黄萎病菌诱导下陆地棉酵母双杂交文库的构建及GhMAPKKK2互作蛋白的筛选  

Construction of Yeast Two-Hybrid Library of Upland Cotton Induced by Verticillium dahliae and Screening of GhMAPKKK2 Interacting Proteins

作　　者：程贯富 张文祥 郭新悦 张国帅 吕晓迪 代培红[1] 雷建峰[2] 李月[1] CHENG Guanfu;ZHANG Wenxiang;GUO Xinyue;ZHANG Guoshuai;LYU Xiaodi;DAI Peihong;LEI Jianfeng;LI Yue(College of Life Sciences,Xinjiang Agricultural University,Urumqi,Xinjiang 830052;College of Agriculture,Xinjiang Agricultural University,Urumqi,Xinjiang 830052)

机构地区：[1]新疆农业大学生命科学学院,新疆乌鲁木齐830052 [2]新疆农业大学农学院,新疆乌鲁木齐830052

出　　处：《核农学报》2025年第1期68-76,共9页Journal of Nuclear Agricultural Sciences

基　　金：国家自然科学基金(32160494);新疆维吾尔自治区自然科学基金资助项目(2023D01E03);新疆维吾尔自治区“天山英才”培养计划“三农”骨干人才-农业技术推广人才项目(2023SNGGNT063);研究生科研创新项目(XJAUGRI2024030)。

摘　　要：为了探究棉花响应黄萎病菌侵染的分子机制并筛选GhMAPKKK2的互作蛋白,以陆地棉XLZ35为材料,基于Gateway克隆技术构建了棉花黄萎病菌诱导的陆地棉酵母双杂交文库,其中初级文库和次级文库的库容分别为1.2×10^(7)和2.0×10^(7)CFU,插入片段重组率均为100%且平均长度大于1000 bp。次级文库质粒浓度为953 ng·μL^(-1),符合构建酵母文库的标准,可用于酵母双杂交筛选互作蛋白。构建了pGBKT7-GhMAPKKK2表达载体,并验证了该载体对酵母细胞无毒性且不存在自激活活性。以GhMAPKKK2为诱饵蛋白,利用酵母文库初步筛选,获得81个阳性互作蛋白,经测序对比选取6个候选蛋白,通过酵母双杂交及双分子荧光互补试验(BiFC)验证,明确了候选蛋白GhFLA与GhMAPKKK2存在互作关系。本研究结果为解析GhMAPKKK2调控棉花抗黄萎病反应分子机制及调控网络提供了重要参考。In order to investigate the molecular mechanism of cotton in response to Verticillium dahliae(V.dahliae)invasion and to identify the interacting proteins of GhMAPKKK2,a yeast two-hybrid library of Gossypium hirsutum XLZ35 induced by V.dahliae was constructed based on Gateway cloning technology.The primary and secondary library capacities were set at 1.2×10^(7)CFU and 2.0×10^(7)CFU,respectively,with a plasmid concentration of 953 ng·μL^(-1).The recombination rate of insertion fragments achieved 100%,and the average length of the inserts sexceeded 1000 bp,which satisfy the prerequisites for yeast library construction and enable efficient screening for interacting proteins.The bait protein vector,pGBKT7-GhMAPKKK2,was successfully constructed and validated as non-toxic to yeast and devoid of self-activation activity.Using this bait vector,81 positive proteins were initially idenified to interact with GhMAPKKK2.Following post-sequencing and alignment analysis,6 promising candidates were selected for further verification.Through rigorous yeast two-hybrid and bimolecular fluorescence complementation(BiFC)experiments,we confirmed a robust interaction between the candidate protein GhFLA and GhMAPKKK2.This study offers crucial insights into the mechanistic and interactional framework of GhMAPKKK2 in mediating cotton resistance to Verticillium wilt in the future.

关 键 词：棉花 黄萎病菌 酵母双杂交 互作蛋白 GhMAPKKK2 

分 类 号：S435.621.2[农业科学—农业昆虫与害虫防治]