臺灣博碩士論文加值系統

臺灣杉（Taiwania cryptomerioides Hayata）為臺灣特有種植物，其精油成分中的萜類化合物是目前研究的一大重點。過去臺灣杉相關之研究多為萜類化合物應用與開發，鮮少著眼於萜類合成酶之生合成機制，為了更了解臺灣杉之揮發性萜類（volatile terpenoids）的生合成，本研究以揮發性萜類合成酶作為研究目標。本研究從本實驗室所建構的臺灣杉次世代定序（next generation sequencing, NGS）資料庫中，獲得五個萜類合成酶的部分基因，再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）的方式獲得基因全長，經由氣相層析質譜儀（GC-MS）的分析，分析出五個萜類合成酶的產物，並依其產物將此五個基因分別命名為Tc-αPin、Tc-Pre/Oci、Tc-βAOL、Tc-COL與Tc-GerD，其中Tc-αPin為單萜合成酶基因，其餘四者則為倍半萜合成酶基因。Tc-αPin、Tc-βAOL與Tc-GerD為單一產物之揮發性萜類合成酶，可分別生合成出單萜α-蒎烯（α-pinene）、倍半萜β-atlantol與倍半萜大根香葉烯 D（germacrene D）；另外，Tc-Pre/Oci與Tc-COL則皆為雙功能萜類合成酶（bifunctional terpenoid synthases），能同時生合成出單萜產物與倍半萜產物。Tc-Pre/Oci之主要產物為倍半萜premnaspirodiene與單萜順式-β-羅勒烯（cis-β-ocimene)，而Tc-COL之主要產物為蓽澄茄醇（cubebol）。在不同組織部位的基因表現量結果發現，五個基因皆在未成熟毬果中表現最多，多數基因在花與葉部中皆有表現，但在根部與木材中則只有Tc-αPin表現。此外，透過甲基茉莉酸（methyl jasmonate, MeJA）創傷處理實驗證實，五個基因皆受到MeJA誘導而使基因表現量增加。

Taiwania cryptomerioides Hayata is an endemic species in Taiwan, and the terpenoidsofTaiwania cryptomerioides Hayata are major focus of current researches.Most previous studies about Taiwania cryptomerioides Hayatahave dealt withthe application and development of terpenoids, rarely focusing on the biosynthesis mechanisms of terpenoid synthases.In order to better understand the biosynthesis of volatile terpenoids of Taiwania cryptomerioides Hayata, this study chose volatile terpenoid synthases as the research target.In this study, weobtain five partial terpenoid synthase genes from the database of next generation sequencing（NGS）of volatile terpenoid synthases in Taiwania cryptomerioides Hayatafrom our lab. Then using polymerse chain reaction（PCR）and Rapid amplification of cDNA ends（RACE）methods, we successfully cloned five volatile terpenoid synthases from Taiwania cryptomerioides Hayata.Through using gas chromatography mass spectrometry（GC-MS）analysis,we find out the products of five terpenoid synthases. According to the compounds that synthases synthesize, they are called asTc-αPin,Tc-Pre/Oci, Tc-βAOL, Tc-COL andTc-GerD,respectively.Tc-αPin,Tc-βAOL and Tc-GerDare single product terpenoid synthases that have only one product, they can synthesizes monoterpenoidα-pinene, sesquiterpenoidβ-atlantol and sesquiterpenoidgermacrene D, respectively.In addition, Tc-COL andTc-Pre/Ociare bothbifunctionalterpenoid synthases, which can producemonoterpenoids and sesquiterpenoid simultaneously.The major product ofTc-Pre/Oci is sesquiterpenoid premnaspirodiene and monoterpenoid cis-β-Ocimene, while the major product of Tc-COL is sesquiterpenoid cubebol.We also report differential expression of genes involved in terpenoid metabolism in several tissues and in response to methyl jasmonate（MeJA）.The expression of fiveterpenoidsynthase geneswere found in the immaturalcones, and all these genes were shown in flowers or leaves, but in the root and wood we only found the Tc-αPin expression.Furthermore,transcript levels of five terpenoidsynthase genes were induced in leaves in response to MeJA.

誌謝 i
中文摘要 ii
Abstract iii
目錄 v
圖目錄 viii
表目錄 xi
第一章、前言 1
第二章、前人研究 3
2.1 萜類合成酶種類 3
2.2 萜類合成酶之分群 3
2.3 揮發性萜類合成酶基因之內含子分布 4
2.4 揮發性萜類合成酶之生合成途徑（Biosynthesis pathway） 6
2.5 揮發性萜類合成酶之胺基酸序列與反應功能區域（Catalysis-initiating motifs） 8
2.6 揮發性萜類合成酶之生合成調控 10
2.6.1 誘導合成 10
2.6.2 器官發育調控 10
2.7 揮發性萜類之應用 11
第三章、材料與方法 13
3.1 實驗試材 13
3.2 臺灣杉總量RNA之萃取 13
3.3 臺灣杉揮發性萜類合成酶基因之選殖 14
3.4 目標蛋白質結構預測 17
3.5 萜類合成酶基因之內含子與外顯子剪接點判定 18
3.6 質體運輸胜肽及蛋白質分子量（MW）、等電點（pI）之預測 19
3.7 蛋白質表現質體之建構（Construction） 20
3.8 西方墨點法（Western Blotting） 21
3.9 重組蛋白質誘導大量表現 22
3.10 目標蛋白質之純化 23
3.11 目標蛋白質活性反應 24
3.11.1 以pET-21a（+）為載體之粗蛋白（crude protein）反應 24
3.11.2 純化蛋白質（purified protein）反應 25
3.12 目標蛋白質萜類產物分析-氣相層析質譜儀（GC-MS） 26
3.13 萜類合成酶序列比對與親緣關係樹狀圖分析 26
3.14 臺灣杉各組織表現量 27
3.15 甲基茉莉酸（Methyl jasmonate, MeJA）處理 28
第四章、結果 29
4.1 臺灣杉揮發性萜類合成酶基因之選殖 29
4.2臺灣杉揮發性萜類合成酶基因序列比對 30
4.3 臺灣杉揮發性萜類合成酶蛋白質結構預測 33
4.4臺灣杉揮發性萜類合成酶基因內含子分布情形 36
4.5 臺灣杉揮發性萜類合成酶表現質體之建構 38
4.6 表現臺灣杉揮發性萜類合成酶重組蛋白質 40
4.7 臺灣杉揮發性萜類合成酶重組蛋白質活性測定與產物分析 48
4.8臺灣杉揮發性萜類合成酶親緣關係樹狀圖分析 58
4.9 臺灣杉各組織的基因表現量 60
4.10 臺灣杉幼葉經MeJA處理後之mRNA表現量 62
第五章、討論 64
5.1 臺灣杉揮發性萜類合成酶之序列與蛋白質特性 64
5.2 臺灣杉揮發性萜類合成酶之親緣關係與演化分析 66
5.3 臺灣杉揮發性萜類合成酶可能之產物生合成機制 69
5.4 臺灣杉揮發性萜類合成酶之各組織基因表現量 73
5.5 臺灣杉揮發性萜類合成酶經MeJA處理後之mRNA表現量 75
5.6 臺灣杉揮發性萜類合成酶產物之生物活性與應用 77
第六章、結論 78
參考文獻 79
附錄 92
附錄表1、片段增幅引子序列 92
附錄表2、3’RACE引子序列 92
附錄表3、5’RACE引子序列 93
附錄表4、全長引子序列 93
附錄表5、蛋白質表現質體建構引子序列 94
附錄表6、RT-PCR（各組織mRNA表現量與MeJA處理）引子序列 95
附錄表7、親緣關係樹狀圖分析所使用之不同植物萜類合成酶胺基酸序列縮寫及登錄碼對照表 96

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