黏虫溶菌酶基因Mswly的克隆与真核表达
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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 20 th and 21 st 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.
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 20 th and 21 st 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.
引文
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[17] JAVAR S,MOHAMED R,SAJAP A S,et al.Expression profiles of lysozyme- and prophenoloxidase- encoding genes in Spodoptera species challenged with entomopathogenic fungus,Metarhizium anisopliae(Metchnikoff) Sorokin using qRT-PCR[J].Invertebr Reprod Dev,2015,59(4):230-236.
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[23] KRYUKOV V Y,YAROSLAVTSEVA O N,WHITTEN M M A,et al.Fungal infection dynamics in response to temperature in the lepidopteran insect Galleria mellonella[J].Insect Sci,2018,25(3):454-466.
[24] SOWA-JASILEK A,ZDYBICKA-BARABAS A,STACZEK S,et al.Studies on the role of insect hemolymph polypeptides:Galleria mellonella anionic peptide 2 and lysozyme[J].Peptides,2014,53:194-201.
[25] CALLEWAERT L,MICHIELS C W.Lysozymes in the animal kingdom[J].J Biosci,2010,35(1):127-160.
[26] KAJLA M K,ANDREEVA O,GILBREATH T M,et al.Characterization of expression,activity and role in antibacterial immunity of Anopheles gambiae lysozyme c-1[J].Comp Biochem Physiol(B:Biochem Mol Biol),2010,155(2):201-209.
[27] ZDYBICKA-BARABAS A,STACZEK S,MAK P,et al.Synergistic action of Galleria mellonella apolipophorin Ⅲ and lysozyme against Gram-negative bacteria[J].Biochim Biophys Acta,2013,1828(6):1449-1456.
[28] 路子云,冉红凡,刘文旭,等.黏虫室内饲养方法的改进[J].河北农业科学,2014,18(6):57-61.
[29] 曹慧,李宗芸,王秋香.果蝇先天性免疫研究进展[J].应用昆虫学报,2009,46(2):196-202.
[30] 孙国庆,哈福,苗永旺,等.动物溶菌酶基因的研究进展[J].中国牛业科学,2012,38(3):51-54.
[31] 柳忠玉,雷健,李晓筱,等.虎杖转录因子pcmyb1基因的克隆及原核表达[J].河南农业科学,2018,47(1):96-102.
[32] JOLLèS P,JOLLèS J.What’s new in lysozyme research?[J].Mol Cell Biochem,1984,63(2):165-189.
[33] VOGEL H,ALTINCICEK B,GL?CKNER G,et al.A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella[J].BMC Genomics,2011,12:308.
[34] 阮班军,代鹏,王伟,等.蛋白质翻译后修饰研究进展[J].中国细胞生物学学报,2014,36(7):1027-1037.
[35] 罗莉,李坤,王保成,等.包涵体变复性技术研究进展[J].中国医药生物技术,2012,7(4):290-293.
[36] 凌同,余黎,白慕群.昆虫杆状病毒表达系统的研究进展与应用[J].微生物学免疫学进展,2014,42(2):70-78.
[37] 梁振普,刘雅静,张小霞,等.昆虫杆状病毒基因组学研究进展[J].河南农业科学,2018,47(9):1-7.
[38] VILCINSKAS A,MATHA V.Antimycotic activity of lysozyme and its contribution to antifungal humoral defence reactions in Galleria mellonella[J].Anim Biol,1997,6:19-29.
[39] BECKERT A,WIESNER J,BAUMANN A,et al.Two c-type lysozymes boost the innate immune system of the invasive ladybird Harmonia axyridis[J].Dev Comp Immunol,2015,49(2):303-312.
[2] 蒋婷,黄芊,蒋显斌,等.3种黏虫幼虫对4种寄主植物的取食选择[J].南方农业学报,2017,48(8):1415-1420.
[3] 张齐凤,杨智超,王振,等.黑龙江省黏虫发生情况及防治措施[J].黑龙江农业科学,2013(4):160-161.
[4] 张云慧,张智,姜玉英,等.2012年三代黏虫大发生原因初步分析[J].植物保护,2012,38(5):1-8.
[5] 宋月芹,王海涛,陈玉国,等.黏虫抗甲氨基阿维菌素苯甲酸盐种群的交互抗性与生化抗性机制[J].农药学学报,2017,19(1):18-24.
[6] HAJEK A E.Ecology of terrestrial fungal entomopathogens[J].Adv Microb Ecol,1997,15(1):193-249.
[7] CHEN J,LAI Y,WANG L,et al.CRISPR/Cas9-mediated efficient genome editing via blastospore-based transformation in entomopathogenic fungus Beauveria bassiana[J].Sci Rep,2017,8:45763.
[8] EVANS H C,ELLIOT S L,BARRETO R W.Entomopathogenic fungi and their potential for the management of Aedes aegypti(Diptera:Culicidae) in the Americas[J].Mem Inst Oswaldo Cruz,2018,113(3):206-214.
[9] FARIA M R,WRAIGHT S P.Mycoinsecticides and mycoacaricides:A comprehensive list with worldwide coverage and international classification of formulation types[J].Biological Control,2007,43(3):237-256.
[10] FERRON P.Biological control of insect pests by entomogenous fungi[J].Ann Rev Entomol,2003,23(1):409-442.
[11] FARENHORST M,MOUATCHO J C,KIKANKIE C K,et al.Fungal infection counters insecticide resistance in African malaria mosquitoes[J].Proc Nat Acad Sci USA,2009,106(41):17443-17447.
[12] 刘春来.昆虫病原真菌在农林害虫生物防治中的应用[J].黑龙江农业科学,2017(3):68-73.
[13] 李世广,林华峰.不同条件下几种虫生真菌对棉铃虫的侵染致病效应[J].生物安全学报,2003,12(1):29-34.
[14] 宋入梅.应用白僵菌和绿僵菌防治甜菜夜蛾的研究[D].保定:河北农业大学,2006:14-18.
[15] LAVINE M D,STRAND M R.Insect hemocytes and their role in immunity[J].Insect Biochem Mol Biol,2002,32(10):1295-1309.
[16] LEMAITRE B,HOFFMANN J.The host defense of Drosophila melanogaster[J].Annu Rev Immunol,2007,25:697-743.
[17] JAVAR S,MOHAMED R,SAJAP A S,et al.Expression profiles of lysozyme- and prophenoloxidase- encoding genes in Spodoptera species challenged with entomopathogenic fungus,Metarhizium anisopliae(Metchnikoff) Sorokin using qRT-PCR[J].Invertebr Reprod Dev,2015,59(4):230-236.
[18] ZHANG W,CHEN J,KEYHANI N O,et al.Comparative transcriptomic analysis of immune responses of the migratory locust,Locusta migratoria,to challenge by the fungal insect pathogen,Metarhizium acridum[J].BMC Genomics,2015,16(1):867.
[19] HUSSAIN A,RIZWAN-UL-HAQ M,AL-AYEDH H A,et al.Susceptibility and immune defence mechanisms of Rhynchophorus ferrugineus(Olivier) (Coleoptera:Curculionidae) against entomopathogenic fungal infections[J].Inter J Mol Sci,2016,17(9):1518.
[20] KONG H,LV M,MAO N,et al.Molecular characterization of a lysozyme gene and its altered expression profile in crowded beet webworm (Loxostege sticticalis) [J].PLoS One,2016,11(8):e0161384.
[21] RAMIREZ J L,DUNLAP C A,MUTURI E J,et al.Entomopathogenic fungal infection leads to temporospatial modulation of the mosquito immune system[J].PLoS Neglected Tropical Diseases,2018,12(4):e0006433.
[22] WOJDA I,KOWALSKI P,JAKUBOWICZ T.Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions[J].J Insect Physiol,2009,55(6):525-531.
[23] KRYUKOV V Y,YAROSLAVTSEVA O N,WHITTEN M M A,et al.Fungal infection dynamics in response to temperature in the lepidopteran insect Galleria mellonella[J].Insect Sci,2018,25(3):454-466.
[24] SOWA-JASILEK A,ZDYBICKA-BARABAS A,STACZEK S,et al.Studies on the role of insect hemolymph polypeptides:Galleria mellonella anionic peptide 2 and lysozyme[J].Peptides,2014,53:194-201.
[25] CALLEWAERT L,MICHIELS C W.Lysozymes in the animal kingdom[J].J Biosci,2010,35(1):127-160.
[26] KAJLA M K,ANDREEVA O,GILBREATH T M,et al.Characterization of expression,activity and role in antibacterial immunity of Anopheles gambiae lysozyme c-1[J].Comp Biochem Physiol(B:Biochem Mol Biol),2010,155(2):201-209.
[27] ZDYBICKA-BARABAS A,STACZEK S,MAK P,et al.Synergistic action of Galleria mellonella apolipophorin Ⅲ and lysozyme against Gram-negative bacteria[J].Biochim Biophys Acta,2013,1828(6):1449-1456.
[28] 路子云,冉红凡,刘文旭,等.黏虫室内饲养方法的改进[J].河北农业科学,2014,18(6):57-61.
[29] 曹慧,李宗芸,王秋香.果蝇先天性免疫研究进展[J].应用昆虫学报,2009,46(2):196-202.
[30] 孙国庆,哈福,苗永旺,等.动物溶菌酶基因的研究进展[J].中国牛业科学,2012,38(3):51-54.
[31] 柳忠玉,雷健,李晓筱,等.虎杖转录因子pcmyb1基因的克隆及原核表达[J].河南农业科学,2018,47(1):96-102.
[32] JOLLèS P,JOLLèS J.What’s new in lysozyme research?[J].Mol Cell Biochem,1984,63(2):165-189.
[33] VOGEL H,ALTINCICEK B,GL?CKNER G,et al.A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella[J].BMC Genomics,2011,12:308.
[34] 阮班军,代鹏,王伟,等.蛋白质翻译后修饰研究进展[J].中国细胞生物学学报,2014,36(7):1027-1037.
[35] 罗莉,李坤,王保成,等.包涵体变复性技术研究进展[J].中国医药生物技术,2012,7(4):290-293.
[36] 凌同,余黎,白慕群.昆虫杆状病毒表达系统的研究进展与应用[J].微生物学免疫学进展,2014,42(2):70-78.
[37] 梁振普,刘雅静,张小霞,等.昆虫杆状病毒基因组学研究进展[J].河南农业科学,2018,47(9):1-7.
[38] VILCINSKAS A,MATHA V.Antimycotic activity of lysozyme and its contribution to antifungal humoral defence reactions in Galleria mellonella[J].Anim Biol,1997,6:19-29.
[39] BECKERT A,WIESNER J,BAUMANN A,et al.Two c-type lysozymes boost the innate immune system of the invasive ladybird Harmonia axyridis[J].Dev Comp Immunol,2015,49(2):303-312.