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標題: | 香水文心蘭中萜類生合成基因的晝夜節律表現與香味合成調控之探討 Regulation of floral scent production and circadian rhythmic expression in terpene synthase of Oncidium Sharry Baby |
作者: | Yung-Feng Ho 何湧峰 |
指導教授: | 葉開溫(Kai-Wun Yeh) |
關鍵字: | 文心蘭,花香,?類,?類生合成?,β-羅勒烯,生理時鐘,CCA1基因, Oncidium,floral scent,terpenoids,terpene synthase,β-ocimene,CBS,circadian clock,CCA1, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 植物的花香釋放通常具有晝夜節律模式。此模式受到外在環境因子-光,或是內在的生理時鐘 (circadian clock) 所調控。然而到目前為止,花香節律表現的分子調控機制仍尚未明瞭。在文心蘭屬 (Oncidium) 中香水文心蘭 (Oncidium Sharry Baby, Onc. SB) 是少數能夠釋放香味的品種。前人的研究中發現,其花香味成分為萜類的β-羅勒烯 (β-ocimene) 與芳樟醇 (linalool),並進一步找到合成香味的關鍵酵素為萜類合成酶 (terpene synthase, TPS)。此外,更發現花香會隨時間變化,在早上時最濃。在本篇研究中,我們發現TPS在正常光照/黑暗變化下,能夠節律地表現數天,且波峰落在早上。進入連續黑暗後,只有第一天有晝夜節律且表現量大幅上升;反之,在連續光照下,TPS的表現量下降許多。由此可知光和生理時鐘在香水文心蘭的花香釋放中皆扮演重要的腳色。比較Onc. SB以及無香味的南西品種 (Onc. GR) 的TPS啟動子,發現數個光反應元件 (Light responsive elements, LREs) 同時存在,但只在Onc. SB中發現了一個植物生物時鐘核心基因CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) 的結合位點 (CCA1-binding site, CBS: AGATTTTT),此順式作用點可調控TPS在早上時段有明顯的表現波峰。利用transactivation的啟動子活性實驗中,選殖出來的OncSBCCA1也確實能夠誘導OncSBTPS啟動子的表現;反之OncGRTPS及突變的OncSBTPS啟動子則無法被誘導。最後,CCA1皆在Onc. SB和Oncsa. GR於每日六時 (ZT0) 中有表現波峰,顯示TPS節律性的表現取決於順反式調控配件的協同作用。總結上述結果,光及生理時鐘皆能夠影響TPS的表現,而啟動子的結構影響了CCA1能否調控TPS。 Floral scent is a crucial factor to attract pollinators for reproduction. To maximize resource efficiency, plants often release floral scent at specific times of day. Rhythmic emission patterns are directly controlled by external factors such as light, or by the endogenous circadian clock. However, the genetic control of floral scent diurnal emission has not been well studied and may differ among species. Oncidium Sharry Baby (Onc. SB) is a fragrant orchid, which is unusual in Oncidium alliance, while Oncidesa Gower Ramsey (Onc. GR), a common Oncidium variety in the cut-flower market, has no scent. In our previous studies, we found that Onc. SB emitted terpenoids β-trans-ocimene and linalool. Besides, the key scent producing enzyme was terpene synthase (TPS). Moreover, we noticed that the scent emission of Onc. SB peaked in the morning. Here, we confirmed that the expression of TPS diurnally oscillates over light/dark cycle several days. Under constant dark, the expression of TPS displayed a diurnal pattern on the first day and dramatically increased the expression level of TPS. In contrast, constant light reduced the expression level of TPS. The evidence suggested that light and internal clock both play important roles in the expression of TPS in Onc. SB. Comparative promoter analysis identified several light responsive elements (LREs), and a potential CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)-binding site (CBS: AGATTTTT), which is important for morning-phased transcription was found in OncSBTPS promoters. Moreover, OncSBCCA1 was able to activate the OncSBTPS promoters in transactivation assay, while no activation occurred in OncGRTPS promoter, and OncSBTPS mutant promoter with mutated CBS. The oscillating effect of CCA1 from both Onc. SB and Onc. GR indicated that cis- regulation playing a key role to affect TPS diurnally expression. Taken together, the internal circadian clock and light factors control Oncidium floral scent biosynthetic gene, whereas the implementation of CCA1 function is dependent on the structure of TPS promoter. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74163 |
DOI: | 10.6342/NTU201903119 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物科學研究所 |
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