小麦E3泛素连接酶TaRING1互作靶标筛选与验证  

Screening and validation of interaction targets for wheat E3 ubiquitin ligase TaRING1

作　　者：王金棉 韩璐昕 康振生[2,3] 刘杰[1,3] WANG Jinmian;HAN Luxin;KANG Zhensheng;LIU Jie(College of Life Sciences,Northwest A&F University,Yangling,Shaanxi 712100,China;College of Plant Protection,Northwest A&F University,Yangling,Shaanxi 712100,China;State Key Laboratory for Crop Stress Resistance and High-Efficiency Production,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]西北农林科技大学植物保护学院,陕西杨凌712100 [3]西北农林科技大学,作物抗逆与高效生产全国重点实验室,陕西杨凌712100

出　　处：《西北植物学报》2024年第9期1411-1419,共9页Acta Botanica Boreali-Occidentalia Sinica

基　　金：“十四五”国家重点研发计划子课题项目(2021YFD1401001-03)。

摘　　要：【目的】为探究TaRING1在小麦与条锈菌互作过程中的作用,解析其作用机理,为小麦条锈病的绿色防控提供理论依据。【方法】利用酵母双杂交技术筛选TaRING1的互作靶标蛋白,并通过荧火素酶互补实验(LCA)及双分子荧光互补(BiFC)验证互作,通过泛素化实验对TaRING1的泛素功能进行验证,通过烟草瞬时表达及小麦原生质体转化分析靶标TaRIP92的亚细胞定位。【结果】以TaRING1作为诱饵,利用酵母双杂交技术筛选到互作靶标TaRIP92,并通过LCA及BiFC验证TaRING1与TaRIP92互作,通过体外泛素化实验证明TaRING1可以泛素化底物TaRIP92,烟草瞬时表达及小麦原生质体转化发现TaRIP92蛋白定位于线粒体。【结论】TaRING1通过与线粒体蛋白TaRIP92互作并对其进行泛素化。[Objective]The study aims to explore the role of TaRING1 in the interaction between wheat and stripe rust,analyze its mechanism and provide theoretical basis for the green prevention and control of wheat stripe rust.[Methods]The interaction target proteins of TaRING1 were screened by yeast two-hy-brid assays and verified by bimolecular fluorescence complementation(BiFC)and luciferase complementa-tion(LCA)assays.The ubiquitylation function of TaRING1 was verified by ubiquitination analysis.The subcellular localization of target TaRIP92 was observed by transient expression in Nicotiana benthamiana and wheat protoplasts.[Results]Using TaRING1 as bait,the target protein TaRIP92 was screened by yeast two-hybrid,and the interaction between TaRING1 and TaRIP92 was verified by LCA and BiFC.In vitro ubiquitination assays proved that TaRING1 ubiquitinated TaRIP92.Transient expression in N.benthamiana and wheat protoplasts showed that TaRIP92 was localized in mitochondria.[Conclusion]TaRING1 interacts with and ubiquitinates the mitochondrial protein TaRIP92.

关 键 词：小麦条锈病 E3泛素连接酶 TaRING1 TaRIP92 

分 类 号：Q78[生物学—分子生物学] S512.1[农业科学—作物学]