Expression of Pinoresinol lariciresinol reductase and Secoisolariciresinol dehydrogdnase in Nicotiana benthaminana hairy roots
Date Issued
2010
Date
2010
Author(s)
Chung, Cheng-Han
Abstract
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.
Subjects
毛狀根
木酚素
綠螢光蛋白
Type
thesis
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