九香虫溶菌酶基因CcLys的克隆与特征分析
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摘要
九香虫Coridius chinensis是我国一种重要的药食两用昆虫,溶菌酶是一种具有抗菌作用的糖苷水解酶。该研究从九香虫中克隆了一种溶菌酶基因CcLys,其cDNA长883 bp,包含一个669 bp的开放阅读框,编码222个氨基酸。九香虫溶菌酶CcLys的N-端包含一段由18个氨基酸组成的信号肽,成熟CcLys形成7个α-螺旋,8个β-折叠片的三维结构。同源性和聚类分析显示CcLys与茶翅蝽Halyomorpha halys溶菌酶的亲缘关系最近。基因表达谱分析表明,CcLys在九香虫的整个发育阶段都有表达,在3龄若虫中表达水平最高;该基因在所检测的成虫组织中都有表达,在脂肪体中表达水平最高。成虫被细菌诱导后,CcLys的表达水平在12 h达到峰值。该研究为进一步明确CcLys基因的功能及开发新型抗菌药物奠定基础。
Coridius chinensis is an important insect used for both medicine and food source in China,and lysozyme is a glycoside hydrolase with antimicrobial activity. In this study,alysozyme gene,designated CcLys,was cloned from C. chinensis. The CcLys cDNA was 883 bp in length which contained a 669 bp open reading frame(ORF) that encoded 222 amino acids. CcLys contained a signal peptide composed of 18 amino acids at N-terminus,and the mature CcLys formed a three-demensional structure consisted of 7 α-helix and 8 β-pleated sheets. Homology and cluster analyses showed that CcLys possesses the closest relationship with that of Halyomorpha haly. Gene expression analyses indicated that CcLys was expressed throughout the developmental stages of C. chinensis with the highest expression level in the 3 rd instar nymph,and this gene was expressed in the adult tissues tested with the highest expression level in the fat body. The mRNA expression level of this gene peaked at 12 h post induction by bacteria. This study will provide a foundation for further understanding the function of CcLys gene and developing new antimicrobial drugs.
Coridius chinensis is an important insect used for both medicine and food source in China,and lysozyme is a glycoside hydrolase with antimicrobial activity. In this study,alysozyme gene,designated CcLys,was cloned from C. chinensis. The CcLys cDNA was 883 bp in length which contained a 669 bp open reading frame(ORF) that encoded 222 amino acids. CcLys contained a signal peptide composed of 18 amino acids at N-terminus,and the mature CcLys formed a three-demensional structure consisted of 7 α-helix and 8 β-pleated sheets. Homology and cluster analyses showed that CcLys possesses the closest relationship with that of Halyomorpha haly. Gene expression analyses indicated that CcLys was expressed throughout the developmental stages of C. chinensis with the highest expression level in the 3 rd instar nymph,and this gene was expressed in the adult tissues tested with the highest expression level in the fat body. The mRNA expression level of this gene peaked at 12 h post induction by bacteria. This study will provide a foundation for further understanding the function of CcLys gene and developing new antimicrobial drugs.
引文
[1] 初源,周帆,张明明,等.昆虫天然免疫反应研究前沿 [J].应用昆虫学报,2013,50(2):311-320.
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[16] 李尚伟,赵柏松,杜娟.九香虫抗菌肽CcAMP1的分离纯化和抗菌活性检测[J].昆虫学报,2015,58(6):610-616.
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[18] Croux C,García JL.Sequence of the lye gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC824:comparison with other lytic enzymes [J].Gene,1991,104(1):25-31.
[19] Henrissat B.A classification of glycosyl hydrolases based on amino acid sequence similarities [J].Biochemical Journal,1991,280(Pt 2):309-316.
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[21] Martinez-Fleites C,Korczynska JE,Davies GJ,et al.The crystal structure of a family GH25 lysozyme from Bacillus anthracis implies a neighboring-group catalytic mechanism with retention of anomeric configuration [J].Carbohydrate Research,2009,344(13):1753-1757.
[22] Korczynska JE,Danielsen S,Schagerlof U,et al.The structure of a family GH25 lysozyme from Aspergillus fumigatus [J].Acta Crystallographica Section F,2010,66(Pt 9):973-977.
[23] Weaver LH,Grütter MG,Remington SJ,et al.Comparison of goose-type,chicken-type,and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution [J].Journal of Molecular Evolution,1985,21(2):97-111.
[24] Abbott DW,Macauley MS,Vocadlo DJ,et al.Streptococcus pneumoniae endohexosaminidase D,structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases [J].Journal of Biological Chemistry,2009,284(17):11676-11689.
[25] Bachali S,Jager M,Hassanin A,et al.Phylogenetic analysis of invertebrate lysozymes and the evolution of lysozyme function [J].Journal of Molecular Evolution,2002,54(5):652-664.
[26] 张波,王林美,叶博,等.柞蚕溶菌酶基因的克隆和序列分析[J].蚕业科学,2009,35(3):539-546.
[27] 于洁,王登杰,雷仲仁,等.烟粉虱溶菌酶基因的鉴定及表达分析[J].中国农业科学,2016,49(13):2534-2543.
[28] 张元臣,刘向东.棉蚜溶菌酶基因AgLYS的克隆与表达分析[J].昆虫学报,2017,60(2):127-135.
[2] Callewaert L,Michiels CW.Lysozymes in the animal kingdom [J].Journal of Biosciences,2010,35(1):127-160.
[3] Dobson DE,Prager EM,Wilson AC.Stomach lysozymes of ruminants [J].The Journal of Biological Chemistry,1984,259(18):11617-11625.
[4] 朱元镇,刘婧仪,于常红.溶菌酶的研究进展及应用 [J].山东医学高等专科学校学报,2018,40(3):207-210.
[5] 尹金凤.溶菌酶对大肠杆菌的抗菌作用机制及其增效技术的研究[D].无锡:江南大学,2011:1-41.
[6] Jolles P,Jolles J.What’s new in lysozyme research [J].Molecular and Cellular Biochemistry,1984,63(2):165-189.
[7] Buonocore F,Randelli E,Trisolino P,et al.Innate immunity in sea bass (Dicentrarchus labrax L.):Molecular characterization,expression and 3D structural analysis and antibacterial activity of a G-type lysozyme [J].Fish & Shellfish Immunology,2013,34(6):1697-1698.
[8] 刘梅,张士璀.溶菌酶C及其生物学功能 [J].生命的化学,2006,26(5):465-467.
[9] Sanz JM,García P,García JL.Role of Asp-9 and Glu-36 in the active site of the pneumococcal CPL1 lysozyme:an evolutionary perspective of lysozyme mechanism [J].Biochemistry,1992,31(36):8495-8499.
[10] 张颖,陈建伟,高源.九香虫资源鉴定、化学、药理与药食应用研究[J].亚太传统医药,2009,5(9):44-47.
[11] 魏超,舒国周,罗会嵩,等.九香虫的形态特征和生物学特性[J].山地农业生物学报,2015,34(4):26-30.
[12] 姚银花.九香虫的生物学特性及其应用价值[J].黔东南民族师范高等专科学校学报,2006,24(6):48-49.
[13] 郭玉红,张庆林.药食昆虫九香虫的生物学及开发利用研究进展[J].时珍国医国药,2015,26(3):692-693.
[14] 国家中医药管理局中华本草编委会.中华本草(第9册)[M].上海:上海科学技术出版社,1999:173-174.
[15] 吴玛莉,金道超.九香虫血淋巴及其纯化蛋白抑菌活性的研究[J].应用昆虫学报,2005,42(3):315-318.
[16] 李尚伟,赵柏松,杜娟.九香虫抗菌肽CcAMP1的分离纯化和抗菌活性检测[J].昆虫学报,2015,58(6):610-616.
[17] Henrissat B,Callebaut I,Fabrega S,et al.Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases [J].Proceedings of the National Academy of Sciences of the United States of America,1995,92(15):7090-7094.
[18] Croux C,García JL.Sequence of the lye gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC824:comparison with other lytic enzymes [J].Gene,1991,104(1):25-31.
[19] Henrissat B.A classification of glycosyl hydrolases based on amino acid sequence similarities [J].Biochemical Journal,1991,280(Pt 2):309-316.
[20] Fouche PB,Hash JH.The N,O-diacetylmuramidase of Chalaropsis species.Identificaiton of aspartyl and glutamyl residues in the active site [J].Journal of Biological Chemistry,1978,253(19):6787-6793.
[21] Martinez-Fleites C,Korczynska JE,Davies GJ,et al.The crystal structure of a family GH25 lysozyme from Bacillus anthracis implies a neighboring-group catalytic mechanism with retention of anomeric configuration [J].Carbohydrate Research,2009,344(13):1753-1757.
[22] Korczynska JE,Danielsen S,Schagerlof U,et al.The structure of a family GH25 lysozyme from Aspergillus fumigatus [J].Acta Crystallographica Section F,2010,66(Pt 9):973-977.
[23] Weaver LH,Grütter MG,Remington SJ,et al.Comparison of goose-type,chicken-type,and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution [J].Journal of Molecular Evolution,1985,21(2):97-111.
[24] Abbott DW,Macauley MS,Vocadlo DJ,et al.Streptococcus pneumoniae endohexosaminidase D,structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases [J].Journal of Biological Chemistry,2009,284(17):11676-11689.
[25] Bachali S,Jager M,Hassanin A,et al.Phylogenetic analysis of invertebrate lysozymes and the evolution of lysozyme function [J].Journal of Molecular Evolution,2002,54(5):652-664.
[26] 张波,王林美,叶博,等.柞蚕溶菌酶基因的克隆和序列分析[J].蚕业科学,2009,35(3):539-546.
[27] 于洁,王登杰,雷仲仁,等.烟粉虱溶菌酶基因的鉴定及表达分析[J].中国农业科学,2016,49(13):2534-2543.
[28] 张元臣,刘向东.棉蚜溶菌酶基因AgLYS的克隆与表达分析[J].昆虫学报,2017,60(2):127-135.
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