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  • 學位論文

山櫻花萜類合成酶基因之選殖與表現

Cloning and Expression of Terpene Synthase Genes From Taiwan Cherry (Prunus campanulata)

指導教授 : 曲芳華

摘要


山櫻花 (Prunus campanulata Maxim.) 又名緋寒櫻,為台灣原生的闊葉樹種,分布於全島海拔500-2200公尺之山地闊葉林中,許多公園校園及山區名勝亦有種植。現有關於山櫻花的研究多與其種子休眠和發芽有關,並未見到有關山櫻花之二次代謝物之研究。本研究著眼於山櫻花二次代謝物中之萜類化合物,特別是分子量較小且具有揮發性的單萜和倍半萜,希望從分子生物學的方面探討山櫻花萜類生合成酵素。我們由資料庫中萜類合成酶的基因序列來設計退化性引子對,並且利用次世代定序技術獲得萜類合成酶之片段序列,以反轉錄聚合酶連鎖反應技術,從樹木成熟葉組織中選殖出五組具有典型合成酶保守序列之基因,接著經由5’ 端及3’端快速增幅cDNA端點 (rapid amplification of cDNA ends, RACE) 選殖,獲得完整基因全長。將所得之基因分別構築於蛋白質表現載體中,轉入大腸桿菌 (Escherichia coli) 中表現,發現其中之兩個合成酶可催化單萜及倍半萜前驅物分別生成芳樟醇 (linalool) 和橙花叔醇 (nerolidol);另外三個萜類合成酶則無活性表現,因此還無法鑑定其生合成之產物。目前已有實驗證實Linalool和Nerolidol皆是與花朵和水果之香味及風味最有關係的萜類之一,除此之外,兩者皆能幫助植物抵抗植食者,並且都具有良好的抗真菌等生物活性。

並列摘要


Prunus campanulata Maxim. also known as Taiwan cherry, is a native broadleaf tree in Taiwan, distributed throughout the island between 500-2200 meters above sea level, and are also planted in many parks, campus and mountain attractions. To date, most of the researches about Prunus campanulata Maxim. are concerned with the seed’s dormancy and germination, but rarely mention about its secondary metabolites. Here we focused on the terpene synthases, especially for those that produce compounds which are small and have volatility. At the beginning, we designed pairs of degenerated primers, and then used it by polymerase chain reaction (PCR) to get the fragments of terpene synthase (TPSs) include the conserve region. Furthermore, we used the next generation sequencing methods to get more fragments that we need, then after 5’ and 3’ Rapid Amplification of cDNA Ends (RACE) done, the full-length cDNA was obtained, with these methods, we obtained five terpene synthase sequences from the leaves of Prunus campanulata Maxim. To identify the function of this five terpene synthases, the recombination protein from Escherichia coli was reacted with gernyl diphosphate (GPP) and farnesyl diphosphate (FPP), they are the substrate of monoterpene and sesquiterpene respectively. Using GC/MS analysis, we revealed that two of these terpene synthases have their major products as linalool and nerolidol. However, the other three terpene synthases do not show any activity. Their functions require further study. Linalool and nerolidol have been confirmed to be the most important terpenes for the fragrance and flavor of flowers and fruits. In addition, these two terpenes both can help plants to own resistance against herbivores, and good bioactivity, including antifungal, anti-inflammatory or anxiolytic etc.

參考文獻


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