動物的物種及食性差異會影響其腸道微生物菌相之組成，而腸道微生物在宿主健康及營養提供上亦扮演了極重要的角色。本研究旨在利用PCR-DGGE技術及DNA定序分析探討壽山動物園內四種不同食性（肉食、雜食、草食反芻及非反芻）動物其腸道微生物菌相組成及其相關菌種對宿主之影響。結果顯示四種不同食性動物菌相構成有顯著差異，菌相豐富度由肉食性至雜食性至草食性逐漸增加，亦發現四種動物中其腸道微生物菌相，以肉食動物菌相與其他動物菌相差異性最大，其次為雜食性動物，而草食性動物不論會反芻與否，彼此菌相的差異性最小。長時間觀察顯示，四種不同食性動物其腸道微生物菌相構成大致穩定，且在不同時間點中，相同物種但不同個體之間其菌相差異不大，顯示外在環境條件雖會影響動物腸道微生物菌相，但宿主物種及食性差異仍是影響腸道微生物菌相組成之主因。另外發現四種不同食性動物鑑定菌種主要皆以Firmicutes門為主，其他菌門皆佔少數，其中Bacteroidetes門的比例遠低於Firmicutes，與人類腸道微生物組成有所差異，推測與動物園內餵食飼料中含較高比例之澱粉類有關。鑑定菌種結果顯示四種不同食性動物皆有大量降解各類碳水化合物、脂類、蛋白質之相關菌種；除肉食性動物中未鑑定到可分解纖維素之Fibrobacteres菌種外，草食性及雜食性動物皆有，顯示腸道微生物確實有協助養份消化及能量代謝的功能。

The animal phylogeny and feeding habits would affect the composition of gastrointestinal tract（GI tract）microbiota. GI tract microbiota plays an important role in host health and nutrient provision. In this study, we used PCR-DGGE and bacterial 16S rDNA sequencing to analyze the GI tract bacterial diversity of four animals with different feeding habits in Shou-Shan zoo, including one carnivore, one omnivore and two herbivores, in which one ruminant and one non-ruminant. The results show a great difference between GI tract bacterial diversity of the four animals. The abundance of GI tract bacterial diversity increased from carnivore, omnivore to herbivore. Comparing the similarity of the GI tract bacterial community structures of these four animals, the carnivore possessed the most different composition, to other animals, the next was the omnivore, while the two herbivores show the highest similarity to each other. Our results also indicated that the GI tract microbiota of these four different animals were very stable during the investigating period. We also found that two individuals of the same species had a very similar bacterial compositions in their GI tracts at different time point. This finding indicated that the bacterial compositions of GI tract in the four animals were affected mostly by the host phylogeny and their feeding habits. Moreover, according to bacterial 16S rDNA sequencing and idencification, results show that the Firmicutes were the dominant bacterial phyum in all four animals GI tracts, the amount of Bacteroides was much less than Firmicutes. This result might caused by the highly starch content in their feed. Large amount of carbohydrate-degrading, protein-degrading, lipid-degrading bacteria were found in all of these different animals. Fiber-degrading bacteria Fibrobacteres were identified in the GI tracts of the herbivores and omnivore, but not the carnivore, showing that GI tract microbiota plays an important role to provide nutrient and assist energy to the host.

摘要...............................................................................................................................I
第一章 前言................................................................................................................1
1.1 腸道微生物.........................................................................................................1
1.1.1 影響腸道微生物菌相的因素......................................................................2
1.1.1.1 飲食.......................................................................................................2
1.1.1.2 年齡.......................................................................................................3
1.1.2 腸道微生物對宿主的影響..........................................................................4
1.1.2.1 養份提供及能量代謝............................................................................4
1.1.2.1.1 碳水化合物....................................................................................5
1.1.2.1.2 脂質................................................................................................7
1.1.2.1.3 蛋白質............................................................................................9
1.1.2.1.4 維生素..........................................................................................10
1.1.2.2 其他.....................................................................................................11
1.1.3 腸道微生物與糞便微生物之差異............................................................12
1.2 動物消化道構造差異對腸道微生物之影響....................................................12
1.2.1 肉食性及雜食性動物消化道構造差異對腸道微生物之影響………..13
1.2.2 草食性動物消化道構造差異對腸道微生物之影響…………………..13
1.3 壽山動物園四種不同食性動物.......................................................................14
1.4 腸道微生物多樣性研究...................................................................................15
1.4.1 從傳統培養技術到分子生物分析............................................................15
1.4.2 16S rDNA 序列........................................................................................16
1.4.3 DGGE 原理..............................................................................................17
1.4.4 PCR-DGGE 技術………………………………………...……………..18
1.5 研究目的...........................................................................................................18
第二章 實驗材料與方法..........................................................................................20
2.1 採集樣本.........................................................................................................20
2.2 DNA extraction................................................................................................20
2.3 PCR.................................................................................................................21
2.3.1 用於DGGE 之反應條件.........................................................................21
2.3.2 用於clone library 欲ligation 之反應條件...............................................22
2.3.3 用於DGGE 單一或小範圍條帶切膠純化後欲ligation 之反應條件…23
2.3.4 用於cloning 藍白篩篩選之反應條件.....................................................23
2.3.5 用於cloning 刪除重複band 之反應條件................................................24
2.3.6 電泳確認..................................................................................................24
vi
2.4 DGGE..............................................................................................................24
2.4.1 DGGE 架設膠台......................................................................................25
2.4.2 DGGE 配製膠片......................................................................................25
2.4.3 DGGE 樣本注入及條件設定..................................................................26
2.5 Cloning and sequencing..................................................................................27
2.5.1 agarose 切膠及純化.................................................................................28
2.5.2 DGGE 單一或小範圍條帶切膠純化......................................................29
2.5.3 Ligation....................................................................................................30
2.5.4 Transformation.........................................................................................30
2.5.5 clone 菌株確認及篩選.............................................................................31
2.5.6 定序鑑定比對..........................................................................................31
第三章 結果與討論..................................................................................................32
3.1 四種不同食性動物腸道微生物DGGE 菌相分析.............................................32
3.2 相同物種不同個體腸道微生物菌相之比較與分析.........................................34
3.3 菌種定序結果.....................................................................................................35
3.4 四種不同食性動物鑑定菌種比較.....................................................................36
3.5 鑑定菌種之生理代謝特性.................................................................................37
3.5.1 碳水化合物代謝..........................................................................................38
3.5.2 脂質代謝......................................................................................................39
3.5.3 蛋白質代謝..................................................................................................40
3.5.4 其他..............................................................................................................41
3.6 動物腸道微生物菌相在時間變化上的分析.....................................................42
3.6.1 孟加拉虎DGGE 時間變化圖及菌種鑑定圖.............................................42
3.6.2 台灣獼猴DGGE 時間變化圖及菌種鑑定圖.............................................44
3.6.3 單峰駱駝DGGE 時間變化圖及菌種鑑定圖.............................................45
3.6.4 查普曼斑馬DGGE 時間變化圖及菌種鑑定圖..........................................46
第四章 結論..............................................................................................................48
參考文獻......................................................................................................................50
圖表..............................................................................................................................63
附錄..............................................................................................................................78

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參考書籍

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