臺灣博碩士論文加值系統

白面鼯鼠分布於台灣中高海拔森林中，主要以各種闊葉樹木的嫩葉為食。膨大的盲腸為其消化道的明顯特徵，內含有大量的共生微生物，可協助宿主進行食物的分解代謝，使得飛鼠能從營養貧乏的葉片中獲取生存所需的能量。儘管消化道微生物對於人體與實驗鼠的重要性已被廣泛驗證，然而對於微生物在野生動物體內的組成結構與其所扮演的功能角色仍然所知有限。因此，本研究採用非傳統培養之分子遺傳工具與新一代的定序技術，針對野生飛鼠的消化道微生物進行詳盡的探索分析。依據實驗方法與討論內容可分為三個研究主軸：一、使用16S rRNA基因序列，研究來自12個消化道樣本(分別為兩隻飛鼠的小腸/盲腸/大腸之腸壁和內容物)的細菌組成結構〈詳見第二章〉。二、使用fosmid 端點定序序列，研究飛鼠盲腸內容物的微生物菌種組成和功能特性〈詳見第三章〉。三、使用多源基因體(metagenome)和多源轉錄體(metatranscriptome)序列，研究飛鼠盲腸微生物之代謝表現特徵〈詳見第四章〉。菌群分析結果顯示：無論採取哪種研究方式，Firmicutes均為飛鼠消化道微生物中主要的優勢菌群。功能比對結果顯示：微生物的代謝功能對於消化道環境內各項營養循環具有重要貢獻，例如：碳水化合物的降解、蛋白質的循環利用、及維生素的生合成。基因表現結果顯示：各項有關醣類代謝利用的基因(包含分解/偵測/運輸醣類之基因)均被大量的表現於飛鼠的盲腸環境中，表示這些居住在小型食葉性哺乳動物體內的微生物菌群，已經適應出能夠有效地從植物葉片中獲取能量之功能特化，與宿主形成共演化關係。

White-faced flying squirrels (Petaurista alborufus lena) inhabiting in subtropical forests of Taiwan, feed on leaves of diverse tree species. The predominant feature of their gastrointestinal tracts is an enlarged cecum that serves as an anaerobic container for microbial fermentation. Symbiotic gut microorganisms providing metabolic activities lacking in the host, are essential for energy extraction from the nutritionally poor diet. Although the importance of gut microbiota has been well demonstrated in humans and lab mice, there is a paucity of knowledge regarding gut microbial constituents and their functional capabilities in wild animals. Therefore, in this research, we applied culture-independent molecular tools and high-throughput sequencing techniques to provide the comprehensive understanding of the gut microbial communities in the wild-caught flying squirrels. Chapter 2 described the bacterial communities of various gut compartments based on 16S rRNA gene sequences. Chapter 3 provided the phylogenetic and functional profiles of the cecal microbiota based on fosmid end-sequences. Chapter 4 revealed the metabolic characteristics of the cecal microbiota based on the comparison of metagenomic and metatranscriptomic sequences. No matter using which approaches, Firmicutes was the predominant group of the gut microbiota. All results indicated that the microbial functions greatly contributed to nutrient cycling (including degradation of carbohydrates, metabolism of proteins, and synthesis of vitamins) in the gut environments. High gene expression for sugar degradation, detection and uptake revealed microbial adaptations for plant biomass usage in small folivorous mammals.

口試委員審定書 i
誌謝 ii
中文摘要 iii
英文摘要 iv
Chapter 1 General Introduction 1
1.1 Digestive systems of mammalian herbivores 1
1.2 Gut microbiota of mammalian herbivores 4
1.3 Plant biomass degradation by microbial enzymes 6
1.4 Molecular approaches for microbial studies 12
1.5 Significance of studies on gut microbiota of flying squirrels 22
1.6 Prokaryotic genome research 27
1.7 Reference 29
Chapter 2 Complexity of intestinal bacterial communities in the leaf-eating flying squirrel implies differentiation in functional properties of the gut microbiota 39
2.1 Abstract 39
2.2 Introduction 40
2.3 Methods 43
2.4 Results 47
2.5 Discussion 52
2.6 References 57
Chapter 3 Metagenomic analysis reveals a functional signature for biomass degradation by cecal microbiota in the leaf-eating flying squirrel (Petaurista alborufus lena) 72
3.1 Abstract 72
3.2 Introduction 73
3.3 Methods 75
3.4 Results 80
3.5 Discussion 92
3.6 Conclusions 98
3.7 References 98
Chapter 4 Integrated metagenome and metatranscriptome reveal adaptive ability for sugar degradation, detection and uptake by the cecal microbiota in the leaf-eating flying squirrel (Petaurista alborufus lena) 117
4.1 Abstract 117
4.2 Introduction 118
4.3 Methods 121
4.4 Results 125
4.5 Discussion 141
4.6 References 150
Chapter 5 Correlation of phylogeny with functional traits reveals the roles of gene evolutionary mechanisms in prokaryotes 178
5.1 Abstract 178
5.2 Introduction 179
5.3 Methods 182
5.4 Results / Discussion 188
5.5 References 200
Chapter 6 Conclusions and perspectives 211
6.1 Conclusions 211
6.2 Perspectives 212
6.3 References 215

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Chapter 4 References
1. Frank DN, Pace NR (2008) Gastrointestinal microbiology enters the metagenomics era. Current Opinion in Gastroenterology 24: 4-10.
2. Ley RE, Hamady M, Lozupone C, Turnbaugh PJ, Ramey RR, et al. (2008) Evolution of mammals and their gut microbes. Science 320: 1647-1651.
3. Xu J, Bjursell MK, Himrod J, Deng S, Carmichael LK, et al. (2003) A genomic view of the human-Bacteroides thetaiotaomicron symbiosis. Science 299: 2074-2076.
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12. Ferrer M, Martinez-Abarca F, Golyshin PN (2005) Mining genomes and ''metagenomes'' for novel catalysts. Current Opinion in Biotechnology 16: 588-593.
13. Gong JH, Si WD, Forster RJ, Huang RL, Yu H, et al. (2007) 16S rRNA gene-based analysis of mucosa-associated bacterial community and phylogeny in the chicken gastrointestinal tracts: from crops to ceca. Fems Microbiology Ecology 59: 147-157.
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19. Glad T, Bernhardsen P, Nielsen KM, Brusetti L, Andersen M, et al. (2010) Bacterial diversity in faeces from polar bear (Ursus maritimus) in Arctic Svalbard. BMC Microbiology 10: 10.
20. Kuo CC, Lee LL (2003) Food availability and food habits of Indian giant flying squirrels (Petaurista philippensis) in Taiwan. Journal of Mammalogy 84: 1330-1340.
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