拟南芥WRKY61转录因子的转录活性与互作蛋白分析

Exploring the Transcriptional Activity and Interacting Proteins of WRKY61 Transcriptional Factor in Arabidopsis thaliana

该研究采用双荧光素酶报告系统、酵母双杂交和双分子荧光互补实验,对拟南芥AtWRKY61的转录活性及与AtWRKY61转录因子的互作蛋白进行了分析,并用qRT-PCR方法分析AtWRKY61对多种非生物逆境的响应特征,为进一步揭示AtWRKY61的功能与分子调控机制奠定基础。绿色荧光蛋白介导的亚细胞定位分析显示,AtWRKY61定位于细胞核内;基于原生质体的双荧光素酶报告系统和酵母实验发现,AtWRKY61具有转录抑制活性。qRT-PCR分析表明,AtWRKY61对多种非生物逆境的处理具有明显的响应,可能是在多条信号通路中发挥作用。酵母双杂交与双分子荧光互补分析表明,AtWRKY61与自身以及同组的AtWRKY9和AtWRKY72存在互作关系,暗示可能通过形成WRKY复合物来行使特定的转录抑制功能。

In this study,we analyzed the transcriptional activity and interacting proteins of AtWRKY61 transcription factor through dual-luciferase（LUC）,yeast two-hybrid（Y2 H）and bimolecular fluorescence complementation（BiFC）experiments.qRT-PCR was used to analyze the response characteristics of AtWRKY61 to various abiotic stress.They lay the foundation for further revealing the function and molecular regulation mechanism of AtWRKY61.Subcellular localization through green fluorescence protein（GFP）shows that AtWRKY61 is localized in the nucleus only.A dual-LUC assay in Arabidopsis protoplasts and yeast experiment reveal that AtWRKY61 has transcriptional repression activity.qRT-PCR results indicate that transcription of AtWRKY61 responded to multiple abiotic stresses and hormone stimuli,suggesting AtWRKY61 may participate in multiple signaling pathways.Y2 Htest demonstrated that AtWRKY61 interacted with itself and two closely related paralogs AtWRKY9 and AtWRKY72,which were further confirmed by BiFC.This implies that AtWRKY61 might perform its transcriptional repression function via forming a complex with itself and two other closely related homologs.