臺灣八角蓮方向蛋白基因之選殖與表現

Cloning and Expression of Dirigent Protein from Podophyllum pleianthum Hance

郭翰蓉(Han-Jung Kuo)

指導教授 ： 李昆達

國立臺灣大學/生命科學院/生化科技學系/碩士(2011年)

https://doi.org/10.6342/NTU.2011.01946

摘要

鬼臼素生合成路徑之起始步驟為兩個分子的松柏醇自由基經由方向蛋白 (dirigent protein, DP) 的作用，進行方向性之雙聚體化 (dimerization)，以形成一分子之松脂醇。自臺灣八角蓮之RNA經反轉錄選殖出一579鹼基對長之方向蛋白基因，其轉譯出之蛋白質預測分子量為22 kDa。該基因在核酸序列上與美洲八角蓮之方向蛋白具97%之一致性，其胺基酸序列則分別有96%之一致性及98%之相似度。此方向蛋白以大腸桿菌進行異源表現時無法產生功能性蛋白質，而在菸草 (Nicotiana tabacum) 毛狀根之植物異源表現系統中，以螢光顯微鏡可觀察到DP-ZsYellow之螢光表現。以粗蛋白進行松柏醇雙聚體化反應，則可觀測到產物松脂醇之生成，且被進一步轉換為下游代謝物。

關鍵字

鬼臼素 ； 臺灣八角蓮 ； 方向蛋白 ； 松柏醇 ； 松脂醇

並列摘要

Podophyllotoxin is an important precursor of anti-tumor drug. Podophylloum pleianthum Hance, an endemic species in Taiwan, is a nature source of podophyllotoxin. The biosynthesis pathway of podophyllotoxin starts from two molecules of coniferyl alcohol radical coupled directionally forming one molecule of (+)-pinoresinol with the involvement of dirigent protein (DP). The cDNA of dirigent protein was cloned from P. pleianthum Hance total RNA by RT-PCR. The length of the dirigent protein cDNA was 579 base pairs and the predicted molecular weight of the dirigent protein was 22 kDa. The cDNA sequence was aligned with DP of Podophyllum peltatum. The identity was 97%. The identity and similarity of amino acid alignment were 96% and 98% respectively. The dirigent protein could not express functionally in recombinant E. coli system. Nicotiana tabacum hairy root for heterologous expression was taken instead. The fluorescent reporter protein ZsYellow was observed by fluorescent microscope. The dimerization reaction of coniferyl alcohol in vitro was performed. Pinoresinol was first converted and was then transferred to other metabolites.

並列關鍵字

Podophyllotoxin ； Podophyllum pleianthum Hance ； Dirigent protein ； Coniferyl alcohol ； Pinoresinol

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