小麦TaNPR1互作蛋白的筛选及其与TaCIPK10互作验证

doi:10.13560/j.cnki.biotech.bull.1985.2026-0251

摘要/Abstract

摘要：

目的针对前期挖掘到的小麦抗叶锈病相关蛋白TaNPR1，筛选其互作蛋白并验证与激酶TaCIPK10的互作关系，为进一步解析TaNPR1调控小麦抗叶锈病的分子机制提供依据。方法构建pGBKT7-TaNPR1诱饵载体，利用酵母双杂交技术筛选其互作蛋白；通过转录组数据分析编码候选互作蛋白基因的表达模式；采用亚细胞定位、蛋白结构对接模拟、酵母双杂交（Y2H）、双分子荧光互补（BiFC）技术对候选互作蛋白TaCIPK10进行亚细胞定位分析和互作验证。结果在小麦酵母双杂交文库中共筛选到86个TaNPR1候选互作蛋白，其中TaCIPK10在抗病品种TcLr26接种叶锈菌24 h后表达显著上调；亚细胞定位显示TaCIPK10主要定位在细胞膜；蛋白结构对接模拟表明二者在结构上具有互作潜力；Y2H和BiFC实验证实TaNPR1和TaCIPK10在体外和体内均存在直接互作。结论 TaNPR1与钙信号相关激酶TaCIPK10直接互作，推测钙信号通路可能通过磷酸化机制参与水杨酸介导的小麦抗叶锈病调控网络。

关键词: 小麦, 叶锈菌, TaNPR1, 互作蛋白, TaCIPK10

Abstract:

Objective To further elucidate the molecular mechanism by which TaNPR1 regulates wheat resistance to leaf rust, we screened for its interacting proteins and confirmed its interaction with the kinase TaCIPK10, thereby laying a solid foundation for understanding this regulatory network. Methods We constructed a pGBKT7-TaNPR1 bait vector and screened for its interacting proteins using yeast two-hybrid (Y2H) technology. Transcriptomic data analysis was performed to characterize the expression patterns of genes encoding these candidate interactors. Subsequently, the candidate interactor TaCIPK10 was selected for further validation. We conducted subcellular localization analysis and verified the interaction through a combination of protein docking simulations, yeast two-hybrid (Y2H) revalidation, and bimolecular fluorescence complementation (BiFC) assays. Results A total of 86 candidate TaNPR1-interacting proteins were identified from a wheat yeast two-hybrid library. Notably, TaCIPK10 was significantly upregulated at 24 hours post-inoculation with Puccinia triticinain the resistant wheat line TcLr26. Subcellular localization analysis revealed that TaCIPK10 is primarily localized to the plasma membrane. Protein docking simulations indicated structural compatibility for their interaction, which was empirically confirmed by Y2H and BiFC assays, demonstrating that TaNPR1 and TaCIPK10 physically interact both in vitro and vivo. Conclusion The direct interaction between TaNPR1 and the calcium signaling-related kinase TaCIPK10 suggests that the calcium signaling pathway may participate in the SA-mediated regulatory network of wheat leaf rust resistance via phosphorylation mechanisms.

Key words: wheat, leaf rust fungus, TaNPR1, interacting proteins, TaCIPK10

牛梓宇, 牛泽霖, 谷冰艳, 贾怡菲, 周晓栋, 刘刚, 王冬梅, 王荣纳. 小麦TaNPR1互作蛋白的筛选及其与TaCIPK10互作验证[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0251.

NIU Zi-yu, NIU Ze-lin, GU Bing-yan, JIA Yi-fei, ZHOU Xiao-dong, LIU Gang, WANG Dong-mei, WANG Rong-na. Screening of TaNPR1-interacting Proteins in Wheat and Validation of Its Interaction with TaCIPK10[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0251.

图/表 9

图1 pGBKT7-TaNPR1诱饵载体构建A：TaNPR1基因扩增产物；B：pGBKT7-TaNPR1诱饵载体双酶切验证

Fig. 1 Construction of the pGBKT7-TaNPR1 bait vectorA: TaNPR1 gene amplification product; B: double digestion verification of pGBKT7-TaNPR1 bait vector

图2 pGBKT7-TaNPR1诱饵载体自激活和毒性检测

Fig. 2 Autoactivation and toxicity detection of pGBKT7-TaNPR1 bait protein

图3 TaNPR1互作蛋白的筛选

Fig. 3 Screening of TaNPR1-interacting proteins

图4 TaNPR1互作蛋白的功能注释及通路分析A：生物过程；B：KEGG通路；C：分子功能

Fig. 4 Functional annotation and pathway analysis of TaNPR1-interacting proteinsA: Biological process; B: KEGG pathway; C: molecular function

图5 TaKIPK2、TaStK24和TaCIPK10在接种叶锈菌后不同时间点表达模式分析不同的小写字母表示显著差异（P<0.05）；FPKM表示每百万个有效测序片段中，每千碱基转录本长度上的片段数目

Fig. 5 Analysis of the expression patterns of TaKIPK2, TaStK24, and TaCIPK10 at different time points after inoculation with leaf rust fungusDifferent lowercase letters indicate significant differences (P<0.05); FPKM indicates the number of fragments per kilobase of transcript length in every million effective sequencing fragments

图6 TaCIPK10的亚细胞定位A：TaCIPK10跨膜结构域预测；B：TaCIPK10亚细胞定位；Bright：明场；GFP：绿色荧光蛋白；mCherry：红色荧光蛋白；Merge：叠加场。比例尺为20 μm

Fig. 6 Subcellular localization of TaCIPK10A: Prediction of the transmembrane domain of TaCIPK10; B: subcellular localization of TaCIPK10. Bright: Bright field. GFP: Green fluorescent protein. mCherry: Red fluorescent protein. Merge: Merged images of the GFP, mCherry and bright field images. Scale bar: 20 μm

图7 蛋白三维结构及互作预测模型

Fig. 7 Protein 3D structure and interaction prediction models

图8 pGADT7-TaCIPK10载体构建及TaNPR1与CIPK10互作的酵母双杂交验证A：TaCIPK10基因扩增产物；B：pGADT7-TaCIPK10载体单酶切验证；C：TaNPR1与TaCIPK10酵母双杂交

Fig. 8 Construction of pGADT7-TaCIPK10 vector and yeast two-hybrid verification of the interaction between TaNPR1 and TaCIPK10A: Amplification product of TaCIPK10 gene; B: single enzyme digestion verification of pGADT7-TaCIPK10 vector; C: yeast two-hybrid of TaNPR1 and TaCIPK10

图9 TaNPR1和TaCIPK10的BiFC互作验证GFP：绿色荧光蛋白；Bright：明场；Merge：叠加。比例尺为20 μm

Fig. 9 BiFC interaction verification of TaNPR1 and TaCIPK10GFP: Green fluorescent protein. Bright: Bright field. Merge: Merged images of the GFP and bright field images. Scale bar: 20 μm

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