食茱萸之倍半萜類合成酶基因選殖與功能鑑定

Cloning and Characterization of Sesquiterpene Synthase Genes from Zanthoxylum ailanthoides

莊竣崴(Chun-Wei Chuang)

指導教授 ： 曲芳華

國立臺灣大學/生物資源暨農學院/森林環境暨資源學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202000487

摘要

食茱萸 (Zanthoxylum ailanthoides) 為臺灣中低海拔常見之花椒屬植物，具有食用與藥用價值。本研究分別選取具有刺小葉與無刺小葉之植株樣本，先以 DNA 條碼序列證實不同型態確實為同種植物，推斷為生長發育產生型態轉變。此外，經氣相層析質譜儀分析了解其揮發性成分的差異，並選定倍半萜類作為研究目標。本研究係以次世代定序 (next generation sequencing, NGS) 獲得轉錄體資料庫，並搭配生物資訊軟體進行註解與功能性分群，以及已發表的芸香科揮發性萜類合成酶基因作為參考序列進行比對，結果共獲得了18 個可能的萜類合成酶序列資料。以此為基礎進行全長基因的選殖，所採用策略包括聚合酶連鎖反應 (polymerase chain reaction, PCR) 以及 cDNA 末端增幅技術 (rapid amplification of cDNA ends, RACE)，共獲得了 5 個倍半萜合成酶基因之全長序列。進一步進行酵素功能鑑定的結果顯示，ZaTPS1、ZaTPS2 與 ZaTPS3 之酵素反應主產物為 Germacrene D，ZaTPS4 則以 trans-α-Bergamotene 與 β-Farnesene 為主要產物，ZaTPS5 則為多產物的倍半萜類合成酶基因。配合表現量分析與葉組織揮發性成分分析，只有 ZaTPS1 的表現量與葉部揮發成分一致，其餘基因表達趨勢未能與植物揮發成分相符合，因此在植物體中各基因的實際產物可能需進一步的酵素作用才可達到終產物。

關鍵字

食茱萸 ； 轉錄體 ； 生物資訊 ； 倍半萜類合成酶 ； Germacrene D

並列摘要

Zanthoxylum ailanthoides grows in the middle and low attitude of Taiwan. It has edible and medicinal value. In this research, plant samples including prickly leaves and non-prickled leaves respectively. The DNA barcode sequence was used to confirm whether the different types are the same plant species, and it was inferred that the growth and development associated with type change.The differences in volatile components were analyzed by gas chromatography mass spectrometry, and those of sesquiterpenes were selected as research targets. The transcriptome data were obtained by next generation sequencing (NGS), and the bioinformatics software was used for annotation and functional clustering. Combined with the published volatile terpene synthase genes belonging to Rutaceae family as a reference database, 18 possible terpene synthetase sequences were selected. the full length of five sesquiterpene synthase genes were obtained by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). Further characterization of enzyme function showed that the main product of ZaTPS1, ZaTPS2 and ZaTPS3 was Germacrene D. ZaTPS4 generated trans-α-Bergamotene and β-Farnesene as the main products, and ZaTPS5 was a terpene synthase gene forming multiple products. Combined with the gene expression level and the analysis of leaf volatile components, only the expression of ZaTPS1 was consistent with the volatile components of the leaves, and the expression trend of the other genes did not correspond to the volatile components of the plants. Therefore, the actual functions of the genes in the plant still need to be further confirm. But the actual function in plants needs more evidence to explain how they works in plant.

並列關鍵字

Zanthoxylum ailanthoides ； transcriptome ； bioinformatics ； sesquiterpene synthase ； Germacrene D

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