臺灣博碩士論文加值系統

Trichoderma harzianum是工業上之纖維水解酵素主要微生物來源之一可將纖維素轉變成葡萄糖，因此對於在二代生物酒精生產上，纖維水解酵素是相當重要的酵素。不僅如此，T. harzianum也產生多功能生物活性的二級代謝物，如蒽醌。經由各種環境因素能影響蛋白質和二級代謝產物生產，而光源便是其中一個。在本次研究中，我們使用客製化波長的光源，red640、green520、blue465和由三原色組成的白光，加上黑暗來處理生長中的T. harzianum，並分析蒽醌類化合物的生產和胞外纖維水解酵素活性以及使用聚合酶鏈鎖反應來檢測纖維水解酵素的RNA不同之處。結果顯示纖維水解酵素的活性在液體培養基中，經由藍光照射培養後擁有最好活性。蒽醌類化合物的生產經過藍光的照射下有顯著的增加，而在其他光源條件下則沒有顯著的差異。在RNA方面我們發現β-葡萄糖苷酶（bgl2），纖維二糖水解酶（cbh1），β-葡聚糖(egl)的基因經由光源來調控。因此我們得出結論，認為不同波長的光源能調控纖維水解酵素的基因表現，也能刺激二級代謝物和蛋白質的產生。

Trichoderma harzianum is a major industrial microorganism source of the cellulases which degraded cellulose into glucose, in the end, so that are the most important enzymes in second generation bio-ethanol production. Meanwhile, T. harzianum also produce versatile bio-active secondary compounds, i.e. anthraquinones. The production of both proteins and secondary metabolites are modulated by various environmental factors, such as light etc. In this study, customer designed with define wavelengths light sources, red640, green520, blue465, bright light, plus dark treated T. harzianum were scrutinized by the anthraquinones production by determiningε436 =11.8 and extracellular cellulase by enzymatic activity as well as detection of cellulase RNA by PCR. The result show cellulase activity in the liquid minimal medium with 1 % cellulose or Mandels-Reese medium with 1 % CMC, the highest is blue light and the lowest is red light. In anthraquinones production, the significant increase is illumination under blue light, and is no significant difference exposed to other lights. We can find β-glucosidase (bgl2), cellobiohydrolases (cbh1) and endo-1,4-beta-glucanas (egl) genes regulation dependent light source in both medium. We conclude different wavelengths light can induce regulate gene of cellulases and stimulate secondary metabolism production.

致謝 I
摘要 II
Abstract III
Contents IV
1. Introduction 1
1.1 Introduction to Fungi 2
1.2 Classification of Fungi 4
1.3 Introduction to Trichoderma 7
1.4 Biofuels 8
1.5 Cellulose and cellulases 11
1.6 Secondary metabolites of anthraquinones 15
1.7 Literature review of light affect fungi 15
2. Purpose 17
3. Materials and methods 19
3.1 Strain 19
3.2 Culture conditions 19
3.3 Monochrome wavelength light sources 21
3.4 Analytical assays 21
3.5 Total RNA Isolation 23
3.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 24
3.7 Primers Design 25
3.8 Polymerase Chain Reaction (PCR) 25
3.9 SDS-PAGE and Zymogram 26
4. Results 29
4.1 Cultivation of T. harzianum strain ETS 323 on PDA 29
4.2 T. harzianum strain ETS 323 cultivation on plate of Minimal Medium and Mandels-Reese Medium 29
4.3 Plate assay for T. harzianum strain ETS 323 production cellulases 30
4.4 T. harzianum strain ETS 323 cultivation in liquid medium 30
4.5 Analytical assay of liquid medium 31
4.6 SDS-PAGE analysis and Enzyme activity stain of extracellular fluid 33
4.7 Expression of cellulase genes by light affection 35
5. Discussion 37
6. Conclusion 41
7. Reference 43
Figure of Results 51
8. Appendix 75

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