聚合醣分解為可發酵的單糖，是生質酒精發酵的首要步驟。為了增進經濟效益，減少能源作物與人類爭糧的爭議，酒精生產原料目前著眼在含量多、取得成本低、利用率低的植物纖維素上。本論文的目的在找出有效分解纖維素的酵素，以提供生產酒精的原料。從纖維素之結構與其在酵素作用下之分解過程來看，酵素至少包含三種主要之纖維酶：內切型纖維素水解酶 (Endo-β-1,4-gulcanase, E.C. 3. 2. 1. 4.)主要作用於纖維素分子內部，隨機水解其中之β-1,4 glucosidic bonds，並將長鏈纖維素分子切斷，產生大量具有還原性末端之小分子纖維素。外切型纖維素水解酶 (Exo-β-1,4-gulcanase, cellobiohydrolases, E.C. 3.2.1.91)將內切型纖維素水解酶作用產生的還原端 (reducing ends) 或非還端(non-reducing ends) 水解成纖維雙糖 (cellubiose)，最後由β－葡萄糖苷酶 (β-glucosidase, E.C. 3.2.1.21)將纖維雙醣水解成葡萄糖。數種微生物被研究能夠藉由內切型纖維素水解酶和β－葡萄糖苷酶來分解纖維素，但是在植物中內切型纖維素水解酶的主要功能和特徵仍然不暸解，只有少數研究討論到其涉及細胞壁代謝的功能。阿拉伯芥 (Arabidopsis) 內切型纖維素水解酶家族九25個基因中，具代表性的12個被轉殖入Pichia pastoris中表現蛋白質，期望能產生具有活性的內切型纖維素水解酶，應用在生質能源上。但是內切型纖維素水解酶活性測試結果顯示重組蛋白缺乏酵素的活性，進一步利用dot blot偵測蛋白質的表現，發現有些重組蛋白有被分泌到細胞外，有些則被留在細胞內。探討缺乏酵素活性的可能原因為蛋白質產生不正確折疊或糖基化。未來，我們可以測試其他的生物反應器。或者利用基因工程修改阿拉伯芥的內切型纖維素水解酶會是另一種生產具功能的纖維素酶與具經濟效率的方法，能夠適應工業規模酒精生成。另一方面，我們想了解阿拉伯芥內切型纖維素水解酶家族九在不同組織間的表現情形。利用半定量PCR方法測試12個選定之阿拉伯芥內切型纖維素水解酶，At1g75680基因的表現，在所有組織中皆可偵測到，At4g39000和At3g43860專一表現在花和花序中，而At1g65610在根部和嫩芽的表現量較多。其它基因在不同組織間則沒有顯著之表現差異。

Generation of alcohol for biofuels from fermentation of sugar or starch has several economic disadvantages such as high cost of sugar processing and land usage competing with staple food. The solution may reside in hydrolysis of cellulose from crop waste such as stalks of rice and corn or non-crop plants such as weeds or wood. Our goal is to identify cellulases that can degrade cellulosic biomass more efficiently. Studies of microbial Family 9 glycoside hydrolase (GH9) proteins, including both endo-glucanases (EC 3.2.1.4) and cellobiohydrolases (EC 3.2.1.91), have shown that they function through an inverting mechanism to cleave the 1, 4-β-glucosidic bond between two unsubstituted Glc units. The main function of plant glycoside hydrolases are involved in polysaccharide metabolism of cell wall during cell growth. Twelve Arabidopsis thaliana (Columbia) endo-1,4-β-glucanases that belong to the GH9, were cloned and expressed in Pichia pastoris in order to produce cellulases to facilitate efficient bio-alcohol production. The recombinant proteins do not show in vitro endo-1, 4-β-glucanase activity, but we can detect the recombinant proteins expression in supernatant or in pellet. The lack of enzymatic activity from recombinant proteins is probably due to improper folding or glycosylation, or fast degradation resulted from the above reasons. Other bioreactor will be tested in the future. Genetic engineering to modify Arabidopsis thaliana (Columbia) endo-β-1, 4-glucanases is another approach to produce functional cellulases with economic efficiency that can be adapted to industrial scale for alcohol generation. On the other hand, we use semi-quantitative PCR method to study the Arabidopsis GH9 genes expression level in different tissue. At4g39000 and At3g43860 were found only in flowers and inflorescence, and At1g65610 expression in roots and shoots of the amount of more. Other genes in different tissues, was no found significant difference.

Acknowledgements.................................................i
摘要.............................................................................ii
Abstract................................................. .....................iii
Contents............................................... .....................iv
Index of tables..................................... .....................vi
Index of figures.................................... .....................vii
Chapter 1: Introduction...........................................1
World wide demands of Biofuels.........................1
Purpose................................................ ....................3
Composition of cell walls and cellulose structure....................................................................3
Structural and functional analysis of cellulases.................................................................4
Functional roles of cellulase in plant...................6
Gene expression pattern of Arabidopsis GH9...7
Phylogenetic analysis and modular structure
of GH9 proteins of Arabidopsis............................8
The Pichia pastoris protein expression system.......................................................................8
Chapter 2：Materials and methods.....................11
Plant materials and growth conditions................11
Phylogeny analysis..................................................11
RNA extraction and reverse transcription............11
Cloning twelve Arabidopsis GH9 genes.............12
Transformation into P. pastoris by electroporation.........................................................13
Identification of positive Pichia recombinants...13
Expression of recombinant proteins from
P. pastoris strains....................................................14
Determination of endo-glucanase activity...........14
Semi- quantitative PCR..........................................15
Chapter 3：Results................................................16
Phylogenetic analysis of family 9 glucanases...16
Expression of recombinant proteins....................16
Determination of endo-glucanase activity...........18
Detection of recombinant proteins.......................18
Expression patterns of Arabidopsis GH9 genes
in different tissues...................................................19
Chapter 4：Discussion..........................................20
Expression of twelve Arabidopsis GH9 proteins
in Pichia Pastoris.....................................................20
Factors affecting expression level........................21
Factors affecting secretion of recombinant
proteins......................................................................22
Expression patterns of Arabidopsis GH9 genes
in different tissues...................................................23
Chapter 5：Conclusions.......................................24
Chapter 6：Tables and figures............................25
Appendix A. Features of expression vector
pPICZαA.....................................................................56
References...............................................................57

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