Expression of Pinoresinol lariciresinol reductase and Secoisolariciresinol dehydrogdnase in Nicotiana benthaminana hairy roots

本篇研究利用選殖自台灣八角蓮 (Podophyllum pleianthum Hance)中鬼臼素(Podophyllotoxin) 合成路徑上的二個酵素Pinoresinol lariciresinol reductase (PLR) 與 Secoisolariciresinol dehydrogdnase (SDH) 之基因 分別與綠色螢光蛋白質 (GFP) 之基因進行基因融合，以探討報導基因 (reporter gene) 在圓葉菸草 (Nicotiana benthaminana) 毛狀根 (hairy root) 中表現效率。選用pCAMBIA 1302做為毛狀根表現的質體，利用CaMV 35S啟動子驅動表現，並設計linker將gfp與所選殖PLR與 SDH的基因連結。將質體建構好後，轉形進入根毛農桿菌 (Agrobacterium rhizogenes) 中，感染菸草葉子，誘導出帶有外源基因的毛狀根。挑選生長狀況良好的毛狀根進行液態培養，利用PCR、螢光顯微鏡與西方墨點法 (Wetsern blot) 確認表現，再以ELISA的方式測定GFP表現量，並將毛狀根進行組織切片，以螢光顯微鏡觀察融合重組蛋白質在毛狀根中的表現位置。結果顯示二組轉形的毛狀根其外源重組蛋白質主要表現在根的中柱。之後，將萃取所得的可溶蛋白質進行in vitro酵素活性的測試，並以LC-MS檢測產物生成，結果顯示融合重組的PLR具有酵素活性，可將pinoresinol 轉變化secoisolariciresinol，而SDH的檢測中無法偵測到產物mataresinol的生成，推測可能是因為所融合的GFP影響到SDH的活性部位，使得其酵素活性降低。此外，發現成功表現融合重組的PLR毛狀根中，其代謝物與未表現PLR的毛狀根具有差異，推測融合重組的PLR與GFP的酵素活性在毛狀根中對其代謝物產生in vivo改變，之後將進一步對此現象進行探討。本研究顯示，毛狀根可發展為代謝工程之表現系統In this study, we used the genes of pinoresinol lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase (SDH), which are in the biosynthesis pathway of podophylltoxin cloned from Podophyllum pleianthum Hance, to fuse with the gene of GFP to investigate the expression of the reporter gene expressing in Nicotiana benthaminana hairy roots. Plasmid pCAMBIA 1302 was chosen to be the vector and the gene was driven by the CaMV 35S promoter, and we designed the linker to combine gfp with the genes of PLR and SDH. The construct was transformed into Agrobacterium rhizogenes used to infect the tobacco leaves and then the hairy roots with foreign gene were induced. We chose the hairy roots which grew well sending to liquid cultures and confirmed the expression of foreign proteins by PCR, fluorescence stereomicroscope observation and Western blot of GFP. And we measured the quantity of GFP by ELISA. We observed the cross section of the hairy root by fluorescence microscopy to find the location of the expression of fusion proteins in hairy roots. The results indicated that the fusion proteins were expressed in the stele of transgenic hairy roots. Protein extracts from transgenic hairy roots were prepared and tested for their bioactivity and the products were detected the by LC-MS. The result indicated that the fusion protein of PLR had bioactivity to biotransform pinoresinol to secoisolariciresinol. However, the product, mataresinol cannot be detected in the bioactivity analysis of the fusion protein of SDH. The reasons might be the active domain on SDH was affected by fusing with GFP, so the bioactivity of SDH declined. Furthermore, we also found the metabolites in the hairy roots which expressed fused PLR to be different from the hairy roots which did not express fused PLR. We speculate that the expression of the fusion protein of PLR and GFP might cause the in vivo change in secondary metabolite pathway of the transgenic hairy roots. We can do the advance analysis of this phenomenon in the future. This study indicates that the hairy roots can be the expression system of metabolic engineering research