- 學位論文
臺灣杉倍半萜合成酶基因之選殖與表現
Cloning and Expression of a Sesquiterpene Synthase Gene from Taiwania cryptomerioides Hayata
摘要
臺灣杉 (Taiwania cryptomerioides Hayata) 為臺灣原生針葉樹種，近年來對其二次代謝萃取產物成分之生物活性有諸多研究；若能從分子生物學角度了解其生合成酵素，將使人為調控代謝產物成分的可能性增加。本研究鎖定萜類產物中之倍半萜，搜尋資料庫中針葉樹倍半萜類合成酶基因序列，設計簡併性引子對，利用反轉錄聚合酶連鎖反應技術，自成熟樹木之葉組織釣取到一具有典型萜類合成酶保守序列之基因，再經由5’及3’端快速增幅cDNA端點 (rapid amplification of cDNA ends, RACE) 選殖，共獲得基因全長1791 bp，轉譯後蛋白質分子量為70.2 kDa。此基因主要於幼葉、雌花及毬果中表現；另外，將葉部經水楊酸浸泡處理後，基因表現量有增加的現象。將此基因構築於蛋白質表現之載體中，轉入大腸桿菌 (Escherichia coli) 表現，發現此蛋白質可催化倍半萜之前驅物，生成倍半萜α-Gurjunene，故將此基因命名為Tc-Gur。為了解該基因在植物的表現情形，以基因轉殖方式表現，發現阿拉伯芥同樣會生成α-Gurjunene，而在臺灣杉毛狀根則生合成不同的倍半萜產物α-Muurolene。
並列摘要
Taiwania (Taiwania cryptomerioides Hayata) is a native conifer species of Taiwan. Many studies have demonstrated the bioactivities of secondary metabolites extracted from Taiwania in recent years. To regulate the biosynthsis of metabolites in Taiwania, it should be illustrate the synthesis based on the molecular level. In this study, we focus on sesquiterpene synthase of Taiwania. First, a pair of degenerated primers was designed for reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from leaves of mature tree. A DNA fragment with the conserved region of the terpene synthases (TPSs) was obtained. After 5’ and 3’ Rapid Amplification of cDNA Ends (RACE), the full-length cDNA was obtained, and it contains an open reading frame of 1791 bp with a predicated molecular mass of 70.2 kDa. The gene was highly expressed in young leaves, female flowers and cones. The expression in leave was enhanced after the treatment of soaking in 100 μM salicylic acid (SA). To identify the function of the obtained TPS, the recombinant protein from Escherichia coli was incubated with farnesyl diphosphate (FPP). After GC/MS analysis and retention time as well as mass spectrum matching with authentic standards revealed that the major product of TPS was sesquiterpene α-Gurjunene. The gene was named as Tc-Gur. To investigate the synthesis of this TPS in plant system, α-Gurjunene is also synthesized in transgenic Arabidopsis. But another sesquiterpene, α-Muurolene, is synthesized in transgenic hairy root of Taiwania.
參考文獻
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