以酵母菌雙雜合系統篩選與RolC 具交互作用之蛋白質

Abstract

毛狀根為植物受到根毛農桿菌感染後，農桿菌將其Ri質體上的T-DNA隨機插入植物染色體產生的根狀組織，具有快速生長、基因組成穩定、能在不含植物賀爾蒙培養基中長期繼代培養、累積高量植物次級代謝物的特性。因為有這些特性，毛狀根被廣泛應用在高價值植物藥與重組蛋白質生產當中。然而，並不是所有的植物都能被根毛農桿菌感染產生毛狀根，因此了解毛狀根分化生成的分子作用機制將能大幅提升毛狀根的應用潛力。在T-DNA當中，rolB與rolC基因被認為在誘導毛狀根扮演重要關鍵的角色。本實驗室先前以酵母菌雙雜合法，發現ORF13a、PHI-2與NtbZIP和RolB蛋白質有交互作用。但以同樣的方法尋找與RolC有交互作用的蛋白質時，發現單獨表現GAL4 DNA-BD/RolC融合蛋白的酵母菌會自動活化下游的報導基因，造成篩選結果幾乎為偽陽性。本研究透過隨機突變rolC基因降低RolC的自活化能力，再進行酵母菌雙雜合實驗，找出在菸草毛狀根當中可能和RolC蛋白質有交互作用的蛋白質。此外，舊有的酵母菌接合轉形方法在本研究中存在接合轉形效率 (mating efficiency) 過低的問題，故本研究改良了接合轉形方法，進一步提升酵母菌的接合轉形效率，並以此新的方法，發現46個可能與RolC蛋白質有交互作用的蛋白質。

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.