利用雙分子螢光互補法研究與 RolB 有交互作用之蛋白

Abstract

RolB對於毛狀根生成具有重要影響，但是目前仍不清楚RolB在植物細胞中的分子作用機制。實驗室先前在酵母菌雙雜合法進行的篩選結果，發現ORF13a、NtbZIP、PHI-2三種蛋白質在酵母菌中與RolB具交互作用。為了在植物細胞內進一步驗證RolB與這三種蛋白質的活體結合，本研究藉由Gateway選殖系統建立雙分子螢光互補法的表現質體，並將質體表現在阿拉伯芥原生質體表現系統中。此外，為了了解本實驗使用的蛋白質各自在細胞中的表現位置，本研究將各蛋白質分別與EYFP結合，並表現在阿拉伯芥原生質體中。實驗結果發現RolB表現在細胞質及細胞核，ORF13a和NtbZIP皆只表現於細胞核，而PHI-2則有可能表現在細胞質及細胞核。雙分子螢光互補法的結果證實，RolB在阿拉伯芥原生質體能分別與ORF13a、PHI-2 和NtbZIP在細胞核中結合。過去文獻曾指出RolB與Nt14-3-3蛋白質具有交互作用，本研究亦使用雙分子螢光互補法驗證這兩種蛋白質之間的結合。結果發現，RolB能與Nt14-3-3於細胞質和細胞核中結合。除此之外，我們還發現Nt14-3-3也能與NtbZIP產生交互作用。根據本研究結果，RolB與ORF13a、NtbZIP、PHI-2以及Nt14-3-3之間的交互作用有可能藉由影響離層酸的訊息傳遞路徑，進而促使毛狀根生長。

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.