摘要：

克隆东方黏虫C型溶菌酶基因MseLYS,构建MseLYS基因的原核表达载体,检测该基因在东方黏虫不同龄期和组织的表达情况,旨在为探究MseLYS基因的功能和结构提供理论参考,也为进一步研究该基因的抗菌功能和生理机制奠定基础。以东方黏虫为研究对象,通过反转录PCR(RT-PCR)和末端快速扩增技术(RACE),获得了溶菌酶基因MseLYS的全长核苷酸序列,之后将去掉信号肽的开放阅读框架(ORF)序列连接到表达载体pET-30a(+)上,并用IPTG进行了诱导表达,最后用荧光定量PCR明确了该基因的时空表达模式。序列分析表明,此基因全长729 bp,ORF全长426 bp,5'和3'非编码区分别为75,228 bp,共编码141个氨基酸残基,其蛋白质的等电点和分子质量分别为7.72,16.13 ku。系统进化树分析结果表明,MseLYS与其他物种的C型溶菌酶聚集在一起,且与其他昆虫的氨基酸序列具有高度一致性,表明MseLYS为C型溶菌酶。SDS-PAGE电泳结果表明,MseLYS在表达菌BL21(DE3)内表达的蛋白质分子质量与预期大小一致,约20 ku,说明MseLYS在大肠杆菌中能够高效表达。龄期表达谱分析表明,MseLYS基因在东方黏虫幼虫、雄虫和雌虫不同发育阶段表达量显著不同,在末龄幼虫、蛹中表达量较高,其他发育时期表达水平较低。组织表达分析结果表明,MseLYS基因在雄虫不同组织表达量存在着显著差异,其中脂肪体和胸内表达量较高;在雌虫不同组织表达量也存在显著差异,其中触角、翅和体壁中表达量较高。综上,成功克隆了东方黏虫C型溶菌酶MseLYS基因的全长序列,构建了该基因的原核表达载体并能够高效表达蛋白质,明确了该基因在不同组织和不同龄期的表达模式。

关键词: 东方黏虫, 溶菌酶, 基因克隆, 原核表达, 定量PCR

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 detected 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)sequence with the signal peptide removed was ligated to the expression vector pET-30a(+),and the inducible expression 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 lysozyme,and was highly consistent with other insect amino acid sequences,suggesting that MseLYS was a C-type lysozyme.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 armyworm,while the expression levels were lower in other developmental stages.The tissue expression analysis results indicated that there were significant differences in the expression levels of this gene among different tissues of male adults,with higher expression levels in the fat body and thorax;this gene expression also showed significant differences among different tissues of female adults,with higher expression levels in antennae,wings,and cuticle.To sum up,the full-length sequence of MseLYS is cloned,its prokaryotic expression vector is successfully constructed,which could efficiently express the target protein,and its expression pattern is clarified in different tissues and ages.

Key words: Mythimna separata, Lysozyme, Gene cloning, Prokaryotic expression, Quantitative PCR