食茱萸單萜合成酶基因選殖與功能鑑定

Cloning and Characterization of Monoterpene Synthase Genes from Zanthoxylum ailanthoides

范瑋琳(Wei-Lin Fan)

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(2022年)

https://doi.org/10.6342/NTU202200383

摘要

萜類化合物屬於植物的一種特殊代謝物，除了對於植物適應環境有重要作用，因其所具有生物活性常被運用於醫療保健用途。食茱萸乃芸香科花椒屬植物，分布於臺灣中低海拔，其葉子具特殊香味，原住民常以其嫩葉為傳統食材。經前人研究已知食茱萸葉部含有多種揮發性萜類化合物，單萜化合物應為其主要香氣成分。本研究以食茱萸葉轉錄體資料，藉由生物資訊軟體與芸香科中已發表之揮發性萜類合成酶基因序列作參考比對，篩選出五個單萜合成酶候選基因，以聚合酶連鎖反應獲得全長序列，並以酵素體外活性反應試驗與大腸桿菌共表現系統得到產物以便進行後續酵素功能鑑定，結果顯示Za_mTPS1主要產物為sabinene，Za_mTPS2主要產物為geraniol，Za_mTPS3、Za_mTPS4主要產物皆為linalool，Za_mTPS5主要產物則為(E)-β-ocimene。此外，亦以反轉錄聚合酶連鎖反應分析不同葉組織之基因表現量，以及嘗試以轉基因毛狀根培養生產目標化合物。

關鍵字

食茱萸 ； 植物香氣成分 ； 萜類合成酶 ； 單萜類化合物 ； 毛狀根培養

並列摘要

Terpenoids refer to specialized metabolites in plants. As well as being important for plants to adapt to the environment, they are also used by humans as bioactive ingredients. Zanthoxylum ailanthoides belongs to the Zanthoxylum genus in the Rutaceae family. It distributed in the middle and low altitudes of Taiwan. Its leaves have a special fragrance, and indigenous peoples often use their tender leaves as traditional food. According to previous studies, it is known that the leaves of Z. ailanthoides contain a variety of volatile terpenoids, and monoterpenoids contribute to their aroma. Using the published volatile terpene synthase gene sequence in Rutaceae as references, we screened the transcriptomes data from the leaves of Z. ailanthoides. Five candidate monoterpene synthase sequences were selected. The full-length sequence was obtained by polymerase chain reaction, and the activities was identified by the enzyme in vitro assay and the E. coli co-expression system. The results showed that the main product of Za_mTPS1 is sabinene, the main product of Za_mTPS2 is geraniol, the main product of Za_mTPS3 and Za_mTPS4 is linalool, and the main product of Za_mTPS5 is (E)-β-ocimene. The gene expressions of different leaf tissues were performed by reverse transcription PCR, and an attempt was made to produce target monoterpenoids in transgenic hairy root cultures.

並列關鍵字

Zanthoxylum ailanthoides ； plant aroma compounds ； terpene synthase ； monoterpenoid ； hairy root

參考文獻

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