許多植物可以散發揮發性化合物作為吸引授粉者或防禦功能。揮發性化合物主要包含萜類、類苯丙醇、苯類、脂肪酸衍生物。萜類合成酶在萜類生合成路徑中是重要的酵素。許多萜類合成酶基因(terpene synthase, TPS)已經在雙子葉植物及少數單子葉植物被分離出來。然而，目前沒有任何報導關於蘭科萜類合成酶基因之研究。蝴蝶蘭是台灣重要的經濟花卉產業，因此，本篇論文主要在具有香味的大葉蝴蝶(Phalaenopsis bellina)及無香味的姬蝴蝶蘭 (Phalaenopsis equestris)分離及鑑定萜類合成酶基因，比較此二種蝴蝶蘭所含萜類合成酶基因序列與其基因之表現。
首先，我們比較兩個物種間花部揮發性化合物之成份。大葉蝴蝶蘭揮發性化合物分析已被報導在2006年蕭郁芸學姊發表之文獻。經分析姬蝴蝶蘭之花部揮發性化合物發現二者皆具有像似量的脂肪酸衍生物，而大葉蝴蝶蘭之萜類化合物含量是顯著於姬蝴蝶蘭並且主要為單萜類化合物。進一步在姬蝴蝶蘭花苞EST（expressed sequence tag）資料庫中分離一個全長complementary DNA (cDNA)的萜類合成酶基因並命名為PeTPS (P. equestris terpene synthase)。萜類合成酶基因之open reading frame (ORF)為1,647 bp可轉譯成549胺基酸。另外，使用姬蝴蝶蘭之一對可增殖PeTPS引子成功選殖大葉蝴蝶蘭萜類合成酶基因並命名為PbTPS (P. bellina terpene synthase)。比較PeTPS及PbTPS二者之轉譯胺基酸序列，發現彼此間有96.1 %序列相似度並具有二價金屬離子結合位置之DDIYD motif及R(H/Q)X8W motif。此外，PeTPS及PbTPS蛋白質並沒有預測導引訊息片段，因此它們的可能作用位置乃位於細胞質。演化分析結果指出姬蝴蝶蘭及大葉蝴蝶蘭之轉譯胺基酸序列被歸類為TPSa群，即屬於被子植物之半萜類合成酶及雙萜類合成酶之分群。南方墨點法分析姬蝴蝶蘭半萜類合成酶在姬蝴蝶蘭基因體中為只含有一個copy number基因。利用北方墨點法分析PeTPS及PbTPS基因之在不同時間的表現模式，發現PeTPS主要表現在姬蝴蝶蘭花苞時期至開花第十天，並以開花當天有最大表現量；而PbTPS在大葉蝴蝶蘭最大表現量卻於花苞時期。在不同的空間表現模式分析結果顯現姬蝴蝶蘭PeTPS基因主要表現在唇瓣及花梗。進一步利用RTS100 kit之小麥胚芽萃取物in vitro系統將PeTPS蛋白質成功表現，經西方墨點法以抗His-tag之抗體證實PeTPS蛋白質為一個65 kDa之蛋白質。利用GC-MS (gas chromatography-mass spectrometry)進行酵素功能分析結果顯示，PeTPS蛋白質之酵素主要會利用geranyl diphosphate (GDP)為受質以形成單萜類如cis-citral, geraniol 及trans-citral，而未偵測到以farnesyl diphosphate (FDP)及geranyl geranyl diphosphate (GGDP)為受質以生成半萜類及雙萜類化合物。這結果顯示PeTPS蛋白質應該是個單萜類合成酶，雖然它的序列分析較相似倍半萜類合成酶。PeTPS基因及PbTPS基因二者在蘭科作物首度被分離。未來之研究可以進行PeTPS基因轉殖植物散發具有香味cis-citral, geraniol 及trans-citral之研究，在應用上可以使無香味植物轉變成香味植物。

Many plants emit volatile compounds as a means of attracting pollinators or defense function. The volatile components mainly consist of terpenoid, phenylpropanoid, benzenoids, and fatty acid derivatives. TPS are key enzymes in terpene biosynthesis pathway. Many TPS genes have been isolated and characterized in dicotyledon and in monocotyledon. However, so far no TPS have been documented in orchids. Phalaenopsis spp. has been developed as an important cash plant in Taiwan ornamental industry. Therefore, I aimed to isolate and functionally characterize TPS gene, and compare the TPS transcripts from both scentless P. equestris and scented P. bellina.
First, floral volatiles emitted form both scentless P. equestris and scented P. bellina were compared. Although the contents of terpene in scented P. bellina were remarkable and mainly composed of monoterpenes (Hsiao et al., 2006), the contents of fatty acid derivatives were similar in both scentless P. equestirs and scented P. bellina. A full-length cDNA encoding sesquiterpene synthase was cloned and sequenced from the EST database of P. equestris flower buds, and thus named as PeTPS. It harbored an ORF cDNA of 1,647 bp and can be translated to a protein of 549 amino acids. An ORF cDNA of terpene synthase (PbTPS) from P. bellina was also cloned using PCR with a pair of primers derived from PeTPS sequence. Alignments for PeTPS and PbTPS deduced amino acid sequences showed a high identity (96.1 %) containing the conserved DDIYD motif involved in the binding of a divalent metal cofactor and an R(H/Q)X8W motif. In addition, no any transit peptides were predicted, suggesting that its subcellular localization was in the cytoplasm. Phylogenetic analysis of plant terpene synthases showed that the PeTPS and PbTPS were classified in the TPSa subfamily, which consists of sesquiterpene synthase and diterpene synthase. The PeTPS gene was a single-copy gene as examined by using Southern blot analysis. Temporal expression of PeTPS transcript was expressed from the first stage of flower buds to day 10 post-anthesis of P. equestris, with a maximal expression on the blooming day. However, the maximal expression of PbTPS was detected at the bud stages of P. bellina. Spatially, PeTPS was highly expressed in lip and pedicle and to a small amount in the vegetative tissues and all other floral organs. The PeTPS protein was expressed by using in vitro system of the RTS 100 Wheat Germ CECF kit. An expected protein size of 65 kDa encoded by PeTPS was detected by using western blot analysis. The recombinant PeTPS protein catalyzed the formation of three monoterpenes, including cis-citral, geraniol and trans-citral, when GDP was added as the substrate in the in vitro functional assay and analyzed by GC-MS. However, little or no sesquiterpenes or diterpenes were produced when FDP and GGDP were added as substrates, respectively. We concluded that the PeTPS was a monoterpene synthase even though its amino acid sequence was similar to sesquiterpene synthase. PeTPS and PbTPS are the first isolated terpene synthase genes in orchids. In future transgenic plants ectopic expressing PeTPS can be generated to emit aromatic volatiles, including cis-citral, geraniol and trans-citral. Therefore, scentless plants species could be changed to scented plant species by introducing the PeTPS or PbTPS gene.

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