臺灣杉Tc-βFar/Lin、Tc-Lin、Tc-Zin/βOci、Tc-βFar/Ger、Tc-αFar/Ger萜類合成酶之基因選殖、活性分析與大腸桿菌生產系統之建立

Gene Cloning and Characterization of Tc-βFar/Lin, Tc-Lin, Tc-Zin/βOci, Tc-βFar/Ger and Tc-αFar/Ger Terpene Synthases from Taiwania cryptomerioides and Establishment of Production System in E. coli

徐子凡(Tzu-Fang Hsu)

指導教授 ： 曲芳華

國立臺灣大學/生物資源暨農學院/森林環境暨資源學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703224

摘要

臺灣杉（Taiwania cryptomerioides Hayata）是臺灣特有種植物，因其木質特性與含有精油成分中的萜類化合物，為臺灣相當重要的針葉樹種。過去已有相當多的研究是針對臺灣杉生成產物的辨認及應用，對生合成的途徑的研究相對較少；本研究的目標是從臺灣杉選殖出重要功能的基因，瞭解各基因的生合成功能，且針對各基因做演化與生產相關的探討，透過氣相層析質譜儀分析，依各基因之主產物進行命名，依序為：Tc-βFar/Lin、Tc-Lin、Tc-Zin/βOci、Tc-βFar/Ger、Tc-αFar/Ger、Tc-Lin為單一產物的單萜合成酶，其餘四者皆屬於倍半萜合成酶，除Tc-Zin/βOci能夠生產出單環的zingiberene外，Tc-βFar/Lin、Tc-βFar/Ger、Tc-αFar/Ger所生成的單萜產物與倍半萜產物皆為線型萜類；在臺灣杉各部位的反轉錄聚合酶連鎖反應（RT-PCR）結果中，五個基因皆會在幼葉與雌毬果中表現，唯一亦會在木材中表現的基因為Tc-βFar/Ger。從親緣樹關係圖的分析結果中，發現Tc-βFar/Lin與臺灣杉中目前已知且親緣較近的倍半萜合成酶基因都有運輸胜肽遺失的現象，表示這群基因在演化的過程中可能先經歷了γ-domain遺失，最後才造成運輸生肽的遺失。透過大腸桿菌代謝工程生產系統，Tc-Zin/βOci能夠大量產生出zingiberene，Tc-βFar/Ger能夠產生出大量的β-farnesene，顯示未來可利用微生物系統來大量生產有效成分。

關鍵字

金合歡烯 ； 芳樟醇 ； 臺灣杉 ； 萜類化合物 ； 揮發性萜類合成酶 ； 薑烯

並列摘要

Taiwania cryptomerioides Hayata is an endemic species in Taiwan. It is an important coniferous species in Taiwan, because of its timber properties and its terpenoids in the essential oil. Most previous researches about the products and applications of T. cryptomerioides Hayata have been discussed, however, the biosynthesis pathway of T. cryptomerioides Hayata has rarely been discussed in the past. This study focuses on cloning the genes and their characteristic. Through using gas chromatography mass spectrometry (GC/MS) analysis, we identified the product of the five terpene synthases. According to the main product that synthase synthesize, they are called Tc-βFar/Lin, Tc-Lin, Tc-Zin/βOci, Tc-βFar/Ger, and Tc-αFar/Ger. Tc-Lin is a monoterpene synthase, the others are sesquiterpene synthases. Except Tc-Zin/βOci synthesizes the single ring zingiberene, all of Tc-βFar/Lin, Tc-βFar/Ger, and Tc-αFar/Ger synthesize the linear monoterpenes or sesquiterpenes; the results of reverse transcription polymerase chain reaction (RT-PCR) demonstrate that these five genes can express in the young leaf and female cone, and only Tc-βFar/Ger can also express in the wood. From the analysis of the phylogenetic tree, we can find that Tc-βFar/Lin and other closer genes of sesquiterpene synthases which are known in Taiwania are all lost the trainsit peptides. This result represents that these genes may have the process which has lost γ-domain at first and lost trainsit peptides finally. Using heterologous expression of diverse synthases in the E. coli, Tc-Zin/βOci can produce large amounts of zingiberene, and Tc-βFar/Ger can produce large amounts of β-farnesene. Thus, large amounts of these sesquiterpenes production system by using microorganism were established in this study.

並列關鍵字

β-farnesene ； linalool ； Taiwania cryptomerioides Hayata ； terpenoids ； volatile terpene synthase ； zingiberene

參考文獻

劉璧綾（2015）五個臺灣杉揮發性萜類合成酶基因選殖與性狀分析。國立臺灣大學生農學院森林環境暨資源學系碩士論文：1-97。

鄭森松、莊閔傑、林群雅、張上鎮、王亞男（2010）臺灣杉不同部位精油及抽出物抗褐根病菌之評估。臺灣大學生物資源暨農學院實驗林研究報告，24（2）：85-95。

Adriana, G.G., Mairim, R.S., Lucindo, J.Q.J. (2014) Terpenes and derivatives as a new perspective for pain treatment: a patent review. Expert Opinion on Theraputic Patents 24(3), 243-265.

Aharoni, A., Giri, A.P., Verstappen, F.W.A., Bertea, C.M., Sevenier, R., Sun, Z., Jongsma, M.A., Schwab, W., Bouwmeester, H.J. (2004) Gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species. The Plant Cell 16, 3110-3131.

Boachon, B., Junker, R.R., Miesch, L., Bassard, J.E., Höfer, R., Caillieaudeaux, R., Seidel, D.E., Lesot, A., Heinrich, C., Ginglinger, J.F., Allouche, L. (2015) CYP76C1 (cytochrome P450)-mediated linalool metabolism and the formation of volatile and soluble linalool oxides in Arabidopsis flowers: a strategy for defense against floral antagonists. The Plant Cell 27, 2972-2990.