三化螟两个乙酰胆碱酯酶基因克隆及序列分析
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摘要
乙酰胆碱酯酶(ACh E)是有机磷(Organophosphate,OP)和氨基甲酸酯(Carbamate,CB)类杀虫剂的作用靶标。昆虫乙酰胆碱酯酶基因(ace)表达量的上升或基因突变导致其对杀虫剂的敏感性下降是昆虫产生抗药性的重要原因。本研究利用RT-PCR技术和c DNA末端快速扩增(RACE)技术成功克隆到三化螟(Scirpophaga incertulas Walker)的两个乙酰胆碱酯酶基因,分别命名为Siace1和Siace2。Siace1的完整开放阅读框(Open reading frame,ORF)长2 085 bp,编码含694个氨基酸的乙酰胆碱酯酶1(Si ACh E1)。Siace2的ORF长1 917 bp,编码含638个氨基酸的乙酰胆碱酯酶2(Si ACh E2)。将三化螟的两个乙酰胆碱酯酶与已报道的各物种的乙酰胆碱酯酶进行相似性比较,结果显示在蛋白水平上,Si ACh E1与台湾稻螟的ACh E1有最高的相似性,达到94%;Si ACh E2与二化螟的ACh E2具有最高的相似性,为98%。本研究为水稻上重要螟虫的乙酰胆碱酯酶基因对比研究提供了基础。
Acetylcholinesterase( ACh E) is the target of organophosphate( OP) and carbamate( CB) insecticides.Mutations in the ACh E gene( ace) leading to decreased insecticide susceptibility is responsible for decreased insecticides resistance in insects. In this study,two Scirpophaga incertulas Walker acetylcholinesterase genes,Siace1 and Siace2,were cloned using RT-PCR and RACE. The open reading frame( ORF) of Siace1 is 2 085 bp in length,encoding acetylcholinesterase 1( Si ACh E1) protein containing a calculated 694 amino acid( aa) residues. Siace2 is 1 917 bp in length,encoding an acetylcholinesterase 2( Si ACh E2) containing 638 aa residues. At the aa level,Si ACh E1 shares the highest similarity( 94%) with the Chilo auricilius ACh E1,and Si ACh E2 shares the highest similarity with the C. suppressalis ACh E2( 98%).This study provides a basis for future comparison of ace genes in different pyralididae insects in rice.
Acetylcholinesterase( ACh E) is the target of organophosphate( OP) and carbamate( CB) insecticides.Mutations in the ACh E gene( ace) leading to decreased insecticide susceptibility is responsible for decreased insecticides resistance in insects. In this study,two Scirpophaga incertulas Walker acetylcholinesterase genes,Siace1 and Siace2,were cloned using RT-PCR and RACE. The open reading frame( ORF) of Siace1 is 2 085 bp in length,encoding acetylcholinesterase 1( Si ACh E1) protein containing a calculated 694 amino acid( aa) residues. Siace2 is 1 917 bp in length,encoding an acetylcholinesterase 2( Si ACh E2) containing 638 aa residues. At the aa level,Si ACh E1 shares the highest similarity( 94%) with the Chilo auricilius ACh E1,and Si ACh E2 shares the highest similarity with the C. suppressalis ACh E2( 98%).This study provides a basis for future comparison of ace genes in different pyralididae insects in rice.
引文
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[13]WEILL M,FORT P,BERTHOMIEU A,et al.A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila[J].Proceedings Biological Sciences,2002,2699(1504):2007-2016.
[14]CHEN M,HAN Z J.Cloning and sequence analysis of 2 different acetylcholinesterase genes in Rhopalosiphum padi and Sitobion avenae[J].Genome,2006,49:239-243.
[15]BAEK J H,KIM J I,LEE D W,et al.Identification and characterization of ace1-type acetylcholine sterase likely associated with organophosphate resistance in Plutella xylostella[J].Pesticide Biochemistry and Physiology,2005,81:164-175.
[16]KIM J I,JUNG C S,KOH Y H,et al.Molecular,biochemical and histochemical characterization of two acetylcholinesterase c DNAs from the German cockroach Blattella germanica[J].Insect Molecular Biology,2006,15:513-522.
[17]LEE D W,KIM S S,SHIN S W,et al.Molecular characterization of two acetylcholinesterase genes from the oriental tobacco budworm,Helicoverpa assulta(Guenee)[J].Biochimica et Biophysica Acta,2006,1760:125-133.
[18]SEONG K M,KIM Y H,KWON D H,et al.Identification and characterization of three cholinesterases from the common bed bug,Cimex lectularius[J].Insect Molecular Biology,2012,21:149-159.
[19]张振飞,黄炳超,肖汉祥,等.4种农业措施对三化螟种群动态的控制作用[J].生态学报,2013,33(22):7173-7180.
[20]罗文辉.9种药剂防治水稻三化螟田间试验[J].农药管理与科学,2008,29(8):34-36.
[21]张振飞,黄炳超,张扬,等.几种药剂对三化螟室内与田间防治效果研究[J].现代农药,2011,10(1):51-54.
[22]LUO G H,LI X H,ZHANG Z C,et al.Cloning of two acetylcholinesterase genes and analysis of point mutations putatively associated with triazophos resistance in Chilo auricilius(Lepidoptera:Pyralidae)[J].Journal of Economic Entomology,2015,108(3):1289-1297.
[2]LOW V L,CHEN C D,LIM P E,et al.First molecular genotyping of insensitive acetylcholinesterase associated with malathion resistance in Culex quinquefasciatus Say populations in Malaysia[J].Pest Managment Science,2013,69:1362-1368.
[3]MUTERO A,PRALAVORIO M,BRIDE J M,et al.Resistanceassociated point mutations in insecticide-insensitive acetylcholinesterase[J].Proceedings of the National Academy of Sciences of the United States of America,1994,91:5922-5926.
[4]WALSH S B,DOLDEN T A,MOORES G D,et al.Identification and characterization of mutations in housefly(Musca domestica)acetylcholinesterase involved in insecticide resistance[J].Biochemical Journal,2001,359:175-181.
[5]王敦,唐振华,尚金燕,等.昆虫乙酰胆碱酯酶基因研究进展[J].昆虫学报,2006,49(3):497-503.
[6]JIANG X,QU M,DENHOLM I,et al.Mutation in acetylcholinesterase1 associated with triazophos resistance in rice stem borer,Chilo suppressalis(Lepidoptera:Pyralidae)[J].Biochemical and Biophysical Research Communications,2009,378:269-272.
[7]FFRENCH-CONSTANT R H,PITTENDRIGH B,VAUGHAN A,et al.Why are there so few resistance-associated mutations in insecticide target genes?[J].Philosophical Transactions of the Royal Society of London,1998,353:1685-1693.
[8]SHI M A,LOUGARRE A,ALIES C,et al.Acetylcholinesterase alterations reveal the fitness cost of mutations conferring insecticide resistance[J].BMC Evolutionary Biology,2004,4:5.
[9]LI B,WANG Y H,LIU H T,et al.Genotyping of acetylcholinesterase in insects[J].Pesticide Biochemistry and Physiology,2010,98:19-25.
[10]KIM Y H,LEE S H.Which acetylcholinesterase functions as the main catalytic enzyme in the Class Insecta?[J].Insect Biochemistry and Molecular Biology,2013,43:47-53.
[11]BOURGUET D,RAYMOND M,FOURNIER D,et al.Existence of two acetylcholinesterases in the mosquito Culex pipiens(Diptera:Culicidae)[J].Journal of Neurochemistry,1996,67(5):2115-2123.
[12]LI F,HAN Z J.Two different genes encoding acetylcholinesterase existing in cotton aphid(Aphis gossypii)[J].Genome,2002,45:1134-1141.
[13]WEILL M,FORT P,BERTHOMIEU A,et al.A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila[J].Proceedings Biological Sciences,2002,2699(1504):2007-2016.
[14]CHEN M,HAN Z J.Cloning and sequence analysis of 2 different acetylcholinesterase genes in Rhopalosiphum padi and Sitobion avenae[J].Genome,2006,49:239-243.
[15]BAEK J H,KIM J I,LEE D W,et al.Identification and characterization of ace1-type acetylcholine sterase likely associated with organophosphate resistance in Plutella xylostella[J].Pesticide Biochemistry and Physiology,2005,81:164-175.
[16]KIM J I,JUNG C S,KOH Y H,et al.Molecular,biochemical and histochemical characterization of two acetylcholinesterase c DNAs from the German cockroach Blattella germanica[J].Insect Molecular Biology,2006,15:513-522.
[17]LEE D W,KIM S S,SHIN S W,et al.Molecular characterization of two acetylcholinesterase genes from the oriental tobacco budworm,Helicoverpa assulta(Guenee)[J].Biochimica et Biophysica Acta,2006,1760:125-133.
[18]SEONG K M,KIM Y H,KWON D H,et al.Identification and characterization of three cholinesterases from the common bed bug,Cimex lectularius[J].Insect Molecular Biology,2012,21:149-159.
[19]张振飞,黄炳超,肖汉祥,等.4种农业措施对三化螟种群动态的控制作用[J].生态学报,2013,33(22):7173-7180.
[20]罗文辉.9种药剂防治水稻三化螟田间试验[J].农药管理与科学,2008,29(8):34-36.
[21]张振飞,黄炳超,张扬,等.几种药剂对三化螟室内与田间防治效果研究[J].现代农药,2011,10(1):51-54.
[22]LUO G H,LI X H,ZHANG Z C,et al.Cloning of two acetylcholinesterase genes and analysis of point mutations putatively associated with triazophos resistance in Chilo auricilius(Lepidoptera:Pyralidae)[J].Journal of Economic Entomology,2015,108(3):1289-1297.