扁柏屬 Beyerene 與 Sandaracopimaradiene 合成?功能鑑定

Abstract

日本扁柏 (Chamaecyparis obtusa) 和臺灣扁柏 (Chamaecyparis obtusa var. formosana) 的木材因為優異的耐久性受到歡迎，其木材的抗腐朽能力往往與萃取物成分中的特殊代謝物 (specialized metabolites) 有關，其中又以雙?類化合物 (diterpenoid) 扮演重要的功用。從前驅物 geranylgeranyl diphosphate (GGPP) 轉變成雙?骨架大多需要經過兩步驟反應，由雙?合成? (diterpene synthase) 催化，class II 雙?合成?負責第一步，class I 雙?合成?負責第二步。過去的研究當中，裸子植物雙?特殊代謝物的生合成，通常由單一個具有雙功能 class II/I 的雙?合成?完成兩步驟的反應，然而近期發現，柏科植物特殊代謝物的生合成與被子植物的生合成模式相同，皆是以兩個單功能的雙?合成?配合達成雙?生合成。由於有關柏科雙?代謝的生合成模式不同於松科，但截至目前其研究相當有限，為證實其確實是柏科獨特的生合成方式，本研究以扁柏屬植物闡明柏科雙?合成?的催化功能和演化關係。本研究建構日本扁柏轉錄組資料，從中獲得 5 條完整雙?合成?基因序列，同時也從臺灣扁柏中選殖 4 條雙?合成?。所有扁柏雙?合成?皆為單功能，親緣關係分析表明柏科雙?合成?經歷多次單功能化。對單功能雙?合成?進行功能鑑定，CovfKSL3 產生 ( ) sandaracopimaradiene，CoKSL2、CovfKSL2 1 和 CovfKSL2 3 能夠產生 ( ) beyerene。( ) beyerene 存在日本扁柏與其他柏科植物萃取物中，而能夠生合成 ( ) beyerene 的雙?合成?在此初次被鑑定。

Ownig to the excellent durability, the wood of Chamaecyparis obtusa and Chamaecyparis obtusa var. formosana is popular in timber production. The contribution of specialized metabolites to decay resistance of Chamaecyparis wood is well known, where diterpenoid plays an important role. The transformation from the precursor, geranylgeranyl diphosphate (GGPP), to majority of the diterpene scaffold goes through dual reactions carried out by diterpene synthases (diTPS). Class II diTPSs initiates the first step, and class I diTPSs finishes the next step. While Class II/I bifunctional diTPSs participate in most of the specialized diterpenoids metabolism in gymnosperms, it was recently discovered that the diterpene biosynthesis in Cupressaceae is completed by collocation of two monofunctional diTPSs. Despite the specificity of diterpenoid metabolism pattern in Cupressaceae, the researches remain limited. More investigation into the catalytic function and evolutionary relationship of the diterpene synthases in Cupressaceae is needed. The transcriptome of C. obtusa is established here, and 5 complete diTPSs were obtained from it. Four diTPSs from C. obtusa var. formosana were cloned as well. All of the Chamaecyparis diTPSs are monofunctional, with phylogenetics analysis suggesting the multiple time monofunctionalization of diTPSs in Cupressaceae. Functional characterization of the monofunctional diTPSs was performed. CovfKSL3 produces ( ) sandaracopimaradiene, while CoKSL2, CovfKSL2 1, and CovfKSL2 3 produces ( ) beyerene, which is found in the extract of C. obtusa and other Cupressaceae species. DiTPSs capable of synthesizing ( ) beyerene were discovered for the first time here.