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| 禾谷镰刀菌木聚糖酶FGSG_03624互作蛋白的筛选及其与小麦通用应激蛋白TaUSP-1A的互作分析 |
| Screening of Fusarium Graminearum Xylanase FGSG_03624 Interacting Proteins and Analysis of Its Interaction with Wheat Universal Stress |
| 投稿时间：2025-09-10 修订日期：2026-01-06 |
| DOI： |
| 中文关键词: 禾谷镰刀菌 木聚糖酶FGSG_03624 小麦 酵母双杂交 通用应激蛋白TaUSP-1A |
| 英文关键词:Fusarium graminarum xylanase FGSG_03624 wheat yeast two-hybrid universal stress protein TaUSP-1A |
| 基金项目:安徽省高校自然科学研究重点项目(2022AH051088)；滁州学院科研启动(2022qd50)；安徽省高校自然科学研究重大项目(2023AH040223) |
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| 中文摘要: |
| 木聚糖酶FGSG_03624在禾谷镰刀菌侵染小麦过程中发挥着双重作用，既能破坏细胞壁的完整性，又能诱导细胞坏死并激活植物免疫反应。为进一步解析FGSG_03624诱导小麦免疫反应的机制，本研究对FGSG_03624进行了亚细胞定位分析，并利用已构建的受禾谷镰刀菌侵染后的小麦穗部cDNA文库，以FGSG_03624为诱饵，通过酵母双杂交技术筛选其互作蛋白，经回转实验、荧光素酶互补实验和双分子荧光互补实验进一步验证互作关系。结果表明，FGSG_03624属于位于细胞膜外的分泌蛋白，可能定位于细胞壁和/或细胞间隙，因此需采用分裂泛素系统酵母双杂交技术筛选互作蛋白。通过自激活检测和功能检测分析，重组载体pBT3STE-FGSG_03624在酵母细胞NMY51中能够正常表达，可作为筛库的重组诱饵载体，但需添加12mM 3-AT抑制其自激活。筛选小麦cDNA文库，最终共获得40个可能与FGSG_03624互作的蛋白。这些蛋白涉及到转录调控、信号传导、胁迫响应、膜运输和植物-病原菌互作等方面。酵母回转实验、荧光素酶互补实验和双分子荧光互补实验验证了与活性氧代谢及胁迫响应相关的通用应激蛋白TaUSP-1A为FGSG_03624的互作蛋白。禾谷镰刀菌侵染小麦后TaUSP-1A上调表达，过表达TaUSP-1A的拟南芥叶片能够增强其对禾谷镰刀菌的抗性。本研究为深入研究FGSG_03624激发小麦免疫反应的分子机制及发掘寄主中更多抗病基因提供了基础。 |
| 英文摘要: |
| The xylanase FGSG_03624 plays a dual role in the infection of wheat by Fusarium graminearum, which can damage the integrity of the cell wall, induce cell necrosis and activate the plant immune response. In order to further understand the mechanism of FGSG_03624 inducing immune response in wheat, this study carried out subcellular localization analysis of FGSG_03624,and the FGSG_03624 was used as bait to screen wheat ear cDNA library infected by Fusarium graminearum through yeast two-hybrid technology. Putative interacting proteins were further validated through rotation assay, luciferase complementation assay (LCA) and bimolecular fluorescence complementation assay (BiFC). The results showed that FGSG_03624 is an extracellular secreted protein, and may be located in the cell wall and/or intercellular space. Therefore, the yeast two-hybrid technology of split ubiquitin system was used to screen the interacting proteins.Autoactivation and function testing assays showed that the recombinant bait vector pBT3STE-FGSG_03632 was expressed normally in yeast strain NMY51 and could be used for library screening, although 12 mM 3-AT was required to suppress autoactivation. A total of 40 proteins that may interact with FGSG_03624 were finally obtained by screening the wheat cDNA library. These proteins were involved in transcriptional regulation, signal transduction, stress response, membrane transport and plant-pathogen interaction in plants. Rotation, LCA, and BiFC assays confirmed that TaUSP-1A, a universal stress protein involved in reactive oxygen species metabolism and stress responses, interacted with FGSG_03624. The expression of TaUSP-1A was up-regulated after F. graminearum infection in wheat, and the Arabidopsis leaves with overexpression of TaUSP-1A could enhance the resistance to F. graminearum. |
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