与拟南芥TR1相互作用的蛋白质筛选和鉴定

Screening and validation of candidate proteins interacting with TR1 in Arabidopsis thaliana

摘要:

TR1是一个定位于质膜上的E3连接酶，前期的研究表明它可赋予多种植物对盐、干旱和热胁迫的耐受性。但是，其发挥功能的分子机制尚不清楚，与其存在相互作用的靶蛋白也未找到。本研究利用酵母双杂交系统以AtTR1-△119为诱饵蛋白筛选拟南芥归一化通用型cDNA文库，结果获得了13个与其有潜在互作的候选蛋白。经酵母正向和反向多次验证发现转化了TR1与CURT1C和TR1与SWEET5的酵母在三缺和四缺培养基中均正常生长，而且在含有X-α-gal的培养基中出现蓝斑。双分子荧光互补技术证明TR1与CURT1C和SWEET5之间也有相互作用，这对进一步研究AtTR1及其相互作用蛋白质在植物抗逆中的作用和分子机制奠定了基础。

Abstract:

TR1 is an E3 ligase localized in the plasma membrane, and the previous studies indicated that it played a key role in salt, drought and heat stress response in many plants.However, the molecular mechanism of its function is unclear, and the target protein interacting with it is not found.In this study, a yeast two-hybrid system screening using TR1-△119 as a bait was performed to screen the Normalized Universal Arabidopsis cDNA library and found 13 candidate proteins with potential interaction. Forward and reverse two-hybrid system multiple validation revealed that diploid yeast containing TR1 and CURT1C or TR1 and SWEET5 will grow on SD/-Trp/-Leu/-His and SD/-Trp/-Leu/-His/-Ade/X-α-gal minimal media and see blue colonies. BiFC technique also demonstrated that TR1 interacts with CURT1C and SWEET5,providing the foundation for further understanding of the functions and molecular reaction tolerance mechanism．