小麦叶锈菌与小麦互作的酵母双杂交cDNA文库构建与应用  

Construction and Utilization of Yeast Two-hybrid cDNA Library of Wheat Interacted by Puccinia triticina

作　　者：温晓蕾 李建嫄 李娜 张娜 杨文香 WEN Xiao-lei;LI Jian-yuan;LI Na;ZHANG Na;YANG Wen-xiang(Wheat Leaf Rust Research Center,College of Plant Protection,Agricultural University of Hebei,Technological Innovation Center for Biological Control of Plant Diseases and Insect Pests of Hebei Province,National Engineering Research Center for Agriculture in Northern Mountainous Areas,Baoding 071001;College of Agronomy and Biotechnology,Hebei Normal University of Science&Technology,Hebei Key Laboratory of Crop Stress Biology,Qinhuangdao 066000;College of Bioscience and Bioengineering,Xingtai University,Xingtai 054000;Hebei Plant Protection Plant Inspection Station,Shijiazhuang 050000)

机构地区：[1]河北农业大学植物保护学院植物病理学系,河北省农作物病虫害生物防治技术创新中心,国家北方山区农业工程技术研究中心,保定071001 [2]河北科技师范学院农学与生物科技学院,河北省作物逆境生物学重点实验室,秦皇岛066000 [3]邢台学院生物科学与工程学院,邢台054000 [4]河北省植保植检总站,石家庄050000

出　　处：《生物技术通报》2023年第9期136-146,共11页Biotechnology Bulletin

基　　金：国家自然科学基金项目(32172367);河北省产业体系小麦创新团队(HCT2018010204)。

摘　　要：为筛选小麦叶锈菌效应蛋白的互作靶标蛋白,解析效应蛋白干扰寄主的防御反应机制。本试验以小麦叶锈菌菌株13-5-28-1(JHKT)与感病品种Thatcher在0-12 d互作的供试样本为材料,利用SMART方法构建三框均一化的、用于筛选与小麦叶锈菌互作的小麦cDNA文库,采用同源重组的方法构建效应因子Pt34084重组诱饵载体pGBKT7-Pt34084,并以其为诱饵蛋白,利用共转化方法筛选互作的靶标蛋白。结果表明,构建的小麦叶锈菌与小麦互作的cDNA文库库容约1.05×10^(7)CFU/mL,滴度为1.2×10^(9)CFU/mL,平均插入片段大于1 kb,重组阳性率为100%,符合cDNA建库要求。构建的诱饵重组载体pGBKT7-Pt34084在SD/-Trp-His-Ade培养基中添加20 mmol/L 3-AT及400 ng/mL ABA可抑制其自激活性。利用该文库共筛选获得16个来自小麦及叶锈菌的不同候选互作蛋白,这些蛋白涉及植物代谢、激素信号转导、抗病反应等多个方面。预示Pt34084可通过多种渠道干扰寄主的防御反应,促进叶锈菌侵染。研究结果有助于解析小麦叶锈菌致病机理,为创造新的有效防控小麦叶锈菌策略奠定基础。In order to screen the target proteins interacting with effector proteins of the wheat leaf rust and to lay a foundation for dissecting the defense response mechanism of the effector proteins interfering with hosts and disease resistance breeding,the samples of interaction between the isolate13-5-28-1(JHKT)and susceptible wheat variety Thatcher at 0-12 d were collected.The three-frame homogenization was constructed by SMART method to screen the wheat cDNA library interacting with the wheat leaf rust.The homologous recombination method was used to construct the recombinant bait vector pGBKT7-Pt34084 of the effector factor Pt34084,and it was used as the bait protein to screen the interaction target protein by co-transformation method.The results showed that the cDNA library structure of the interaction between the wheat leaf rust and wheat was successfully constructed.The library capacity was about 1.05×10^(7)CFU/mL,the titer was 1.2×10^(9)CFU/mL,the average insertion fragment was more than 1 kb,and the recombination positive rate was 100%.The cDNA library met the requirement.The constructed bait recombinant vector pGBKT7-Pt34084 inhibited its self-activation after the addition of 20 mmol/L 3-AT and 400 ng/mL ABA to SD/-Trp-His-Ade medium.By this library a total of 16 different candidate interacting proteins from the wheat and the leaf rust were screened.These proteins were involved in plant metabolism,hormone signal transduction,plant disease resistance and other aspects.It indicated that Pt34084 could interfere with the host defense response and promote leaf rust infection through multiple pathways.The results are conducive to analyzing the pathogenic mechanism of wheat leaf rust and lay a foundation for creating new effective strategies to prevent and control wheat leaf rust.

关 键 词：小麦叶锈菌 小麦叶锈病 CDNA文库 酵母双杂交 效应蛋白 互作蛋白 筛选 

分 类 号：S435.121.43[农业科学—农业昆虫与害虫防治]