小麦E3泛素连接酶TaRING1互作靶标筛选与验证
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西北农林科技大学

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“十四五”国家重点研发计划子课题(2021YFD1401001-03)


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

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    【Objective】 In order 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 protein of TaRING1 was screened by yeast two-hybrid technique and verified by bimolecular fluorescence complementation and luciferase complementation assays. The ubiquitin function of TaRING1 was verified by ubiquitination experiments. The subcellular localization of target TaRIP92 was observed by transient expression in Nicotiana benthamiana and protoplast transformation in wheat. 【Results】 Using TaRING1 as bait, the interaction target TaRIP92 was screened by yeast two-hybrid technology, and the interaction between TaRING1 and TaRIP92 was verified by LCA and BiFC. In vitro ubiquitination experiments proved that TaRING1 could ubiquitinate TaRIP92. Transient expression of Nicotiana benthamiana and wheat protoplast transformation showed that TaRIP92 protein was localized in mitochondria. 【Conclusion】 TaRING1 interacts with mitochondrial protein TaRIP92 and ubiquitinates it.

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王金棉,韩璐昕,康振生,等.小麦E3泛素连接酶TaRING1互作靶标筛选与验证[J].西北植物学报,2024,44(9):1411-1419

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  • 收稿日期:2024-05-06
  • 最后修改日期:2024-05-16
  • 录用日期:2024-05-17
  • 在线发布日期: 2024-07-15
  • 出版日期: 2024-09-05
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