玉米大斑病菌效应因子StSse19基因的克隆、表达模式分析及在原核系统中的表达  

Cloning,prokaryotic expression and expression analysis of StSse19 gene,an effector of Seosphaeria turcica

作　　者：杨俊芳 尹贵波 周启慧 刘玉卫 巩校东[1] 谷守芹[1,2] YANG Junfang;YIN Guibo;ZHOU Qihui;LIU Yuwei;GONG Xiaodong;GU Shouqin(College of Life Sciences/Hebei Bioinformatic Utilization and Technological Innovation Center for Agricultural Microbes/State Key Laboratory of North China Crop Improvement and Regulation,Hebei Agricultural University,Baoding 071001,China;Collaborative Innovation Center for Wetland Conservation and Green Development of Hebei Province,Hengshui 053010,China)

机构地区：[1]河北农业大学生命科学学院/华北作物改良与调控国家重点实验室/河北省农业微生物生物信息利用技术创新中心,河北保定071000 [2]河北省湿地保护与绿色发展协同创新中心,河北衡水053010

出　　处：《河北农业大学学报》2024年第3期66-71,104,共7页Journal of Hebei Agricultural University

基　　金：国家自然科学基金(31671983,31701741);中央引导地方科技发展资金(236Z6507G);河北省自然科学基金(C2023204100);河北省湿地保护与绿色发展协同创新中心开放基金(2023hbxczx1-3).

摘　　要：玉米大斑病是由玉米大斑病菌引起的玉米叶部真菌性病害。本课题组前期通过对病菌侵染玉米的转录组分析发现,效应因子StSse19为重要的致病因子,但对其结构及功能尚不清晰。本研究以野生型菌株01-23的cDNA为模版,克隆StSse19并对其结构进行生物信息学预测;结合对病菌侵染玉米的RNA-Seq数据分析明确该基因的表达模式,进而利用qRT-PCR技术证实其表达模式;构建StSse19的融合表达载体pET28a-StSse19,在大肠杆菌BL21(DE3)中以IPTG诱导表达该目的基因,通过SDS-PAGE技术检测其表达情况。结果表明,StSse19的CDS序列由330个核苷酸组成,编码109个氨基酸残基,序列比对发现该蛋白无同源蛋白,表明其为大斑病菌的特异效应蛋白;对StSse19的表达模式分析发现,该基因在侵染玉米24 h的表达量显著提高,72 h表达量下降,推测该基因参与病菌的侵染过程;将StSse19在原核系统中进行表达,SDS-PAGE分析结果显示,该蛋白大小为28 kD,用Ni柱层析法结合Western blot检测显示获得了纯化的目的蛋白。该研究明确了StSse19基因的结构特征及表达模式、获得了StSse19在原核体系中的表达蛋白,为深入揭示其功能及作用机制奠定了基础。Northern leaf blight of corn is a fungal disease caused by Seosphaeria turcica.In the primary research of the transcriptome analysis of the early infection stage of maize by S.turcica showed that the effector StSse19 was a significant pathogenic factor.However,its structure and function have not been reported.Analysis of gene expression pattern during infection of maize by the pathogen showed that the expression level of StSse19 peaked after 24 hours of incubation,followed by a decrease at 72 hours.In the present research,we cloned the specific effector StSse19 using the cDNA of S.turcica wild-type strain 01-23 as the template and analyzed its structure using bioinformatics technology.The prokaryotic expression vector pET28a-StSse19 was constructed and the target gene was induced by IPTG to express in Escherichia coli BL21(DE3).Further,the target protein was confirmed by SDS-PAGE analysis.Moreover,qRT-PCR was employed to investigate the expression level of the StSse19 gene during infection periods.The results revealed that the CDS of the specific effector,StSse19 was consisted of 330 nucleotides,encoding a protein of 109 amino acids.Sequence comparison showed that this protein was unique in S.turcica as it has no homologue compared to other fungus.The results indicated that this gene played a key role in the process of pathogen infection.Furthermore,the StSse19 protein was 28 kD.The purified target protein was verified through purification by Nickel column chromatography and identification by Western blot.This study will not only clarify the structural characteristics of the StSse19 and its expression pattern during the infection stage of the pathogen,but also lay the foundation for further understanding its functions in S.turcica.

关 键 词：玉米大斑病菌 效应因子 StSse19 原核表达 QRT-PCR 

分 类 号：S435.131.4[农业科学—农业昆虫与害虫防治]