臺灣博碩士論文加值系統

臺灣杉（Taiwania cryptomerioides Hayata）為臺灣原生種，含有豐富的萜類化合物，藉由研究萜類合成酶能更瞭解萜類化合物的生合成途徑。本研究利用聚合酶連鎖反應（polymerase chain reaction, PCR）與cDNA末端快速增幅（rapid amplification of cDNA ends, RACE）等技術，自臺灣杉選殖出Tc-Lon、Tc-Ced/Ger、Tc-αCub/Ger、Tc-Ger1和Tc-Ger2萜類合成酶基因。經功能分析，Tc-Lon可合成longifolene，Tc-Ced/Ger和Tc-αCub/Ger則具有雙功能性，能同時合成倍半萜類產物和單萜類產物。Tc-Ced/Ger之主要產物為倍半萜cedrol與單萜geraniol，Tc-αCub/Ger之主要產物為倍半萜α-cubebene與單萜geraniol。分析胺基酸序列的差異性，發現Tc-Ger1缺失了DDXXD功能區塊（motif），因此利用點突變實驗將Tc-Ger1的胺基酸轉換為典型的DDXXD motif序列，其兩個突變株（mutant）A359D和A359D/H363D也如野生型（wild type）的功能一樣能產生geraniol，但GC-MS分析偵測訊號減弱。Tc-Ger1和Tc-Ger2的產物geraniol構造簡單，因此推測可能是受到大腸桿菌中的磷酸酶影響而生成，因此設計實驗證實Tc-Ger1和Tc-Ger2的確具有催化產生geraniol的能力。本研究也成功利用代謝工程技術建立萜類化合物的生合成系統，期盼未來能提供大量生產。

Taiwania (Taiwania cryptomerioides Hayata) is a conifer species native to Taiwan, which is known for several bioactive secondary metabolites from it. Studying terpene synthases is the key way to understand the pathway of terpene biosynthesis. In this study, Tc-Lon, Tc-Ced/Ger, Tc-αCub/Ger, Tc-Ger1 and Tc-Ger2 were isolated from Taiwania by using polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. Tc-Lon is a sequiterpene synthase and it can synthesize longifolene. Tc-Ced/Ger and Tc-αCub/Ger are bifunctional terpene synthases, which can produce monoterpenes and sesquiterpenes simultaneously. Tc-Ced/Ger can produce sesquiterpene cedrol and monoterpene geraniol. Tc-αCub/Ger can produce sesquiterpene α-cubebene and monoterpene geraniol. DDXXD motif is absent in Tc-Ger1, so the two mutants A359D and A359D/H363D were made to clarify these two residues how them work. Interestingly, A359D and A359D/H363D have the same function like the wild type of Tc-Ger1, and it was confirmed that Tc-Ger1 and Tc-Ger2 can produce geraniol. In this study, terpene synthesis pathways were built by using metabolic engineering. Using the strategy of the metabolic engineering for terpene overproduction should also work in other synthetic pathways in the future.

目錄
誌謝 i
中文摘要 ii
英文摘要 iii
目錄 iv
圖目錄 vii
表目錄 x
一、前言 1
二、文獻探討 3
2.1萜類合成酶種類與生合成途徑 3
2.2萜類合成酶之分群 5
2.3萜類合成酶之反應功能區域與蛋白質結構 7
2.4單萜類合成酶合成途徑 9
2.5倍半萜類合成酶合成途徑 11
2.6利用代謝工程技術製造大量萜類化合物 13
2.7萜類化合物之生物活性及其應用 16
三、試驗材料與方法 17
3.1實驗試材 17
3.2臺灣杉RNA萃取及cDNA之合成 17
3.3臺灣杉揮發性萜類合成酶基因之選殖 17
3.4蛋白質表現質體之構築（construction） 23
3.5質體運輸胜肽及蛋白質分子量（MW）、等電點（pI）之預測 26
3.6重組蛋白質表現 27
3.7重組蛋白質純化 29
3.8目標蛋白質活性反應 31
3.9目標蛋白質之萜類產物分析（GC-MS） 32
3.10裸子植物萜類合成酶親源關系樹狀圖（phylogenetic tree）分析 33
3.11臺灣杉萜類合成酶基因在各組織表現情況 33
3.12蛋白質結構模擬 34
3.13點突變試驗 35
3.14大腸桿菌代謝工程技術in vivo試驗 36
3.15小圓葉菸草in vivo試驗 38
3.16臺灣杉萜類合成酶基因之內含子與外顯子 40
四、結果 41
4.1臺灣杉萜類合成酶基因之選殖 41
4.2臺灣杉萜類合成酶親緣關係樹狀圖分析 46
4.3臺灣杉萜類合成酶表現質體之建構 47
4.4五個萜類合成酶於大腸桿菌之表現－Western blot分析 49
4.5五個萜類合成酶基因的純化分析 53
4.6臺灣杉萜類合成酶重組蛋白質活性測定與產物分析（GC/MS） 57
4.7臺灣杉各組織的基因表現量 68
4.8利用代謝工程技術製造大量萜類化合物 70
4.9小圓葉菸草表達萜類合成酶蛋白之實驗 72
五、討論 76
5.1萜類合成酶之序列分析 76
5.2萜類合成酶之親緣關係 78
5.3萜類合成酶之產物探討 80
5.4萜類合成酶之各組織基因表現量 86
5.5 Geraniol的合成探討 89
5.6代謝工程技術製造大量萜類化合物 94
5.7小圓葉菸草表達萜類合成酶蛋白之實驗困難 94
5.8臺灣杉萜類合成酶產物之生物活性與應用 96
六、結論 97
參考文獻 98
附錄 106

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