紅檜葉部雙萜合成酶之基因選殖與功能鑑定

Abstract

紅檜 (Chamaecyparis formosensis Matsum.) 是臺灣珍貴的針葉樹五木之一，亦是臺灣特有種，目前已鑑定出豐富的雙萜類化合物 (diterpenoids)，這些雙萜類不只參與生理調控與防禦機制，許多已被證實具有潛在的生物活性或醫藥價值，藉由雙萜合成酶 (diterpene synthases) 的研究能更深入探討雙萜類的生合成途徑。 本研究自紅檜葉部轉錄體資料庫選殖出3個Class II與 5個Class I單功能的雙萜合成酶，以cDNA 末端快速擴增實驗獲得基因全長，利用蛋白質表現質體構築與純化獲得目標蛋白質，進行Class II雙萜合成酶單獨反應與 Class II、Class I 雙萜合成酶配對反應，利用氣相層析質譜儀 (gas chromatography-mass spectrometry, GC/MS) 分析產物。Class II雙萜合成酶單獨反應產物鑑定出(+)-copalyl diphosphate；將Class II與Class I的雙萜合成酶進行配對組合反應，獲得levopimaradiene、isophyllocladene和biformene等雙萜產物。本研究亦利用臺灣杉 (Taiwania cryptomerioides) Class II的labd-13-en-8-ol diphosphate synthase與紅檜Class I的雙萜合成酶進行配對反應，獲得sclareol。同時也透過大腸桿菌 (Escherichia coli) in vivo coexpression的雙萜生產系統，產生與蛋白質配對反應相同的產物。 目前已知苔蘚類 (Bryophytes)、石松類 (Lycophytes)、松科 (Pinaceae) 具有雙功能 Class II/I的雙萜合成酶，少數已發表的裸子植物具有Class I單功能的雙萜合成酶；被子植物透過單功能的Class II與Class I雙萜合成酶配對產生雙萜產物。紅檜雙萜合成酶在演化歷程中扮演過渡的角色，它們保有完整的三個domain，但無論是一次或二次代謝途徑皆已趨向單功能化，目前研究推測單功能的雙萜合成酶透過不同配對組合，可以增進雙萜產物的結構多樣性。透過內含子分布分析，發現紅檜雙萜合成酶的基因結構同時存在祖先型與過渡期的內含子組成，顯示隸屬柏科的紅檜與現今已發表的裸子植物雙萜合成酶存在演化差異與結構特異性。

Taiwan red cypress (Chamaecyparis formosensis Matsum.) is an endemic and precious conifer of Taiwan, which is rich in several bioactive diterpenoids. These diterpenoids not only participate in plant physiological regulation and plant defense but also have been verified having potential biological activities or medical values. Studying diterpene synthases (diTPSs) is the key way to understand the pathway of diterpenoid biosynthesis. In this study, three class II diTPSs and five class I diTPSs candidate genes of Taiwan red cypress were identified by analyzing the transcriptome of leaves. The complete coding sequences (CDS) of each gene were obtained by rapid amplification of cDNA ends (RACE). CDS of each gene were cloned into expression plasmids and target recombinant proteins were acquired by purification. Class II diTPSs GGPP single assay and class II + class I diTPSs GGPP couple assay were conducted, and products were analyzed by gas chromatography-mass spectrometry (GC/MS). Through class II diTPSs GGPP single assays, (+)-copalyl diphohpate was identified. Levopimaradiene, isophyllocladene and biformene were identified from class II + class I diTPSs GGPP couple assays. Besides, sclareol was obtained by labd-13-en-8-ol diphosphate synthase from Taiwania cryptomerioides coupled with class I diTPSs from C. formosensis. This research also exploited the Escherichia coli in vivo coexpression system to acquire target diterpene products. Bifunctional class II/I diTPSs are so far identified from bryophytes, lycophytes and Pinaceae. Besides, few class I diTPSs are found from gymnosperms. Angiosperms require pairs of monofunctional class II and I diTPSs. DiTPSs from C. formosensis play transitional roles during the evolution of diTPSs, which consist of three domains but evolve into monofunctionalization. The current study speculates that monofunctional diTPSs can increase diterpene product structural diversity through different pairs of combinations. By analyzing intron distribution of diTPSs, it was found that diTPSs of C. formosensis co-exist ancestral type and transitional type, which indicate diTPSs of C. formosensis (Cupressaceae) have differences and specificity from published conifer diTPSs.