Cloning and Expression of Dirigent Protein from Podophyllum pleianthum Hance

• Main Authors: Han-Jung Kuo, 郭翰蓉
• Other Authors: Kung-Ta Lee
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/56936677970780375355

碩士 === 國立臺灣大學 === 生化科技學系 === 99 === 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.