山胡椒單萜合成酶基因之選殖與功能定性

Abstract

山胡椒 (Litsea cubeba (Lour) Persoon)，為台灣常見的闊葉植物，其精油可應用於食品以及醫藥製品，具消腫、止痛等功效。據前人研究指出，山胡椒精油的單萜成分具有良好的抗發炎活性。本試驗採集山胡椒不同組織，利用基因資料庫中木本闊葉植物之單萜合成酶基因序列設計退化性引子對，並藉由聚合酶連鎖反應及 cDNA 末端快速擴增法，得到三個山胡椒單萜合成酶基因全長，分別命名為LcTPS1、LcTPS2與LcTPS3，其轉譯區序列長度分別為 1750 bp、1743 bp、與1743 bp，其中LcTPS2與LcTPS3兩個核酸序列具高達 97％ 的相似度。為了解該基因在植物體之表現型態，本研究利用反轉錄聚合酶連鎖反應與即時聚合酶連鎖反應觀察三個單萜合成酶基因在山胡椒各部位組織的表現狀況，結果顯示LcTPS1在雄樹的葉子中表現量最高，於未成熟果的表現量最低；LcTPS2在成熟果的表現量最高，於雌樹的葉子表現量最低；而LcTPS3則在未成熟果表現量最高，於枝條的表現量最低。由鄰近連接演化樹分析結果，三個 LcTPS 基因皆坐落在TPS-b的分群之中，其中 LcTPS2 與 LcTPS3 被分類在同一組次分群中而與LcTPS1區分開來。而由蛋白質預測分析結果，LcTPS1之蛋白質大小約為67.1 kDa，而LcTPS2與LcTPS3則約為67.4 kDa。將LcTPS1、LcTPS2與LcTPS3基因分別構築於大腸桿菌 (Escherichia coli BL21) 用之載體 pTYB12 得到可溶部蛋白質，以體外反應的方式驗證 LcTPS1 產物為反式-蘿勒烯；LcTPS2 產物為α-側柏烯；LcTPS3 則具雙重功能，其產物為α-側柏烯與 (+)-香檜烯。目前已有研究指出α-側柏烯與(+)-香檜烯具有良好的上真菌活性，反式-蘿勒烯具有良好的抗氧化活性。

Mountain spicy tree (Litsea cubeba (Lour) Persoon) is a primitive evergreen tree in Taiwan. Previous studies had showed that the fruit of L. cubeba possess the dispels rheumatism, detumescence, detoxify, and analgetic effect, and in the essential oil of L. cubeba, the dominant component is monoterpenes, which have anti-inflammatory activity. To elucidate the biosynthesis of monoterpenes which have bioactivity in L. cubeba, three cDNAs corresponding to monoterpene synthase genes expressed in different tissues of L. cubeba were isolated, and the corresponding enzymes were functionally characterized in vitro. To isolate cDNAs corresponding to monoterpene synthases of L. cubeba, two pairs of primers were designed according to the monoterpene synthases of woody plant. Monoterpene DNA sequences were collected from NCBI database, and polymerase chain reaction (PCR), rapid amplication of cDNA end (RACE) were used for getting full sequence of L. cubeba monoterpene synthases. These genes obtained were named LcTPS1, LcTPS2, and LcTPS3, which have 1749 bp (base pair), 1743 bp and 1743 bp coding region respectively. To investigate expression levels of LcTPS1, LcTPS2, and LcTPS3 at different tissues of L. cubeba, reverse transcription PCR and real-time PCR were used for detection of transcript levels for all three genes at different tissues. The results showed that LcTPS1 genes were most prominent in the leaf of the male trees, LcTPS2 genes were most prominent in development fruit tissue, and LcTPS3 genes were prominent in mature fruit tissue. Phylogenetic analyses indicated that LcTPS1, LcTPS2, and LcTPS3 all belonged to TPS-d terpene family, which represents the angiosperm monoterpene synthase genes. To functionally characterize corresponding enzymes of LcTPS1, LcTPS2, and LcTPS3 in vitro, LcTPS1, LcTPS2, and LcTPS3 were expressed in E. coli protein expression system for pure protein. Recombinant LcTPS1 converted geranyl diphosphate to trans-ocimene, LcTPS2 converted geranyl diphosphate to α-thujene while LcTPS3 is a bifunctional enzyme that converted geranyl diphosphate to α-thujene and (+)-sabinene respectively. According to previous studies, α-thujene and sabinene possess great anti-fungal activity and trans-ocimene possesss antioxidant activity.