台灣八角蓮 Pinoresinol lariciresinol reductase 與 Secoisolariciresinol dehydrogenase 之基因選殖與表現

Abstract

鬼臼素 （Podophyllotoxin） 對癌細胞具強效毒殺作用，其化學衍生物已應用於臨床抗癌藥物之使用。本研究從台灣八角蓮 （Podophyllum pleianthum Hance）中選殖出鬼臼素生合成路徑中之pinoresinol lariciresinol reductase 基因 PLR （將 pinoresinol 先轉化為 lariciresinol，再轉化為 secoisolariciresinol） 以及 secoisolariciresinol dehydrogenase 基因 SDH （可將 secoisolariciresinol 轉化為 matairesinol）。選殖 sdh 是以台灣八角蓮 RNA為材料，經反轉錄-聚合酶連鎖反應 （RT-PCR） 得到 cDNA 序列，此序列與美洲八角蓮Podophyllum peltatum 之 sdh cDNA 序列進行比對後，達到 98.1% 的一致性，其胺基酸序列的一致性亦達 98.2%。選定在25 oC、0.01 mM isopropyl--D-1-thiogalactopyranoside （IPTG） 誘導 9 小時為重組蛋白 SDH 於大腸桿菌表現之最適條件。變性膠體電泳 （Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE） 分析可以清楚看到與 SDH-His tag 蛋白質 （34 kDa） 分子量大小相同之重組蛋白之生成。重組蛋白轉化的結果以高效能液相層析 （High performance liquid chromatography, HPLC） 系統進行分析，比較其反應產物 matairesinol 之滯留時間 (23.97分鐘) 及其UV全波長吸收光譜，皆和標準品相吻合。故可確認台灣八角蓮 sdh 之cDNA序列已成功選殖，並可在大腸桿菌內進行具有酵素活性之表現。此外，由台灣八角蓮經由cDNA末端快速擴增技術（Rapid amplification of cDNA ends, RACE）PCR 與 RT-PCR得到之 plr 序列，與美國金鐘連翹 （Forsythia intermedia） 之 plr cDNA 序列比對後，達到 68.9% 的一致性，其胺基酸序列的一致性亦達 75.2% 一致性與 85% 的相似度。西方墨點結果顯示目標蛋白質已表現於大腸桿菌，可以清楚看到與 PLR-His tag 蛋白質 （39 kDa） 分子量大小相同之重組蛋白之生成。

Podophyllotoxin possesses strong tumor-specific cytotoxicity, and its chemical derivatives have been employed in clinical cancer treatment. From Podophyllum pleianthum Hance, pinoresinol lariciresinol reductase (PLR) gene was cloned, which can convert pinoresinol to lariciresinol and consequently to secoisolariciresinol, and secoisolariciresinol dehydrogenase (SDH) gene, which can convert secoisolariciresinol to matairesinol. The total RNA of Podophyllum pleianthum Hance was used as the material for sdh cloning by using reverse transcriptase PCR (RT-PCR). The cDNA sequence of sdh which got from Podophyllum pleianthum Hance aligned with sdh cDNA sequence from Podophyllum peltatum. It reached to 98.1% identity. The alignment of amino acid sequence reached to 98.2%. The optimum conditions was used 0.01 mM IPTG inducting 9 hours at 25oC for expression of SDH in E. coli. The result showed a 34 kDa of protein in SDS-PAGE, the same size for sdh with a SDH-Histag design. The conversion of recombinant enzyme reaction was further analyzed by HPLC, the retention time (23.97 min) and the UV absorption spectrum matched with the characters of authentic matairesinol. It indicated that the cDNA sequence of sdh was cloned from Podophyllum pleianthum Hance and expressed in E.coli functionally. Meanwhile, cDNA of plr got from Podophyllum pleianthum Hance by using rapid amplification of cDNA ends PCR (RACE PCR) and RT-PCR, was aligned with plr cDNA sequence from Forsythia intermedia, and it reached to 68.9% identity. Alignment of amino acid sequence also reached to 75.2% identity and 85% similarity. Western blotting proved the expression of target protein PLR in E. coli. The results showed a 39 kDa of recombinant protein expressed in SDS-PAGE, the same size for sdh with a PLR-His tag design.