大豆膜系统cDNA文库的构建及大豆疫霉效应子PsAvr3a互作蛋白的筛选  

Construction of the Soybean Membrane System cDNA Library and Interaction Proteins Screening for Effector PsAvr3a

作　　者：侯筱媛 车郑郑 李姮静 杜崇玉 胥倩 王群青 HOU Xiao-yuan;CHE Zheng-zheng;LI Heng-jing;DU Chong-yu;XU Qian;WANG Qun-qing(College of Plant Protection,Shandong Agricultural University,Tai'an 271018;State Key Laboratory of Crop Biology,Tai'an 271018)

机构地区：[1]山东农业大学植物保护学院,泰安271018 [2]作物生物学国家重点实验室,泰安271018

出　　处：《生物技术通报》2023年第4期268-276,共9页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31972249,32172387);山东省优秀青年基金(ZR2021YQ20)。

摘　　要：植物质膜是响应侵染的第一道屏障,其受体蛋白是感知病原菌入侵的雷达,是植物免疫系统的重要组成部分。大豆疫霉(Phytophthora sojae)通过分泌效应子干扰寄主免疫反应以促进自身侵染定殖。构建大豆膜系统酵母双杂交cDNA文库,为进一步探究大豆疫霉效应子与寄主膜蛋白靶标互作机制奠定基础。以大豆疫霉无毒效应子PsAvr3a为诱饵蛋白筛选文库,初步获得198个候选寄主靶标。酵母一对一验证试验表明,有5个候选靶标蛋白与PsAvr3a互作,包括酪氨酸分解酶、囊泡转运蛋白、乙烯合成调控酶、细胞代谢结合蛋白、抗逆的渗透蛋白等。通过双分子荧光互补试验(bimolecular fluorescence complementation, BIFC)与荧光素酶互补试验(luciferase complementation assay, LCA)验证得出GmACO、GmSec61与PsAvr3a均存在互作。确定2个寄主大豆膜蛋白乙烯前体ACC氧化酶GmACO、三聚体易位子GmSec61是大豆疫霉无毒效应子PsAvr3a的互作靶标。The plant plasma membrane is the first barrier in response to pathogen infection,and the receptor proteins on it are the radar for sensing pathogen invasion,which is an important part of the plant immune system.Phytophthora sojae disrupts the host immune activities of plants by secreting effectors,promoting Phytophthora colonization and spread.The construction of soybean split-ubiquitin based membrane yeast two-hybridization cDNA library is significant for exploring the interaction mechanisms between effectors and host cell membrane protein targets.The avirulent effector PsAvr3a was used as a bait protein to screen the library,and 198 candidate host targets were preliminarily obtained.The yeast two-hybrid technique between the interested target proteins and PsAvr3a revealed that five of them interacted with the bait vector.The enzyme that regulates tyrosine breakdown,the vesicle transport protein,the ethylene biosynthesis regulatory enzyme,the cell metabolic binding protein,the stress-resistant osmotic protein,and others were among the prospective host targets.The interactions amony GmACO,GmSec61 and PsAvr3a were verified by Bimolecular Fluorescence Complementation(BIFC)and Luciferase Complementation Assay(LCA).Therefore,two soybean membrane proteins,ethylene precursor ACC oxidase GmACO and trimeric transposon GmSec61,were identified as the host target of Phytophthora sojae avirulent effector PsAvr3a.

关 键 词：大豆 效应子 大豆疫霉 分裂泛素酵母双杂交 膜蛋白 无毒基因 

分 类 号：S435.651[农业科学—农业昆虫与害虫防治]