基共分離867株細菌，其中301株顯示具脫氮活性，經16S rRNA定序確定其中188株為假弧菌屬菌株。親緣關係分析顯示，大部分菌株皆屬於Pseudovibrio denitrificans (157株，83.51%)。然而，有些分離株很難辨識到種，顯示其中有些菌株可能為新種。針對5株測試目標細菌與1株真菌進行抗菌活性分析，發現有52株(27.96%)假弧菌屬分離株對至少一株病原菌具有抗菌活性。吾人研究結果顯示海綿可能是發現具潛力藥物分子的假弧菌屬菌株的重要來源。

Denitrifying bacteria of the genus Pseudovibrio (Alphaproteobacteria: Rhodobacterales: Rhodobacteraceae) are widely distributed in the in the marine environment, especially within sponges. They were first isolated and described in 2004 from seawater of Nanwan Bay, Kenting National Park, Taiwan. These facultatively anaerobic marine bacteria were repeatedly isolated from marine invertebrates such as ascidians, tunicates, algae, tube worms, corals and sponges. Pseudovibrio is in fact believed to play an important role in symbiotic relationships with their hosts. Moreover, antimicrobial activities within Pseudovibrio sp. toward broad-spectrum of pathogens has been described many times, suggesting they could be used as a novel source of bioactive compounds. In the present study, 9 sponge samples were used to screen for Pseudovibrio strains by using a culture-based method. Sponge samples were collected from either the husbandry center, National Museum of Marine Biology and Aquarium (NMMBA) or Nanwan Bay, Taiwan. A total of 867 bacterial strains were isolated from three different selective media, among which 301 strains displayed denitrifying activity and within which 188 strains were confirmed to be Pseudovibrio based on 16s rRNA gene sequences. Phylogenetic analysis of these strains based on 16S rDNA fragments indicated that most of the isolates belong to Pseudovibrio denitrificans (157 isolates, 83.51%). However, many isolates are difficult to resolve to species suggesting some of them might be new Pseudovibrio species. All the Pseudovibrio isolates were screened for their antimicrobial activity against five test bacteria and a fungus. Fifty-two (27.96%) of the isolates showed activity against at least one test microbes. Our results demonstrate that marine sponge could be a source of Pseudovibrio strains which have a great potential in discovery of usefully medical molecules.

圖目錄 VI
表目錄 VII
第一章 緒論 1
1.1前言 1
1.2假弧菌屬的簡介 2
1.3假弧菌屬的分離和其生物活性 4
1.4假弧菌屬之二次代謝產物 5
1.5假弧菌屬菌生理學相關研究 6
1.6使用分子生物學方法鑑定菌株 7
1.7研究動機與目的 8
第二章 實驗材料與方法 15
2.1 藥品、儀器與培養基 15
2.1.1藥品 15
2.1.2微生物培養基 16
2.1.3實驗儀器 16
2.1.4生物活性實驗測試菌株 17
2.2 培養基配置 18
2.2.1菌種培養基 18
2.2.2選擇性培養基 20
2.3 膠體幾丁質製備方法 21
2.4 採樣及細菌分離培養 22
2.4.1海綿樣本來源 22
2.4.2樣本處理 22
2.4.3平板培養與活菌計數 22
2.4.4脫氮能力測試 23
2.5 16S rDNA序列鑑定假弧菌屬細菌 23
2.5.1萃取與純化細菌之基因體DNA 23
2.5.2聚合酶連鎖反應增幅16S rDNA片段 24
2.5.3 DNA凝膠電泳分析 24
2.5.5 DNA純化 25
2.5.6 DNA序列處理與Blast序列比對 26
2.5.7分子親緣關係分析 26
2.6 假弧菌屬菌株洋菜錠抗菌活性試驗 26
2.6.1抗菌活性測試目標菌株培養 26
2.6.2抗菌活性篩選 27
2.7利用隨機擴增多形態DNA區分假弧菌屬分離株 27
2.7.1以RAPD引子隨機增幅假弧菌屬分離株基因體DNA片段 27
2.7.2 RAPD電泳分析 28
2.7.3 RAPD圖譜群聚分析 29
第三章 結果與討論 31
3.1海綿樣本採集與型態鑑種 31
3.2海綿分離菌株平板培養結果 32
3.3 脫氮能力測試 32
3.4 16S rDNA序列鑑定假弧菌屬分離株分析與培養 33
3.5 16S rDNA序列之分子親緣關係分析 34
3.6假弧菌屬菌株抗菌活性試驗 35
3.7隨機擴增多形態DNA分析結果 36
第四章 結論與建議 61
4.1結論 61
4.2建議 61
參考文獻 63
附錄 73

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