Screening of the Interaction Proteins of RolC by Use of Yeast Two-hybrid System

• Main Authors: Ke-Jin Lin, 林科錦
• Other Authors: Kung-Ta Lee
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/rvjg9x

碩士 === 國立臺灣大學 === 生化科技學系 === 106 === Hairy roots are root-like tissues, which are resulted from the infection of Agrobacterium rhizogenes. During infection, Agrobacterium would transfer the T-DNA from its Ri plasmid to plant cell and randomly insert into plant chromosome. Hairy roots are characterized by high growth rate, genetic stability, and high accumulation of plant secondary metabolite, and they could be maintained in hormone-free medium. Due to these features, hairy roots are widely applied in production of high-value pharmaceutical molucules and recombinant proteins. However, still many plants are resistant to the infection of A. rhizogenes, the utilization of hairy root culture in these plants is limited. Understanding the molecular mechanism during hairy root formation increases applicability of medical herbs hairy root. Our previous study found that RolB protein can interact with ORF13a, NtbZIP, and PHI-2 by using yeast two-hybrid assay. But when applying the same method to screen the interacting proteins of RolC, we found that RolC would autoactivate the the reporter genes in Y2HGold, resulting too many false positive. In this study, we randomly mutated rolC gene by error-prone PCR to reduce autoactivity of RolC, and used these mutant RolC as bait to screen the interactors in tobacco hairy roots. Besides, we applied new mating method to increase the mating efficiency of yeasts. Using the new mating method, we found 46 potential interacting proteins of RolC.