河北农业大学学报2025，Vol.48Issue(3)：22-29,8.DOI:10.13320/j.cnki.jauh.2025.0035
玉米大斑病菌m6A甲基转移酶基因的克隆及功能分析
Cloning and function analysis of the m6A methyltransferase gene StMETTL4 in Setosphaeria turcica
摘要
Abstract
Corn northern leaf blight,caused by the fungal pathogen Setosphaeria turcica,is a major disease affecting maize leaves.The present study investigated the homologous gene StMETTL4 of the m6A methyltransferase METTL4,which has previously been identified in S.turcica.The structural and functional characteristics of StMETTL4 were analyzed in detail.Using cDNA from the wild-type strain 01-23 as a template,the StMETTL4 gene was successfully cloned,and its structural features and expression patterns at different developmental stages were predicted through bioinformatics analysis.The mRNA levels of StMETTL4 in maize were quantified using qRT-PCR during S.turcica infection.Additionally,a fusion expression vector,pET28a-StMETTL4,was constructed,and the recombinant protein was expressed in Escherichia coli BL21(DE3)cells following IPTG induction.The expression of the target protein was confirmed via SDS-PAGE.The StMETTL4 protein was purified using Ni-affinity chromatography,and its m6A methyltransferase catalytic activity was subsequently assessed.The results revealed that the coding sequence of StMETTL4 contained 1 518 nucleotides,encoding a protein of 506 amino acids that contained a typical MT-A70 domain.Its expression was significantly upregulated during the germ tube and penetration peg development stages of S.turcica,and notably increased during the infection process in maize with a peak at 24 h post-inoculation,which suggested that it may play an important role in the development of infection structures and the pathogenicity of S.turcica.Furthermore,SDS-PAGE analysis showed that the purified protein was successfully obtained with an approximate molecular weight of 72.85 kD,and its m6A methyltransferase activity was experimentally confirmed.The study not only clarified the structural characteristics of the StMETTL4 gene and its expression pattern during pathogen growth,development,and host infection,but also confirmed that the StMETTL4 protein has m6A methyltransferase catalytic activity,laying a foundation for further exploration of its function.关键词
玉米大斑病菌/m6A甲基转移酶/StMETTL4/原核表达/qRT-PCRKey words
Setosphaeria turcica/m6A methyltransferase/StMETTL4/prokaryotic expression/qRT-PCR分类
农业科技引用本文复制引用
朱蒙芳,刘志航,李依纯,薛启鑫,巩校东,谷守芹,刘玉卫..玉米大斑病菌m6A甲基转移酶基因的克隆及功能分析[J].河北农业大学学报,2025,48(3):22-29,8.基金项目
中央引导地方科技发展资金项目(236Z6508G) （236Z6508G）
河北省自然科学基金项目(C2023204100) （C2023204100）
石家庄市驻冀高校基础研究项目(241791197A). （241791197A）
