植物二次代謝物是自然界中最主要的天然產物，其中萜類（terpenoid）更是為數最多的植物二次代謝物。萜類在植物體扮演著許多角色，如：吸引授粉者，或防止草食動物及病原菌的攻擊。大葉蝴蝶蘭（Phalaenopsis bellina）是一種具有香味的蝴蝶蘭。先前研究發現，它的香味來自於花部器官所釋放的單萜類物質（monoterpenes），其中包含香葉醇（geraniol）、芳樟醇（linalool）以及兩者之衍生物。為找出大葉蝴蝶蘭的萜類合成酶（terpene synthase, TPS）基因，我們以其他植物中已經被鑑定功能的TPS基因與現有的姬蝴蝶蘭基因體序列進行序列比對，在姬蝴蝶蘭中找到13個單萜類合成酶相似（monoterpene synthase-like, MTPS-like）基因。排除4個基因長度小於1000 bp之序列，將其餘9個基因進行演化樹的分析。其中有6個基因被分群到屬被子植物單萜類合成酶之b亞群（TPS-b subfamily），且被註記為松油醇合成酶（(α)-terpineol synthase）或蒎烯合成酶（(α)-pinene synthase）。另外3個基因分群在目前研究較少的TPS-f亞族中，且被註記為芳樟醇合成酶（linalool synthase），分別被命名為PeMTPS6，PeMTPS7及PeMTPS8。經過基因表現分析，只有PeMTPS7及PeMTPS8會在大葉蝴蝶蘭中表現。進一步利用rapid amplification of cDNA ends (RACE)技術擴增出大葉蝴蝶蘭中的PbMTPS7及PbMTPS8之全長cDNA序列。PbMTPS7從開花前五天（D-5）即有基因表現且持續到開花後十天（D+10），以開花後三天（D+3）及後五天（D+5）有較高表現量。PbMTPS8的基因表現量較PbMTPS7低，但仍然在開花後三天（D+3）及後五天（D+5）有較高表現量。此外PbMTPS7亦在葉子表現，且其表現量為花中之4倍高。而PbMTPS8在營養組織的表現量較花部低。總的來說，PbMTPS7與PbMTPS8的基因表現模式與花香釋放的模式具正相關性。為進一步確認PbMTPS7及PbMTPS8的酵素功能，我們利用大腸桿菌（E. coli）表現異源性酵素並進行酵素功能分析。PbMTPS7以geraniol diphosphate (GDP)為受質合成羅勒烯（(β)-cis-Ocimene）及芳樟醇，而PbMTPS8則以GDP為受質合成芳樟醇，但二者皆無法以FDP及GGDP為受質合成萜類物質。此外，根據羅勒烯與芳樟醇在植物體內的生合成路徑推測，PbMTPS7和PbMTPS8會座落於葉綠體胞器（chloroplast）之中，但其胺基酸序列中皆缺乏典型的轉運胜肽（transit peptide），以致無法以軟體準確預測其次細胞方位。為此，以綠螢光融合蛋白之次細胞定位分析（subcellular localization），確認PbMTPS7與PbMTPS8皆座落於葉綠體當中。綜合以上，本篇論文的研究結果將有助於我們了解大葉蝴蝶蘭的單萜類生合成相關酵素。

Plant secondary metabolites are the major nature products, and terpenoid is the largest group among all. Terpenoids play many roles in plants, such as attracting pollinators, and against herbivores or pathogenic bacteria. Phalaenopsis bellina is a scented Phalaenopsis orchid with charming floral fragrance. Previous studies have shown that the major floral compounds of P. bellina are monoterpenes including geraniol, linalool and their derivatives. To clone the terpenoid synthase (TPS) from P. bellina, we used known TPS genes of other plants to search the genome database of P. equestris. Thirteen monoterpene synthase-like (MTPS-like) genes in P. equestris were identified. Among them, four genes were less than 1000 bp and were excluded from the analysis. Phylogenetic analysis of rest nine putative orchid TPS genes indicated that six genes were claded in angiosperm MTPS specific clade (TPS-b), and three were in the TPS-f subfamily. The six TPS-b genes were annotated as either (α)-terpineol or (α)-pinene synthase, and the three TPS-f genes were annotated as linalool synthase, and designated PeMTPS6, PeMTPS7 and PeMTPS8. Gene expression analysis indicated that only PeMTPS7 and PeMTPS8 were expressed in the scented P. bellina. Rapid amplification of cDNA ends (RACE) was used to isolate the full length cDNA sequences of PeMTPS7 and PeMTPS8 in P. bellina, and named as PbMTPS7 and PbMTPS8. The temporal expression of PbMTPS7 indicated that it was expressed from five days before anthesis (D-5) to ten days post anthesis (D+10), with the highest expression level on three days post (D+3) and five days post (D+5) anthesis. PbMTPS8 also showed high expression on D+3 and D+5. Spatially, PbMTPS7 was not only expressed in the flower but also in the leaf, and the expression level was up to a 4-fold higher level as compared to that in flower. The spatial expression of PbMTPS8 indicated that expression level in vegetative organs was much lower than in flower. Overall, the expression level of PbMTPS8 was lower than PbMTPS7. In addition, the expression patterns of PbMTPS7 and PbMTPS8 showed positive correlation with the volatile emission pattern of P. bellina. For enzyme functional assay, both PbMTPS7 and PbMTPS8 were ectopically expressed in E. coli were performed. PbMTPS7 could use geranyl diphosphate (GDP) as a substrate for producing (β)-cis-ocimene and linalool, while PbMTPS8 used GDP as a substrate for producing linalool. Neither farnesyl diphosphate (FDP) nor geranylgeranyl diphosphate (GGDP) could be catalyzed by the two enzymes as substrates. Moreover, according to biosynthesis pathway of monoterpene in plants, both PbMTPS7 and PbMTPS8 were expected to be localized in the chloroplast, but they lack of typical transit peptide. Thus, subcellular localization prediction softwares could not precisely predict their subcellular localization. For this, green fluorescent protein was fused at either N- or C- terminus of both PbMTPS7 and PbMTPS8 and indicated that both PbMTPS7 and PbMTPS8 were localized in the chloroplast. Taken together, the knowledge gained from this study may lead to a better understanding of the monoterpene biosynthesis in P. bellina.

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