玉米大斑病菌cDNA文库的构建及转录因子StMR1互作蛋白的筛选  

Construction of cDNA Library of Setosphaeria turcica and Screening of Transcription Factor StMR1 Interacting Proteins

作　　者：王秋月 段鹏亮 李海笑 刘宁[1] 曹志艳[1,2] 董金皋 WANG Qiu-yue;DUAN Peng-liang;LI Hai-xiao;LIU Ning;CAO Zhi-yan;DONG Jin-gao(College of Plant Protection,Hebei Agricultural University,Baoding 071000;State Key Laboratory of North China Crop Improvement and Regulation,Baoding 071000)

机构地区：[1]河北农业大学植物保护学院,保定071000 [2]华北作物改良与调控国家重点实验室,保定071000

出　　处：《生物技术通报》2024年第6期281-289,共9页Biotechnology Bulletin

基　　金：国家自然科学基金项目(32072370);河北省自然科学基金项目(C2021204136);国家现代农业产业技术体系(CARS-02)。

摘　　要：【目的】筛选玉米大斑病菌(Setosphaeria turcica)转录因子的互作蛋白,解析黑色素调控转录因子StMR1调控玉米大斑病菌致病性的分子机制。为解析玉米大斑病菌侵染过程中转录因子的调控网络,阐明病菌的致病机理提供参考。【方法】收集玉米大斑病菌菌丝和孢子不同萌发阶段作为试验材料,采用Gateway方法构建玉米大斑病菌cDNA文库,使用同源重组的方法构建转录因子StMR1的诱饵载体,采用酵母双杂交技术筛选其互作蛋白并进行一对一验证。【结果】构建的玉米大斑病菌文库插入的平均片段长度大于1000 bp,初级文库及次级文库的库容量为1.2×107和1.04×107CFU,重组率为100%,可以用于酵母双杂交筛选。成功构建可以用于筛库的诱饵载体pGBKT7-StMR1,经初筛与复筛得到3个互作蛋白,一对一验证短链脱氢酶、糖基转移酶、富含亮氨酸重复序列蛋白均与转录因子StMR1存在互作。【结论】成功构建了丰富度高且质量好的玉米大斑病菌cDNA文库并筛选到了与转录因子StMR1互作的蛋白。【Objective】To screen the interaction proteins of transcription factors of Setosphaeria turcica,and to analyze the molecular mechanism of melanin-regulated transcription factor StMR1 in regulating the pathogenicity of S.turcica.This study provides a reference for elucidating the regulatory network of transcription factors in the infection process of S.turcica and elucidating the pathogenic mechanism of the pathogen.【Method】Different germination stages of hyphae and spores of S.turcica were collected as test materials,and the cDNA library of S.turcica was constructed by gateway method,The bait vector of transcription factor StMR1 was constructed by homologous recombination,and its interacting proteins were screened by yeast two-hybrid technology and verified one-to-one.【Result】The average fragment length of the constructed library was greater than 1000 bp,the library capacity of the primary library and the secondary library were 1.2×107 and 1.04×107 CFU,respectively,and the recombination rate was 100%,which could be used for yeast two-hybrid screening.The bait vector pGBKT7-StMR1 that could be used for screen library was successfully constructed,and three interacting proteins were obtained through primary screening and rescreening,and one-to-one verification of the interaction between short-chain dehydrogenase,glycosyltransferase and leucine-rich repeat protein and transcription factor StMR1 were verified.【Conclusion】A high quality and abundant cDNA library was successfully constructed and the protein interacting with StMR1 was screened.

关 键 词：玉米大斑病菌 CDNA文库 转录因子 酵母双杂交 互作蛋白 

分 类 号：S435.13[农业科学—农业昆虫与害虫防治]