利用雙分子螢光互補法研究與 RolB 有交互作用之蛋白

Abstract

RolB is crucial for the formation of hairy roots, but how it works in plant tissue is still unknown. Our previous work found that RolB protein might interact with ORF13a, NtbZIP, and PHI-2 by using yeast two-hybrid assay. In this study, we further confirmed the interactions between RolB and those three proteins in living plant cells. We constructed plasmids for bimolecular fluorescence complementation (BiFC) assay by Gateway cloning system, and applied BiFC assay in Arabidopsis protoplasts. Besides, to figure out the subcellular localization of each protein used in this study, we constructed and expressed EYFP-fusion proteins in Arabidopsis protoplasts. We found RolB was expressed in the nucleus and the cytoplasm, ORF13a as well as NtbZIP were expressed only in the nucleus, and PHI-2 might be expressed both in the cytoplasm and the nucleus. The BiFC assay show that RolB can interact with ORF13a, NtbZIP, and PHI-2 in the nucleus in Arabidopsis protoplasts. We also confirmed the interaction between RolB and Nt14-3-3, which had been reported previously, and found that they can interact in both the cytoplasm and the nucleus in Arabidopsis protoplasts. Moreover, we discovered that Nt14-3-3 can interact with NtbZIP in the nucleus. Our results infer that the protein-protein interactions between RolB, ORF13a, NtbZIP, PHI-2, and Nt14-3-3 might modulate ABA signaling pathway to enhance root growth.