臺灣博碩士論文加值系統

分解能力之族群，可作為高活性纖維分解酵素基因重要來源之一，極具發展潛力。然而在本研究之前，台灣對此重要主題及其相關基礎研究如瘤胃真菌在台灣地區反芻動物中的分佈、瘤胃真菌在人工培養下是否能維持基因穩定性、生長於台灣反芻動物中的瘤胃真菌是否為特殊族群與是否具有獨特的纖維分解酵素基因等許多主題並未曾進行整體性研究。因此本研究中，長期調查台灣水牛等反芻動物瘤胃真菌菌相並以核糖體基因建立相關菌株分類及類源關係、確認人工培養條件下基因的穩定性與建構自有菌株基因庫篩選纖維分解酵素基因等主題為研究目的。菌相調查結果顯示台灣水牛瘤胃中真菌族群約為105 cfu/ml，可觀察鑑定出具有Neocallimastix、Piromyces、Caecomyces、Orpinomyces與Anaeromyces等五個屬的厭氣性真菌菌株。菌株分離培養實驗結果顯示，現存的十一株分離株中，除八株已確認學名的菌株外，尚包括一株已命名完成的新種菌株Piromyces polycephalus W-33與分別在形態和核糖體基因與目前已知菌株具明顯差異的兩菌株W-98與W-1，為疑似新種。以PCR方式擴增核糖體基因經定序後與基因資料庫中相關基因進行類源關係分析，經由PAUP演算結果顯示，台灣水牛瘤胃中厭氣性真菌族群與目前已知其他菌種有不同類源關係，因此，從菌相的多樣性與種源的岐異性都可證明台灣水牛的瘤胃的確是獨特的分離源。由於完整生活史尚未被確認，因此無法僅以形態指標判定瘤胃真菌在人工培養時基因的穩定性，故以基因多形性圖譜(RAPD)與ITS1(Internal transcriptional space1)序列作為基因變異程度的評估指標，並以高等擔子菌靈芝作為有性世代的參考模式，結果顯示，RAPD圖譜與ITS1基因經50及150繼代培養後仍保持不變，因此確認厭氣性真菌人工長期繼代培養過程中基因的穩定性。在確認分離株屬性後，為分離完整片段的纖維分解酵素基因，因此本研究中建構Neocallimastix sp. W-1與Anaeromyces sp. W-98等菌株的纖維誘導cDNA library，在所建構的基因庫中，篩選出四段纖維分解酵素基因，其中12-8、10-2可能因趨同演化的關係，因此在ORF區域與其他已知同屬菌株序列有極高的相同度，但自另一株形態差異較大Anaeromyces sp. w-98菌株的 cDNA library所分離出的基因中98-9、98-7則與已知序列的相似度較低，為兩條新的纖維分解酵素基因。

Anaerobic fungi survive in the gut of herbivores and have shown diverse plant polysaccharide hydrolytic activities. These characteristics make anaerobic fungi a potential source of cellulase genes. However, the research about anaerobic fungi was not extensively investigated before. The goals of this thesis focus on the anaerobic fungal distribution in the rumen of Taiwan water buffalo, the heredity stability of anaerobic fungi after long-term subculturing in vitro, the relatedness of anaerobic fungal isolates, and the characteristics of the anaerobic fungal cellulase genes. In this study, diversity of anaerobic fungi were isolated from the rumen of Taiwan water. A new species Piromyces polycephalus W-33 and two strains with specific morphology were obtained during isolation. The Internal transcriptional space 1 phylogenetic analysis showed ITS1 of anaerobic fungi obtained from water buffalo showed the diversity relatedness with the other reference sequences. These results suggested the rumen fungi colonized in the water buffalo are a special group in phylogeny. To ensure the heredity stability of anaerobic fungal strain subcultures frequently for long periods, the genetic features of different generations of N. frontalis SK were examined. The identical RAPD patterns and invarious ITS1 sequences indicated the heredity stability among different generations of anaerobic fungi in artificial frequently subculture. Two cellulase genes were isolated from W-1 cDNA library and sequences showed high homology with known sequence. Another two sequence isolated from w-98 cDNA library showed lower homology with known sequence.

中文摘要……………………………………………………………….1
英文摘要…………………………………………………………..2
第一章、緒言…………………………………………………………...3
一、研究背景……………………………….……………..…………………………3
1. 纖維分解酵素的重要性及應……………..………………….……………………3
2. 纖維分解酵素的來源………………….….…………….…………………….…4
3瘤胃環境與瘤胃微生物………………………….…………………………….…5
4絕對厭氣性真菌………………………………………….………….……..……..8
4.1 絕對厭氣性分佈與種類……….……………………………..…………….…8
4.2 絕對厭氣性的特異性……………………………………………………….…8
4.3 絕對厭氣性真菌的分類鑑定………………………………………………..11
4.4 厭氣性真菌生活史…………………………………………………………..14
4.5 絕對厭氣性真菌纖維分解酵素基因………………………………………..15
二、研究目的………………………………………………………………...….17
三、預定工作項目………………………………………………………………17
第二章、材料與方法……………………………….………………………………18
1. 菌株分離培養與形態鑑定…………………………….……………………….18
1.1 分離源……………………………………………………..………………...18
1.2 培養基與厭氣環境建立………………………………………..……………18
1.3真菌族群密度估算……………………………………………..……………19
1.4 真菌菌株分離…………………………………………..………….……..….19
1.5 菌株形態鑑定……………………………..…………………………………..20
2. 核糖體基因鑑定………………..………………………………………………21
2.1 厭氣性真菌菌體置備…………………………..………………………….…21
2.2 Genomic DNA 萃取………………………………….……………………...21
2.3 核糖體基因PCR擴增反應……………………………………………..…...22
2.4 PCR擴增產物選殖至 pGEM-T easy 載體……………………..………….22
2.5 ITS1基因定序……………………………...……………………………….…23
2.6 基因序列排序與親源關係分析…………………………………………….23
3. 厭氣性真菌遺傳穩定性試驗………………………………………………….…24
3.1 靈芝培養與genomic DNA萃取………………………………………..…..24
3.2 不同世代厭氣性真菌菌體收集………………………………...…………….24
3.3 DNA萃取方式……………………………………………………………..…..24
3.4 基因多形性圖譜分析………….……………………………………………..24
3.5 ITS 1序列分析………………………………………………………………25
4. 自分離株建構cDNA library………………………………………………….…26
4.1 RNAse-free操作環境建立………………………………….…………….…..27
4.2 RNA的誘導生成與菌體回收…………………………………...…………….27
4.3 全RNA 萃取……………………………………………………..………..….27
4.4 mRNA 分離……………………………………...…………………….………28
4.5 cDNA合成……………………………………………………………………..29
4.6 cDNA端點Sfi I限制脢截切…………………………..………………..……..29
4.7 cDNA 片段大小分離…………………………………………………….….29
4.8 cDNA 與載體的黏合…………………………………………………….……29
5. 纖維分解酵素基因篩選………………………………………………………..31
5.1 λ噬菌體填裝……………………………….………………………………31
5.2 溶菌斑計數…………………………………………….………………….….31
5.3 cDNA library放大與基因篩選…………………………………………….…..31
5.4 纖維分解酵素基因篩選………………………………………….……….32
5.5 In vivo excision…………………………………………………………...…….32
5.6 受質專一性測試…………………………………..………………………….32
5.7 菌種保存……………………………………………………………………..32
5.8 基因定序………………………………………………………………..……..32
第三章、結果與討論………………………….………………………………….…..33
1、台灣本土厭氣性真菌菌種資源調查……………………………………………33
1.1真菌數目變化與季節關係…………………………………………………….33
1.2 厭氣性真菌菌相分佈…………………...…………………………………….34
1.3 結論……………………………………………………………………………35
2. 分離株的形態鑑定……………………….………………………………………36
2.1 H-3分離株….………………………………………………………………..36
2.2 H-16分離株……….……………………………...……………………………36
2.3 W-1分離株………………………………………………………….………….37
2.4 Sk 分離株……………………………………….…………………………..…37
2.5 J-6/J-8分離株………………………………………………………………..…39
2.6 J-18分離株.………………………………….…………………………………39
2.7 W-98分離株……………………………………….…………………………...39
2.8 W-72 分離株………………………………...…………………………...……41
2.9 W-33分離株………………………………………...………………………….41
2.10 結論…………………………….…………………………………………..43
3. 絕對厭氣性真菌核糖體基因序列分析……………………………………….…44
3.1 核糖體基因序列排序(alignment)與分析………………….…………..……44
3.2 ITS1核糖體基因親源關係分析…………………….……………………....48
3.3 結論………………………………………………………….…………….48
4. 厭氣性真菌人工培養下遺傳穩定性分析…………………………………….…52
4.1形態、碳源耐受性分析……………………………………..……………..…52
4.2 RAPD 分析……………………………………………………...………….…52
4.3 ITS1 區域序列分析………………………………………………………....53
4.4 結論………………………………………………………………………….55
5. W-1與W-98 cDNA-library建構與篩選………………….………………….…..56
5.1 cDNA-library的建構………………………………………………………...56
5.2 纖維分解酵素基因篩選………………………………..………………….…58
5.3 12-8 基因序列分析………………………………………….……………....58
5.4 10-2基因序列分析……………………………………….…………………..60
5.5 98-7基因序列分析………………………….……………………………….62
5.6 98-9基因序列分析…………………………………………………………...65
5.8 結論……………………………………………………………………….….65
6. 綜合討論…………………………………………….…….………………….…..67
6. 總結………………….……………………………………………..………….…..68
參考文獻…………………………………………………….……………………..69
附錄一…………………………………………………………………………………..74
附錄二…………………………………………………………………………………..75

1. Ethanol Vehicles Benefit Everyone. Biofuels Update 4:1-4.
2. www.banking.state.ny.us/.
3. Akhmanova, A., F. G. Voncken, H. Harhangi, K. M. Hosea, G. D. Vogels, and J. H. Hackstein. 1998. Cytosolic enzymes with a mitochondrial ancestry from the anaerobic chytrid Piromyces sp. E2. Mol Microbiol 30:1017-27.
4. Akhmanova, A., F. G. Voncken, K. M. Hosea, H. Harhangi, J. T. Keltjens, H. J. op den Camp, G. D. Vogels, and J. H. Hackstein. 1999. A hydrogenosome with pyruvate formate-lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism. Mol Microbiol 32:1103-14.
5. Alexopoulos, C. J., C. W. Min, and M. Blackwell. 1997. Phylum Chytridiomycota. Introductory Mycology 4th Ed:86-123.
6. Aylward, J. H., K. S. Gobius, G. P. Xue, G. D. Simpson, and B. P. Dalrymple. 1999. The Neocallimastix patriciarum cellulase, celD, contains three alomost identical catalytic domains with high specific activities on Avicel. Enzyme Microbiol. Technol. 24.
7. Bayer, E. A., H. Chanzy, R. Lamed, and Y. Shoham. 1998. Cellulose, cellulases and cellulosomes. Curr Opin Struct Biol 8:548-57.
8. Bayer, E. A., E. Morag, and R. Lamed. 1994. The cellulosome--a treasure-trove for biotechnology. Trends Biotechnol 12:379-86.
9. Belaich, J. P., C. Tardif, A. Belaich, and C. Gaudin. 1997. The cellulolytic system of Clostridium cellulolyticum. J Biotechnol 57:3-14.
10. Belghith, H., S. Ellouz-Chaabouni, and A. Gargouri. 2001. Biostoning of denims by Penicillium occitanis (Pol6) cellulases. J Biotechnol 89:257-62.
11. Bowman, B. H., J. W. Taylor, A. G. Brownlee, J. Lee, S. D. Lu, and T. J. White. 1992. Molecular evolution of the fungi: relationship of the Basidiomycetes, Ascomycetes, and Chytridiomycetes. Mol Biol Evol 9:285-96.
12. Breton, A., A. Bernalier, F. Bonnemoy, G. Fonty, B. Gaillard, and P. Gouet. 1989. Morphological and metabolic characterization of a new species of strictly anaerobic rumen fungus: Neocallimastix joyonii. FEMS Microbiol Lett 49:309-14.
13. Breton, A., A. Bernalier, M. Dusser, G. Fonty, B. Gaillard-Martinie, and J. Guillot. 1990. Anaeromyces mucronatus nov. gen., nov. sp. A new strictly anaerobic rumen fungus with polycentric thallus. FEMS Microbiol Lett 58:177-82.
14. Breton, A., M. Dusser, B. Gaillard-Martinie, J. Guillot, L. Millet, and G. Prensier. 1991. Piromyces rhizinflata nov. sp., a strictly anaerobic fungus from faeces of the Saharian ass: a morphological, metabolic and ultrastructural study. FEMS Microbiol Lett 66:1-8.
15. Brookman, J. L., G. Mennim, A. P. Trinci, M. K. Theodorou, and D. S. Tuckwell. 2000. Identification and characterization of anaerobic gut fungi using molecular methodologies based on ribosomal ITS1 and 185 rRNA. Microbiology 146:393-403.
16. Bryant, M. P. 1972. Commentary on the Hungate technique for culture of anaerobic bacteria. Am J Clin Nutr 25:1324-8.
17. Callaway, T. R., and S. A. Martin. 1997. Effects of cellobiose and monensin on in vitro fermentation of organic acids by mixed ruminal bacteria. J Dairy Sci 80:1126-35.
18. Chen, H., X. L. Li, D. L. Blum, and L. G. Ljungdahl. 1998. Two genes of the anaerobic fungus Orpinomyces sp. strain PC-2 encoding cellulases with endoglucanase activities may have arisen by gene duplication. FEMS Microbiol Lett 159:63-8.
19. Chen, H., X. L. Li, and L. G. Ljungdahl. 1997. Sequencing of a 1,3-1,4-beta-D-glucanase (lichenase) from the anaerobic fungus Orpinomyces strain PC-2: properties of the enzyme expressed in Escherichia coli and evidence that the gene has a bacterial origin. J Bacteriol 179:6028-34.
20. Chen, Y. C., C. Y. Chien, and R. S. Hseu. 2002. Piromyces polycephalus (Neocallimastigaceae), a new rumen fungus. Nova Hedwigia:(Accepted).
21. Coleman, G. S. 1980. Rumen ciliate protozoa. Adv Parasitol 18:121-73.
22. Coutinho, P. M., and B. Henrissat. 1999. Carbohydrate-Active Enzymes server at URL: http://afmb.cnrs-mrs.fr/~cazy/CAZY/index.html. Last updated on 2002 May 14.
23. Csiszar, E., A. Losonczi, G. Szakacs, I. Rusznak, L. Bezur, and J. Reicher. 2001. Enzymes and chelating agent in cotton pretreatment. J Biotechnol 89:271-9.
24. Dehority, B. A., and C. G. Orpin. 1997. Development of, and natural fluectuationin, rumen microbial populations. The Microbiol ecosystem. P.N. Hobson, C. S. Stewart (Eds). Blackie Academic & Professional:197-245.
25. del Campillo, E. 1999. Multiple endo-1,4-beta-D-glucanase (cellulase) genes in Arabidopsis. Curr Top Dev Biol 46:39-61.
26. Delmer, D. P., and Y. Amor. 1995. Cellulose biosynthesis. Plant Cell 7:987-1000.
27. Denman, S., G. P. Xue, and B. Patel. 1996. Characterization of a Neocallimastix patriciarum cellulase cDNA (celA) homologous to Trichoderma reesei cellobiohydrolase II. Appl Environ Microbiol 62:1889-96.
28. Dongowski, G., and S. Sembries. 2001. Effects of commercial pectolytic and cellulolytic enzyme preparations on the apple cell wall. J Agric Food Chem 49:4236-42.
29. Dore, J., and D. A. Stahl. 1991. Phylogeny of anaerobic rumen Chytridiomycetes inferred from small subunit ribosomal RNA sequence comparison. Canad. J. Bot. 69:1964-1971.
30. Endo, K., Y. Hakamada, S. Takizawa, H. Kubota, N. Sumitomo, T. Kobayashi, and S. Ito. 2001. A novel alkaline endoglucanase from an alkaliphilic Bacillus isolate: enzymatic properties, and nucleotide and deduced amino acid sequences. Appl Microbiol Biotechnol 57:109-16.
31. Faichney, G. J. 1996. Rumen physiology: the key to understand the conversion of plants into animal products. Aust. J. Agri. Res. 47:163-174.
32. Fanutti, C., T. Ponyi, G. W. Black, G. P. Hazlewood, and H. J. Gilbert. 1995. The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain. J Biol Chem 270:29314-22.
33. Fonty, G., K. N. Joblin, and A. Brownlee. 1990. Contribution of anaerobic fungi to rumen function. The rumen ecosystem. S. Hoshino, R. O. Onodera (Eds). Japan Scientific Press. Tokyo, Tapan:93-100.
34. Freelove, A. C., D. N. Bolam, P. White, G. P. Hazlewood, and H. J. Gilbert. 2001. A novel carbohydrate-binding protein is a component of the plant cell wall-degrading complex of Piromyces equi. J Biol Chem 276:43010-7.
35. GAILLARD-MARTINIE, B., A. BRETON, M. DUSSER, and V. JULLIAND. 1995. Piromyces citronii sp. nov., a strict anaerobic fungus from the equine caecum: a morphological metabolic and ultrastructural study. FEMS Microbiol. Lett. 130:321-326.
36. Goda, S. K., and N. P. Minton. 1995. A simple procedure for gel electrophoresis and northern blotting of RNA. Nucleic Acids Res 23:3357-8.
37. Gold, J. J., I. B. Heath, and T. Bauchop. 1988. Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equi gen. nov. sp. nov., assigned to the Neocallimasticaceae. BioSystems 21:403-415.
38. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis.
39. Harchand, R. K., and S. Singh. 1997. Extracellular cellulase system of a thermotolerant streptomycete: Streptomyces albaduncus. Acta Microbiol Immunol Hung 44:229-39.
40. Heath, B., T. Bauchop, and R. A. SKIPP. 1983. Assignment of the rumen anaerobe Neocallimastix frontalis to Spizellomycetale (Chytridiomycetes) on the basis of its polyflagellate ultrastructure. Canad. J. Bot. 61:295-307.
41. Ho, Y. W., and D. J. Barr. 1995. Classification of anaerobic gut fungi from herbivores with emphasis on rumen fungi from Malaysia. Mycologia 87:655-677.
42. Ho, Y. W., D. J. S. Barr, N. Abdullah, S. Jalaludin, and H. Kudo. 1993. A new species of Piromyces from the rumen of deer in Malaysia. Mycotaxon 47:285-293.
43. Ho, Y. W., D. J. S. Barr, N. Abdullah, S. Jalaludin, and H. Kudo. 1993. Piromyces spiralis, a new species of anaerobic fungus from the rumen of goat. Mycotaxon 48:59-68.
44. Ho, Y. W., and T. Bauchop. 1991. Morphology of three polycentric rumen fungi and description of a procedure for the induction of zoosporogenesis and release of zoospores in cultures. J Gen Microbiol 137:213-7.
45. Horn, G. T., and R. D. Wells. 1981. The leftward promoter of bacteriophage lambda. Structure, biological activity, and influence by adjacent regions. J Biol Chem 256:2003-9.
46. Hseu, R. S., and H. H. Wang. 1996. A study on sexuality of the Ganoderma Species. Memoirs of the College of Agriculture, Natonal Taiwan University 36:342-49.
47. Hseu, R. S., H. H. Wang, H. F. Wang, and J. M. Moncalvo. 1996. Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences. Appl Environ Microbiol 62:1354-63.
48. Imai, T., C. Boisset, M. Samejima, K. Igarashi, and J. Sugiyama. 1998. Unidirectional processive action of cellobiohydrolase Cel7A on Valonia cellulose microcrystals. FEBS Lett 432:113-6.
49. Ito, S. 1997. Alkaline cellulases from alkaliphilic Bacillus: enzymatic properties, genetics, and application to detergents. Extremophiles 1:61-6.
50. J., F. H. Degradation of plant cell wall polysaccharides by rumen bacteria. Micro-organisms in ruminant nutriention. Prins R.A. (eds). Nottingham University Press. The Nertherland.:49-68.
51. Klyosov, A. A. 1990. Trends in biochemistry and enzymology of cellulose degradation. Biochemistry 29:10577-85.
52. Leschine, S. B. 1995. Cellulose degradation in anaerobic environments. Annu Rev Microbiol 49:399-426.
53. Li, J., B. Heath, and T. Bachup. 1992. Piromyces mae and Piromyces dombonica, two new species of uniflagellate anaerobic chytridmycetes fungi from hind-gut of horse and elephans. Can. J. Bot. 68:1021-1033.
54. Li, J., B. Heath, and L. Packer. 1992. The phylogenetic relationships of anaerobic chytridiomycetous gut fungi (Neocallimastigaceae) and the Chytridiomycota. I. Cladistic analysis of rRNA sequences. Canad. J. Bot. 70:1738-1740.
55. Lowe, S. E., G. G. Griffith, A. Milne, M. K. Theodorou, and T. A. P. J. 1987. The life cycle and growth kinetics of an anaerobic rumen fungus. J. Gen. Microbiol. 133:1815-27.
56. Martin, S. A. 1998. Manipulation of ruminal fermentation with organic acids: a review. J Anim Sci 76:3123-32.
57. Marvin-Sikkema, F. D., M. N. Kraak, M. Veenhuis, J. C. Gottschal, and R. A. Prins. 1993. The hydrogenosomal enzyme hydrogenase from the anaerobic fungus Neocallimastix sp. L2 is recognized by antibodies, directed against the C-terminal microbody protein targeting signal SKL. Eur J Cell Biol 61:86-91.
58. Marvin-Sikkema, F. D., T. M. Pedro Gomes, J. P. Grivet, J. C. Gottschal, and R. A. Prins. 1993. Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2. Arch Microbiol 160:388-96.
59. Muthumeenakshi, S., P. R. Mills, A. E. Brown, and D. A. Seaby. 1994. Intraspecific molecular variation among Trichoderma harzianum isolates colonizing mushroom compost in the British Isles. Microbiology 140:769-77.
60. Nicol, F., I. His, A. Jauneau, S. Vernhettes, H. Canut, and H. Hofte. 1998. A plasma membrane-bound putative endo-1,4-beta-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis. Embo J 17:5563-76.
61. Nitsan, Z., S. Mokady, and A. Sukenik. 1999. Enrichment of poultry products with omega3 fatty acids by dietary supplementation with the alga Nannochloropsis and mantur oil. J Agric Food Chem 47:5127-32.
62. Ohara, H., J. Noguchi, S. Karita, T. Kimura, K. Sakka, and K. Ohmiya. 2000. Sequence of egV and properties of EgV, a Ruminococcus albus endoglucanase containing a dockerin domain. Biosci Biotechnol Biochem 64:80-8.
63. Orpin, C. G. 1977. The rumen flagellate Piromonas communis: Its life history and invasion of plant material in the rumen. J. Gen. Microbiol. 99:107-117.
64. Orpin, C. G. 1975. Studies on the rumen flagellate Neocallimastix frontalis. J Gen Microbiol 91:249-62.
65. Orpin, C. G. 1976. Studies on the rumen flagellate Sphaeromonas communis. J Gen Microbiol 94:270-80.
66. Ouhida, I., J. F. Perez, J. Gasa, and F. Puchal. 2000. Enzymes (beta-glucanase and arabinoxylanase) and/or sepiolite supplementation and the nutritive value of maize-barley-wheat based diets for broiler chickens. Br Poult Sci 41:617-24.
67. Overmeyer, C., uuml, S. nnemann, C. von Wallbrunn, and F. Meinhardt. 1996. Genetic Variability Among Isolates and Sexual Offspring of the Plant Pathogenic Fungus Calonectria morganii on the Basis of Random Amplification of Polymorphic DNA (RAPD) and Restriction Fragment Length Polymorphism (RFLP). Curr Microbiol 33:249-55.
68. Ozcan, N., C. Cunningham, and W. J. Harris. 1996. Cloning of a cellulase gene from the rumen anaerobe Fibrobacter succinogenes SD35 and partial characterization of the gene product. Lett Appl Microbiol 22:85-9.
69. Ozkose, E., B. J. Thomas, D. R. Davies, G. W. Griffith, and M. K. Theodorou. 2001. Cyllamyces aberensis gen.nov. sp.nov., a new anaerobic gut fungus with branched sporangiophores isolated from cattle. Can. J. Bot. 79:666-673.
70. PANWAR, R., SINGH U. S., and S. R. S. 1987. Fluorescent staining of nuclei in filamentous fungi. Stain Technol 62:205-208.
71. Percudani, R., A. Trevisi, A. Zambonelli, and S. Ottonello. 1999. Molecular phylogeny of truffles (Pezizales: Terfeziaceae, Tuberaceae) derived from nuclear rDNA sequence analysis. Mol Phylogenet Evol 13:169-80.
72. Qiu, X., B. Selinger, L. Yanke, and K. Cheng. 2000. Isolation and analysis of two cellulase cDNAs from Orpinomyces joyonii. Gene 245:119-26.
73. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
74. Straatsma, G., and R. A. Samson. 1993. Taxonomy of Scytalidium thermophilum, an important thermophilic fungus in mushroom compost. Mycological Res. 97:321-328.
75. Theodorou, M. K., G. Mennim, D. R. Davies, W. Y. Zhu, A. P. Trinci, and J. L. Brookman. 1996. Anaerobic fungi in the digestive tract of mammalians herbivores and their potential for exploitation. Proc. Nutr. Soci. 55:913-926.
76. Tomme, P., H. Van Tilbeurgh, G. Pettersson, J. Van Damme, J. Vandekerckhove, J. Knowles, T. Teeri, and M. Claeyssens. 1988. Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis. Eur J Biochem 170:575-81.
77. Trinci, A. P., D. R. Davies, K. Gull, M. I. Lawrance, and M. K. Theodorou. 1994. Anaerobic fungi in herbivorous animals. Mycol. Res. 98:129-152.
78. van der Giezen, M., K. A. Sjollema, R. R. Artz, W. Alkema, and R. A. Prins. 1997. Hydrogenosomes in the anaerobic fungus Neocallimastix frontalis have a double membrane but lack an associated organelle genome. FEBS Lett 408:147-50.
79. Van Rensburg, P., W. H. Van Zyl, and I. S. Pretorius. 1998. Engineering yeast for efficient cellulose degradation. Yeast 14:67-76.
80. van Solingen, P., D. Meijer, W. A. van der Kleij, C. Barnett, R. Bolle, S. D. Power, and B. E. Jones. 2001. Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake. Extremophiles 5:333-41.
81. Webb, J., and M. K. Theodorou. 1991. Neocallimastix hurleyensis sp. nov., an anaerobic fungi from the ovine rumen. Can. J. Bot. 69.
82. Williams, A. M., and C. G. Orpin. 1987. Polysaccharide-degrading enzymes formed by three species of rumen anaerobic fungi grown on a range of carbondrate substrates. Can. J. Microbiol. 33:418-426.
83. Wood, T. M. 1992. Fungal cellulases. Biochem Soc Trans 20:46-53.
84. Wubah, D. A., M. S. Fuller, and D. E. Akin. 1991. Resistant body formation in Neocallimastix sp., an anerobic fungus from the rumen of a cow. Mycologia 81:40-47.
85. Xue, G. P., K. S. Gobius, and C. G. Orpin. 1992. A novel polysaccharide hydrolase cDNA (celD) from Neocallimastix patriciarum encoding three multi-functional catalytic domains with high endoglucanase, cellobiohydrolase and xylanase activities. J Gen Microbiol 138 ( Pt 11):2397-403.
86. Yarlett, N., C. G. Orpin, E. A. Munn, N. C. Yarlett, and C. A. Greenwood. 1986. Hydrogenosomes in the rumen fungus Neocallimastix patriciarum. Biochem J 236:729-39.
87. Zuo, J., Q. W. Niu, N. Nishizawa, Y. Wu, B. Kost, and N. H. Chua. 2000. KORRIGAN, an Arabidopsis endo-1,4-beta-glucanase, localizes to the cell plate by polarized targeting and is essential for cytokinesis. Plant Cell 12:1137-52.