水稻轉錄因子OsERF106交互作用蛋白的分離及鑑定

Abstract

APETALA2/Ethylene-Responsive Factor (AP2/ERF)轉錄因子為一多基因家族，除了參與調節植物生長發育，亦可影響植物對逆境的耐受性。本研究室先前的研究成果指出，水稻OsERF106的表現受鹽逆境誘導。過量表現OsERF106的水稻幼苗地上部導致生長遲緩，體內蓄積過量的鈉離子、CAT活性較低，且植株不耐鹽逆境，OsERF106可能做為水稻鹽逆境的調控因子。在本試驗中，我們注意到過量表現OsERF106的水稻幼苗還具有較長的根系。然而，我們對於OsERF106如何影響水稻生長發育及鹽逆境的耐受性，仍所知有限。由於AP2/ ERF可能藉由與其它蛋白質的相互結合，進而影響其蛋白的次細胞定位、穩定性、轉錄活性及標的特異性，從而改變或調控植物的生長發育及非生物性逆境反應。是以找到與OsERF106交互作用之蛋白質並鑑定明瞭其生理功能至為重要。因此，我們利用IP-LC-MS/MS (Immunoprecipitation (IP)/liquid chromatography-tandem mass spectrometry)的方法，篩選與OsERF106交互作用的可能蛋白質，試圖釐清OsERF106的作用機制。藉由西方墨點法 (Western blot)可測得OsERF106-GFP融合蛋白存在於過量表現株的地上部及地下部。IP-LC-MS/MS分析及文氏圖 (Venn diagram)比對顯示，無論是在正常或鹽 (150 mM NaCl)處理條件下，有77和22個蛋白分別可在水稻地上部和地下部與OsERF106交互作用，此些蛋白主要和逆境反應、生長發育及細胞組件相關。針對此些蛋白質，本試驗選取已知和鹽逆境及生長發育相關之蛋白質OsMBF1 (multiprotein bridging factor1) 及Os03gBTF3 (basic transcription factor 3)進行後續研究。OsMBF1是一種轉錄共因子 (transcriptional co-factor)已證實廣泛參與在非生物逆境反應中，而BTF3目前已知會調節植物逆境反應相關基因的表現，以及參與植物生長發育的過程。利用雙分子螢光互補法(BiFC, Bimolecular fluorescence complementation)實驗發現OsMBF1、Os03gBTF3可能與OsERF106具有交互作用。後續將對OsMBF1和Os03gBTF3在OsERF106調控水稻鹽逆境耐受性或生長發育所扮演的角色多作探討。

APETALA 2/Ethylene-Responsive Factors (AP2/ ERF) belong to a multiple gene family, and involved in the regulation of plant growth, development and stress response. In our previous study, we showed that the expression of OsERF106 was induced under salt stress. The OsERF106-overexpression transgenic rice displayed retarded growth, Na+ overaccumulation, low CAT activity and increased salt sensitivity. These results suggested that OsERF106 may serve as a negative regulator in response to salinity stress tolerance of rice. In this study, we noticed that the OsERF106-overexpression transgenic rice had a longer primary root. However, it’s still not clear how OsERF106 affect rice plant growth and tolerance to salt stress. AP2/ERFs may physically interact with other proteins, which affect its localization, stability, abundance, transcriptional activity and target specificity to regulate plant growth and development and abiotic stress response. Thus, in order to find the interacting proteins of OsERF106 and further understand its role and physiological function related to rice plant growth and salinity stress tolerance, we screened OsERF106 interacting protein by LC-MS/MS. The result showed that under normal growth condition (0 mM) and 150 mM NaCl treatment, there were 77 and 22 putative proteins interacting with OsERF106 in shoot and root, respectively. Most of these proteins are known to be involved in stress response, plant growth regulation and cellular components. Among them, we selected two known major transcription factors, including OsMBF1 (multiprotein bridging factor1) and Os03gBTF3 (basic transcription factor 3). For further studies, OsMBF1 is transcription co-factor participating in abiotic stress, and BTF3 regulates stress related gene and is involved in plant growth and development. BiFC analysis revealed potential interactions between OsERF106 and OsMBF1, OsERF106 and Os03gBTF3. In the future, we will take more action on OsMBF1 and Os03gBTF3 genes to address their role on rice plant growth and stress response.