黏虫溶菌酶基因Mswly的克隆与真核表达  

Cloning and Eukaryotic Expression of Mswly in Oriental Armyworm,Mythimna separata

作　　者：张元臣[1] 蔡新 张国强[1] 董丽丽 王景顺[1] ZHANG Yuanchen;CAI Xin;ZHANG Guoqiang;DONG Lili;WANG Jingshun(Anyang Institute of Technology,Anyang 455000,China)

机构地区：[1]安阳工学院

出　　处：《河南农业科学》2019年第5期70-77,共8页Journal of Henan Agricultural Sciences

基　　金：安阳工学院博士科研启动基金项目(BSJ2016011);河南省自然科学基金重点项目(182300410003)

摘　　要：为了研究溶菌酶在害虫抵抗虫生真菌中的作用及分子机制,以黏虫为试验材料,利用RT-PCR和RACE技术获得黏虫溶菌酶基因 Mswly的全长序列,同时采用体外真核表达系统对Mswly基因进行蛋白质表达。克隆、测序后分析表明,Mswly 基因的cDNA全长为652 bp(GenBank登录号为MG692501),开放阅读框序列长度为426 bp,编码141个氨基酸。其编码蛋白质分子质量为 16.26 ku ,理论等电点为6.57;含有信号肽,剪切位点在第20个和21个氨基酸之间;含有溶菌酶活性所需要的谷氨酸和天冬氨酸活性位点。系统进化树分析表明,Mswly与其他物种的C型溶菌酶聚集在一起,说明Mswly为C型溶菌酶。Western blotting 结果显示,Mswly重组蛋白大小约17 ku,与预期分子质量大小一致,说明 Mswly 基因在昆虫Sf9细胞系能够高效表达。综上,从黏虫脂肪体中克隆得到了1个C型溶菌酶基因 Mswly 的完整cDNA序列,并将其在草地贪夜蛾细胞系Sf9中进行了高效表达。In order to study the role and molecular mechanism of lysozyme in the resistance of insect pests to entomogenous fungi,using RT-PCR and RACE technologies the full-length sequence of Mswly gene was acquired from the oriental armyworm, Mythimna separata ,and protein expression of the Mswly gene was performed by in vitro eukaryotic expression system(Sf9 cell line).The online prediction results after cloning and sequencing showed that the cDNA of Mswly gene was 652 bp(GenBank accession number:MG692501) and the sequence length of its ORF was 426 bp,encoding 141 amino acids.The molecular weight and theoretical isoelectric point(pI) of the Mswly protein were predicted to be 16.26 ku and 6.57 ,respectively.The Mswly protein harbored a signal peptide whose shear site was between the 20th and 21st amino acids,and the active sites of glutamic acid and aspartic acid residues required for lysozyme activity.Phylogenetic tree analysis showed that Mswly was clustered with C-type lysozymes of other species,suggesting that Mswly was C-type lysozyme.Western blotting result showed that the size of recombinant protein of Mswly gene was approximately 17 ku,which was consistent with the expected molecular weight,indicating that Mswly gene could be efficiently expressed in insect Sf9 cell line.In this study,the complete cDNA sequence of a C-type lysozyme gene Mswly was cloned from the fat body of oriental armyworm,which was highly expressed in the Sf9 cell line of Spodoptera frugiperda.

关 键 词：黏虫 溶菌酶 基因克隆 RACE 系统发育 真核表达 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]