华北农学报2024，Vol.39Issue(4)：206-214,9.DOI:10.7668/hbnxb.20194652
东方黏虫溶菌酶基因MseLYS的克隆与表达分析
Cloning and Expression Analysis of MseLYS from Oriental Armyworm,Mythimna separata(Lepidotera:Noctuidae)
摘要
Abstract
;A lysozyme gene,MseLYS,was cloned from oriental armyworm,and then,its prokaryotic expression vector was constructed by the prokaryotic expression system,and finally,the expression pattern of this gene was de-tected in different stages and tissues of oriental armyworm,to provide theoretical reference for exploring the function and structure of the MseLYS and lay a foundation for further study of the antibacterial function and physiological mechanism.We cloned the sequence of the lysozyme gene MseLYS from the midgut of Mythimna separata by reverse transcription PCR(RT-PCR)and rapid amplification of cDNA ends(RACE).The open reading frame(ORF)se-quence with the signal peptide removed was ligated to the expression vector pET-30a(+),and the inducible ex-pression was carried out by IPTG.Quantitative PCR was used to detect the spatiotemporal expression pattern of this gene.Sequence analysis showed that the full length cDNA of MseLYS was 729 bp,and its ORF was 426 bp,encoding a total of 141 amino acid residues.Meanwhile,the 5'non-coding region and 3'non-coding region included 75,228 bp,respectively.The isoelectric point and molecular weight of the protein encoded by MseLYS were 7.72 and 16.13 ku,respectively.Phylogenetic tree demonstrated that MseLYS was clustered with other species'C-type lyso-zyme,and was highly consistent with other insect amino acid sequences,suggesting that MseLYS was a C-type lyso-zyme.SDS-PAGE showed that the expressed protein was consistent with the expected size,which was about 20 ku,indicating that MseLYS can be expressed efficiently in BL21(DE3).Instar expression profiling analysis illustrated that there were significant differences in the expression levels of MseLYS gene among different developmental stages of larvae,males,and females.The expression levels of MseLYS gene were higher in the late larval and pupal stages of oriental army worm,while the expression levels were lower in other developmental stages.The tissue expression anal-ysis results indicated that there were significant differences in the expression levels of this gene among different tis-sues of male adults,with higher expression levels in the fat body and thorax;this gene expression also showed signif-icant differences among different tissues of female adults,with higher expression levels in antennae,wings,and cuti-cle.To sum up,the full-length sequence of MseLYS is cloned,its prokaryotic expression vector is successfully con-structed,which could efficiently express the target protein,and its expression pattern is clarified in different tissues and ages.关键词
东方黏虫/溶菌酶/基因克隆/原核表达/定量PCRKey words
Mythimna separata/Lysozyme/Gene cloning/Prokaryotic expression/Quantitative PCR分类
农业科技引用本文复制引用
张元臣,苏圣盈,张家祺,薛爽,王景顺..东方黏虫溶菌酶基因MseLYS的克隆与表达分析[J].华北农学报,2024,39(4):206-214,9.基金项目
国家自然科学基金项目(32100390) （32100390）
河南省研究生教育改革与质量提升工程项目(YJS2024AL137) （YJS2024AL137）
