【摘要】 小麦叶锈病严重危害小麦生产，系统研究抗病相关蛋白作用机制对保障粮食安全具有重大意义。本研究分析了14-3-3蛋白编码基因TaGRF3在小麦与叶锈菌互作过程中的表达模式，通过构建小麦抵御叶锈菌侵染的cDNA文库，筛选TaGRF3的互作蛋白并进行验证。研究结果表明，荧光定量PCR和数字PCR分析发现TaGRF3能够被叶锈菌侵染诱导表达，且在抗病组合接种叶锈菌后18 h达到最高峰。同时，以抗病组合小麦Tc Lr26为材料，采用重组技术构建了高质量的小麦抵御叶锈菌侵染的酵母cDNA文库；利用诱饵表达载体pGBKT7-TaGRF3对cDNA文库进行筛选，获得198个阳性克隆，通过测序和序列比对，共获得75个候选互作蛋白；功能富集分析表明TaGRF3与其互作蛋白主要参与氨基酸代谢、氧化还原反应、糖异生及去除超氧化物自由基等过程；结合转录组数据分析和功能注释信息选取TaPRXⅥ进行一对一互作验证；通过酵母双杂交（Y2H）和双分子荧光互补（BiFC）发现两者存在相互作用。本研究初步探索了在小麦抵御叶锈菌侵染中14-3-3蛋白TaGRF3的表达模式和互作蛋白，对下一步研究其功能和分子调控机制奠定了理论基础。更多还原

【Abstract】 Wheat leaf rust seriously endangered wheat production. The systematic study of the action mechanism of disease-related proteins is of great significance to ensuring food security. This paper explored the expression pattern of the 14-3-3 protein-encoding gene TaGRF3 during the interaction between wheat and leaf rust bacteria. By constructing and screening the cDNA library of wheat that resists leaf rust bacteria infection, TaGRF3 interacting protein was obtained. Through fluorescence quantitative PCR and digital PCR analysis, it was found that TaGRF3 expression could be induced by infection of leaf rust bacteria, and peaked at 18 hours after inoculation of leaf rust bacteria. At the same time, the leaves of disease-resistant wheat TcLr26 were inoculated with leaf rust and sampled for construction of a high-quality yeast cDNA library using recombinant technology. The cDNA library was screened using the bait expression vector pGBKT7-TaGRF3, and a total of 198 positive clones were obtained. Through sequencing and sequence comparison, a total of 75 candidate interaction proteins were obtained. Functional enrichment analysis showed that these proteins were mainly involved in amino acid metabolism, redox reaction, glucogenesis and removal of superoxide radicals. TaPRXⅥ was selected as the candidate interaction protein of TaGRF3 based on transcriptome data analysis and functional annotation information. Their interaction was further confirmed through yeast two-hybrid(Y2H) and bimolecular fluorescence complementation(BiFC). This study initially explored the expression pattern and interacting protein of the 14-3-3 protein TaGRF3 in wheat against leaf rust infection, laying a theoretical foundation for the next step of studying its functional and molecular regulatory mechanism.更多还原