臺灣杉 Pinoresinol-Lariciresinol Reductase之選殖與特性分析

Abstract

木酚素 (lignan) 為2個C6-C3單元結合為骨架之植物酚類化合物，具有多樣的生物活性，如臺灣杉 (Taiwania cyptomerioides Hayata) 木材中之dimethylmatairesinol、taiwanin A及taiwanin E 等，即具有顯著毒殺腫瘤細胞之生物活性，可望發展為新的抗癌藥物。Pinoresinol-lariciresinol reductase (PLR) 為木酚素生合成途徑上游酵素，可催化pinoresinol還原成lariciresinol，再將lariciresinol還原成secoisolariciresinol。Secoisolariciresinol經secoisolariciresinol dehydrogenase (SDH) 氧化，生成之產物matairesinol，一般認為是下游木酚素生合成之前驅物。為了解臺灣杉木酚素生合成酵素，本研究建立臺灣杉木材形成層、邊材、移行帶及心材等木材組織轉錄體資料庫，透過分析基因在此4個組織中之表現量相關性，選取3個臺灣杉PLR及6個SDH候選基因，並以cDNA末端快速擴增實驗獲得基因全長。將臺灣杉PLR及SDH基因於大腸桿菌中異源表現，萃取粗蛋白進行酵素反應實驗，其中TcPLR1可催化 (+)-pinoresinol還原為lariciresinol，TcPLR2.2及TcPLR3可催化 (+)-pinoresinol及lariciresinol之還原反應，產生 secoisolariciresinol。RT-PCR結果顯示TcPLR1之表現量於形成層最高，其次為心材，TcPLR2.2及TcPLR3則皆只於形成層及邊材表現，又前者在形成層表現量較高，後者主要於邊材表現。本研究完成臺灣杉PLR之鑑定，未來透過臺灣杉PLR基因之表現趨勢，期望有助於找尋下游木酚素生合成酵素，以建立臺灣杉木酚素之生合成途徑。

Lignans, which are synthesized by dimerization of two C6-C3 units, exist abundantly in the wood of Taiwania cryptomerioides Hayata. Lignans in Taiwania, such as dimethylmatairesinol, taiwanin A and taiwanin E, were reported to have siginificant cytotoxic activity against tumor cells. As the key enzyme of lignan biosynthesis, pinoresinol-lariciresinol reductase (PLR) catalyzes the two-step reduction of pinoresinol to form lariciresinol and then to secoisolariciresinol. Secoisolariciresinol would next be oxidized by secoisolariciresinol dehydrogenase (SDH), and the product matairesinol is suggested to be the precursor for other downstream ignans. In this study, three PLR and six SDH candidate genes of T. cryptomerioides were identified by analysing the transcriptome of wood cambium, sapwood, transition zone and heartwood. The complete coding sequences (CDS) of each gene were obtained by rapid amplification of cDNA ends (RACE). Using Escherichia coli as the host, the heterologously expressed TcPLRs exhibited PLR activity, but the TcSDHs did not exhibited SDH activity. Among the three TcPLRs, TcPLR1 reduced pinoresinol to lariciresinol, while TcPLR2.2 and TcPLR3 reduced both pinoresinol and lariciresinol. The results of RT-PCR revealed that TcPLR1 most highly expressed in the wood cambium, and next in the heartwood. TcPLR2.2 and TcPLR3 both expressed in the wood cambium and the sapwood, while TcPLR2.2 showed slightly higher expression level in the cambium, and TcPLR3 mostly expressed in the sapwood. This study characterizes three PLRs of T. cryptomerioides, which involve in the biosynthesis of upstream lignans, and the expression pattern of TcPLR genes may provide information for identifying downstream lignan biosynthetic genes in the future.