臺灣扁柏雙萜合成酶功能鑑定與柏科雙萜合成酶演化途徑分析

Abstract

為了瞭解柏科植物雙萜合成酶的演化途徑，本研究使用具高經濟價值的臺灣原生樹種，柏科 (Cupressaceae) 扁柏屬 (Chamaecyparis)的臺灣扁柏 (C. obtusa (Sieb. and Zucc.) Endl. var. formosana (Hayata) Rehder)，以及與其親緣關係非常相近的日本扁柏 (C. obtusa (Sieb. and Zucc.) Endl.) 作為研究對象，進行雙萜合成酶的選殖，並分別自臺灣扁柏與日本扁柏成功選殖出 7 個及 5 個雙萜合成酶。透過 in vivo 大腸桿菌 (Escherichia coli) 共表現系統 (co-expression system)，輔以 in vitro 重組純化蛋白表現系統，鑑定了 4 個臺灣扁柏雙萜合成酶及 2 個日本扁柏雙萜合成酶的功能：CovfCPS、CoCPS 為 (+)-copalyl diphosphate synthase ((+)-CPS)；CovfLS及CoLS 與 (+)-copalyl diphosphate ((+)-CPP) 反應合成 levopimaradien，也能與 LPP (labda-13-en-8-ol diphosphate) 反應，合成 manoyl oxide；CovfPAP 與 GGPP 反應，合成 geranylgeraniol；CovfTBS 與 GGPP 反應，合成 thunbergol。接著利用親緣關係分析、模體 (motif) 結構分析、基因結構 (gene structure) 分析松科 (Pinaceae)、柏科雙萜合成酶的演化關係，從中發現柏科雙萜合成酶的演化路徑十分多樣：單功能 class II 雙萜合成酶為單獨一個演化路徑，基因結構上缺少第一個內含子；單功能 class I 雙萜合成酶則分成三個演化路徑，group 1 缺少第一、二個內含子及第一、二個外顯子，group 2 大部分都保有 14 個內含子，group 3 缺少第一、二個內含子，且與紅豆杉科 taxadiene synthase 親緣關係相近。經由基因的比對，發現臺灣扁柏及日本扁柏相較於其他扁柏屬或其他柏科的植物，在 genomic DNA 基因的結構上之相似性非常高有完全相同之處，未來將可做為種間及變種之分類參考資料。

In order to understand the evolutionary pathway of diterpene synthases in Cupressaceae plants clearly, this study used Chamaecyparis obtusa var. formosana, which is Taiwan native tree species with high economic value, as the research object. At the same time, Chamaecyparis obtusa was selected as the research object as well, because the relationship between C. obtusa var. formosana and C. obtusa is close. Finally, for diterpene synthase, 7 and 5 diterpene synthases were cloned respectively in C. obtusa var. formosana and C. obtusa. The functions of 5 diterpene synthases from C. obtusa var. formosana and 2 diterpene synthases from C. obtusa were identified by in vivo co-expression system in Escherichia coli and supplemented by in vitro recombinant purified protein expression system. CovfCPS and CoCPS are (+)-copalyl diphosphate synthase ((+)-CPS). CovfLS and CoLS convert (+)-copalyl diphosphate ((+)-CPP) to levopimaradien, and use LPP (labda-13-en-8-ol diphosphate) to form manoyl oxide. CovfPAP converts GGPP to geranylgeraniol. CovfTBS converts GGPP to thunbergol. Then, using the relationship analysis, motif structure analysis, and gene structure to analyze the evolutionary relationship of the diterpene synthases of Pinaceae and Cupressaceae. It is found that the evolution pathway of Cupressaceae diterpene synthases is very diverse. The evolution pathway of class II diterpene synthases are a single pathway, lacking the first intron in gene structure. The monofunctional class I diterpene synthase can be divided into three evolutionary pathways. Class I diterpene synthases in group 1 lack intron 1, 2 and exon 1, 2. Most of class I diterpene synthases in group 2 have all 14 introns. Class I diterpene synthases in group 3 lack intron1, 2 and they are closely related to taxadiene synthase. From the above analysis results, it can also be found that the same functional diterpene synthase of C. obtusa var. formosana and C. obtusa has a very high similarity in genomic DNA compared with other Chamaecyparis species or Cupressaceae plants. Therefore, it will be used as a reference for the classification between C. obtusa var. formosana and C. obtusa in the future.