大葉蝴蝶蘭的花因具有優雅外形及甜美香味，因此十分受大眾歡迎。雖然花香的生合成路徑已在多種植物被廣泛研究，但調控機制幾乎未知。大葉蝴蝶蘭的花香經由本實驗室過去的研究發現主要由單萜類構成，單萜類生合成路徑亦被推導出，其中PbGDPS (geranyl pyrophosphate synthase)因可合成單萜類的前驅物GDP而被視為是蘭花香味生合成路徑中的關鍵酵素。PbGDPS的表現模式與大葉蝴蝶蘭單萜類釋放曲線一致，皆在開花後第五天達到最大量。利用本實驗室所建立的蘭花Oligo-microarray實驗結果，搜尋到三個與PbGDPS的表現模式相似的MYB轉錄因子，分別為PbMYB1，PbMYB2及PbMYB6。PbMYB1的表現量於開花後第五天達到最高，主要在花瓣及唇辦中表現。PbMYB2會在開花當天及開花後第五天有大量的表現，而在花萼及花瓣中皆可偵測到其表現。PbMYB6會持續表現，但仍然在開花後第五天有較大的表現量。經由多重序列比對確認PbMYB1，PbMYB2屬於R2R3-MYB轉錄因子，PbMYB6因含有一個coiled-coil區域，被歸類於MYB-CC轉錄因子。演化分析顯示PbMYB1，PbMYB2可能與phenylpropanoids生成及花藥發育有關，而關於MYB-CC轉錄因子的研究目前主要發現和磷缺乏時的訊息傳遞有關。為確認PbMYB是否可以調控PbGDPS，首先選殖出PbGDPS的2515-bp啟動子，經由序列分析發現其上具有MYB結合區位。藉由雙螢光酵素系統確認PbMYB1，PbMYB2與PbMYB6具有和PbGDPS啟動子結合之能力，並進一步利用酵母菌單雜交系統發現PbMYB轉錄因子可結合上PbGDPS啟動子上約-1000至-700及-150至轉錄起始點的兩個小片段上。經由比較PbGDPS與不具香味的姬蝴蝶蘭PeGDPS的啟動子序列後，發現於-1000至-700的小片段中有一段11個核甘酸大小的片段差異(TTATTAACTTT)，此11個核甘酸大小的片段也可在其它三種具有香味的蝴蝶蘭(P. amboinensis, P. lueddemanniana, P. gigantea)GDPS啟動子上發現。此外，利用酵母菌單雜交系統發現不具香味的PeGDPS啟動子無法被PbMYB轉錄因子結合，進一步證實PbMYB轉錄因子可直接結合上11個核甘酸大小的片段。因此，此片段極有可能是PbMYB1，PbMYB2與PbMYB6調控的cis-element，且極有可能與蝴蝶蘭的香味性狀有關。由於PbMYB1，PbMYB2與PbMYB6在空間及時間上有不同的表現模式，推測此三個PbMYB轉錄因子，特別是PbMYB1與PbMYB2，會在不同的花部器官與時期中影響PbGDPS的表現，進而影響單帖類物質的合成，並影響香味物質的合成量或參與在防禦機制中。本研究的貢獻在於利用比較不同的蝴蝶蘭品種，找出可能與花香合成調控有關的影響因子，使我們對蘭花花香調控有更進一步的了解。

Phalaenopsis bellina is very popular for its elegant form and sweet fragrances. Although the floral scent biosynthesis pathway has been studied in various species, their regulation mechanism are almost unknown. Previously P. bellina geranyl diphosphate synthase (PbGDPS) was identified to be one of the key enzyme for supplying geranyl diphosphate for monoterpenes biosynthesis. To further study the regulation of PbGDPS expression, three MYB transcription factors, including PbMYB1, PbMYB2, and PbMYB6, were identified from the Phalaenopsis TF-Oligo microarray. PbMYB1 and PbMYB2 have typical R2R3 MYB DNA binding domain, while PbMYB6 is a MYB-CC (coil-coiled) type protein. Phylogenetic analysis results place PbMYB1 and PbMYB2 in the same group involved in regulation of phenylpropanoid biosynthesis and anther development. Study on the MYB-CC type transcription factors is mainly focused on controlling of phosphate metabolism in plants. Transcription level of PbMYB1, PbMYB2 and PbMYB6 all increased at D+5 (day 5 post-anthesis), consistent with both maximal emission of monoterpenes and expression of PbGDPS. Expression of PbMYB1 was detected in petals and lips, while PbMYB2 expressed in sepals and petals. To study the regulation of PbGDPS expression by these three MYB transcription factors, a 2515-bp promoter region of PbGDPS was isolated and consensus DNA-binding sites for MYB transcription factors were predicted. All three MYB transcription factors revealed the ability of driving the expression of both 2-kb and 1-kb PbGDPS upstream regulatory sequences in the dual-luciferase assay. The yeast one-hybrid assay of dissected PbGDPS 1-kb promoter region has shown PbMYBs could bind on the furthest subfragment and minimal promoter. According to the comparison between PbGDPS and PeGDPS promoter sequences, a putative cis-element (TTATTAACTTT) was identified from the furthest subfragment of PbGDPS promoter. This 11-nt element also existed in the GDPS promoters of three other scented Phalaenopsis species (P. amboinensis, P. lueddemanniana, P. gigantea). The furthest region of PbGDPS promoter, as well as the 11-nt cis-element, were bound by PbMYBs, in contrast, PbMYBs could not bind on the PeGDPS promoter. The 11-nt unique sequences, is a potential cis-element bound by PbMYBs, and might related to the scent phenotype in Phalaenopsis. The differentially temporal and spatial expression of PbMYB1, PbMYB2 and PbMYB6 was responsible for regulating PbGDPS expression in right organs at right timing. In this study, the regulation floral scent in P. bellina is initially unraveled by comparing the promoter of GDPS among scent and scentless Phalaenopsis species.

Chen WH（陳文輝）(2007) 台灣如何成為蝴蝶蘭王國？ 科學人雜誌 第69期
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