植物二次代謝物為自然界裡重要的產物，其中萜類 (terpenoid) 擁有為數最多的化合物。萜類物質能在植物與外界環境中扮演多重角色，如: 吸引授粉者或抵禦病原體的攻擊。這些萜類化合物是透過萜類合成酶 (terpene synthase) 來催化合成，透過其序列的差異分成八個亞群 (TPS-a~h)。為了探討蘭科植物萜類合成酶之演化，由Apostasia shenzhenica, Vanilla fragrans, Dendrobium catenatum 以及 Phalaenopsis equestris之基因庫調取共10、14、35及19個TPS基因，另外來自P. aphrodite及 P. bellina之transcriptome各取得22及16個TPS。這些蘭科的TPS與其他植物的TPS進行演化分析，並針對具有基因庫的四個物種進行TPS之間的排列關係 (gene cluster) 探討。發現蘭科TPS隨著時間推移其數目透過基因複製增加，並且gene cluster之形成數目和複雜度也隨演化過程增加。大葉蝴蝶蘭 (Phalaenopsis bellina)為少數具有香味的蝴蝶蘭品種，先前研究發現其香味分子主要為芳樟醇 (geraniol) 、香葉醇 (linalool) 以及兩者之衍生物。為找出大葉蝴蝶蘭和香味合成相關的TPS基因，透過已知的TPS保留序列進行RT-PCR及real-time PCR找到兩個TPS相似基因分別命名為PbTPS5和PbTPS10。透過暫時性轉殖表現這兩個基因在煙草 (N. tabacum) 以及一心維納斯 (P. I-Hsin Venus)發現皆會產生香葉烯 (myrcene) 衍生物，且在轉殖一心維納斯中亦偵測到香葉醇的生成量大幅上升。然而，利用大腸桿菌表現異源性酵素PbTPS5並進行功能性分析結果顯示PbTPS5以 geranyl diphosphate (GDP) 為受質產生芳樟醇 (geraniol)。本篇研究讓我們更深入了解蘭科植物的萜類合成酶演化以及大葉蝴蝶蘭單萜生合成，在未來可透過轉殖單萜合成酶基因於無香味的蝴蝶蘭中生產具有香味的品種。

Plant secondary metabolites are the major nature products, the emitted volatile compounds as a means of attracting pollinators or defense function, including terpenoids, phenylpropenoids, benzeniods, and fatty acid derivatives. Phalaenopsis bellina is a scent orchid with the main floral compounds of monoterpenes for linalool, geraniol and their derivatives. The enzymes catalyzing the formation of terpenes from geranyl diphosphate (GDP) are terpene synthase (TPS). Based on the sequences, TPSs can be divided into eight subfamilies, TPS-a, -b, -c, -d, -e/f, -g, and -h. To analyze the evolution of TPS genes in Orchidaceae, 10, 14, 35 and 19 TPS genes were first identified from the whole genome sequences of Apostasia shenzhenica, Vanilla fragrans, Dendrobium catenatum and Phalaenopsis equestris, respectively distributing among three subfamilies of Orchidaceae. Phylogenetic relationship of these genes was analyzed, and functional gene clusters were built to assess whether duplication events have occurred with tandem or non-tandem duplication. To functionally characterize the floral scent related monoterpene synthase (MTPS) genes in P. bellina, PbTPS5 and PbTPS10 classified in TPS-b subfamily were cloned. Transient overexpression of these genes in both Nicotiana tabacum and P. I-Hsin Venus was performed. The volatiles emitted from the transient overexpressing plants were collected and analyzed by using GC-MS. Tetrahydromyrcenol was the major product from overexpression of both PbTPS5 and PbTPS10 in N. tabacum. In contrast, linalool production was highly increased for the overexpression of PbTPS10 in P. I-Hsin Venus, about four-fold higher than the overexpression of PbTPS5. However, in vitro enzyme assay of PbTPS5 showed the production of geraniol after fed with geranyl diphosphate (GDP). Taken together, the knowledge gained from this study will enhance our knowledge evolution of TPS gene in Orchidaceae as well as the monoterpene biosynthesis in P. bellina. In future, transgenic plants ecotopically expressing MTPS can be generated to modify the scentless orchids to scent one by introducing MTPS genes.

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