HUANG Lei,PENG Yingchuan,HAN Zhaojun.Cloning and polymorphism analysis of acetylcholinesterase genes from Sesamia inferens[J].Journal of Nanjing Agricultural University,2019,42(6):1050-1058.[doi:10.7685/jnau.201903046]





Cloning and polymorphism analysis of acetylcholinesterase genes from Sesamia inferens
黄磊1 彭英传2 韩召军1
1. 南京农业大学植物保护学院/农作物生物灾害综合治理教育部重点实验室, 江苏 南京 210095;
2. 江西农业大学昆虫研究所, 江西 南昌 330045
HUANG Lei1 PENG Yingchuan2 HAN Zhaojun1
1. College of Plant Protection/Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, China;
2. Institute of Entomology, Jiangxi Agricultural University, Nanchang 330045, China
Sesamia inferensacetylcholinesterasegene cloningpolymorphism
[目的] 本研究旨在通过克隆大螟的乙酰胆碱酯酶基因ace,进行多态性分析,以便寻找抗药性相关的突变位点,为建立高通量药剂筛选方法和适宜的抗药性分子检测方法提供新途径。[方法] 利用PCR和cDNA末端快速扩增(RACE)技术克隆大螟的ace全长序列,通过分子特征和构建系统发育树对其进行验证。通过序列比对,分析不同地理种群大螟ace基因的多态性,寻找经典抗药性突变位点,同时测定相应种群的三唑磷抗性,联合分析相关的抗药性突变位点。[结果] 克隆获得了大螟2个ace基因的全长cDNA,分别将其命名为Sinace1Sinace2(GenBank登录号分别为MK641586和MK641585)。它们均含有乙酰胆碱酯酶家族的保守结构特征,系统发育树分析表明它们和鳞翅目近缘种的ace基因亲缘关系最近。关键靶标基因Sinace1在4个地理种群的97条基因序列中共发现了比率不同的107种核苷酸多态性位点和21种氨基酸突变类型,但未发现经典抗药性位点的突变。生物测定结果表明:不同地理种群的大螟对三唑磷的敏感性相差较小(1~5倍),对比分析也没有发现种群之间突变比率与抗性水平一致的突变位点。[结论] 大螟ace1基因尚未产生可测定的抗药性突变,但本研究率先克隆报道了大螟的2个ace基因全长序列,并揭示了大螟不同地理种群ace1基因的主要多态性位点。
[Objectives] This study tries to seek a new molecular approach substitute by cloning the acetylcholinesterase genes(ace)from the Sesamia inferens and analyzing their polymorphism and resistance related mutations. It provides a new way to establish high-throughput insecticide screening and suitable insecticide resistance molecular detection methods.[Methods] Acetylcholinesterase genes were cloned by polymerase chain reaction(PCR)and rapid amplification of cDNA ends(RACE),and confirmed by their molecular characteristics and phylogenetic tree analysis. The polymorphism frequencies were analyzed for the key gene ace1 among different geographical populations. The corresponding resistance levels to triazophos were also tested,and finally the resistance mutations were pursued.[Results] Two full-length cDNA sequences from S.inferens were cloned,characterized,and designated as Sinace1 and Sinace2(GenBank accession No.:MK641586 and MK641585),respectively. They both shared all the conserved structural features of the ace family,and the phylogenetic tree analysis indicated that they had the closest relationship with the corresponding ace genes from lepidopeterians. Polymorphism analysis of the 97 Sinace1 sequences showed 107 nucleotide polymorphism sites and 21 amino acid mutations all with varied frequencies among the four geographical populations. No resistant mutations previously reported in different ace genes were found. Four of the geographical populations were tested and showed that their sensitivities to triazophos varied within 5 times. Comparison analysis found no amino acid mutations with their occurrences consistent with triazophos resistance among the geographical populations.[Conclusions] No detectable resistance mutations have happened in Sinace1 of S.inferens. However,the two ace genes and the polymorphism firstly reported in this work should lay a foundation for the study of the resistance mechanism for organophosphate resistance,and technique development for selecting efficient insecticides by enzyme activity inhibition.


[1] Soreq H,Seidman S. Acetylcholinesterase:new roles for an old actor[J]. Nature Reviews Neuroscience,2001,2(4):294-302.
[2] Casida J E,Durkin K A. Neuroactive insecticides:targets,selectivity,resistance,and secondary effects[J]. Annual Review of Entomology,2013,58(1):99-117.
[3] Oppenoorth F J,van Asperen K. Allelic genes in the housefly producing modified enzymes that cause organophosphate resistance[J]. Science,1960,132(3422):298-299.
[4] Newcomb R D,Campbell P M,Ollis D L,et al. A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly[J]. Proc Natl Acad Sci USA,1997,94(14):7464-7468.
[5] Newcomb R D,Campbell P M,Russell R J,et al. cDNA cloning,baculovirus-expression and kinetic properties of the esterase,E3,involved in organophosphorus resistance in Lucilia cuprina[J]. Insect Biochemistry & Molecular Biology,1997,27(1):15-25.
[6] Newcomb R D,Gleeson D M,Yong C G,et al. Multiple mutations and gene duplications conferring organophosphorus insecticide resistance have been selected at the rop-1 locus of the sheep blowfly,Lucilia cuprina[J]. Journal of Molecular Evolution,2005,60(2):207-220.
[7] Hall L M,Spierer P. The Ace locus of Drosophila melanogaster:structural gene for acetylcholinesterase with an unusual 5’ leader[J]. The EMBO Journal,1986,5(11):2949-2954.
[8] Arpagaus M,Fournier D,Toutant J P. Analysis of acetylcholinesterase molecular forms during the development of Drosophila melanogaster:evidence for the existence of an amphiphilic monomer[J]. Insect Biochemistry,1988,18(6):539-549.
[9] Zhu K Y,Brindley W A. Enzymological and inhibitory properties of acetylcholinesterase purified from Lygus hesperus Knight(Hemiptera:Miridae)[J]. Insect Biochemistry and Molecular Biology,1992,22(3):245-251.
[10] Zhu K Y,Marshall Clark J. Purification and characterization of acetyl-cholinesterase from the Colorado potato beetle,Leptinotarsa decemlineata(say)[J]. Insect Biochemistry and Molecular Biology,1994,24(5):453-461.
[11] Jiang X J,Qu M J,Denholm I,et al. Mutation in acetylcholinesterase1 associated with triazophos resistance in rice stem borer,Chilo suppressalis(Lepidoptera:Pyralidae)[J]. Biochemical & Biophysical Research Communications,2009,378(2):269-272.
[12] 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. Wang D,Tang Z H,Shang J Y,et al. Advances in the studies of acetylcholinesterase genes from insects[J]. Acta Entomologica Sinica,2006,49(3):497-503(in Chinese with English abstract).
[13] 张海艳,李海东,韩召军. 大螟田间种群对不同杀虫剂敏感性的差异[J]. 中国稻米,2012,18(1):29-33. Zhang H Y,Li H D,Han Z J. Difference in susceptibility of field populations of Sesamia inferens(Walker)to various insecticides[J]. China Rice,2012,18(1):29-33(in Chinese with English abstract).
[14] 吕亮,常向前,杨小林,等. 湖北水稻蛀秆螟虫越冬情况调查[J]. 环境昆虫学报,2018,40(5):1051-1057. Lü L,Chang X Q,Yang X L,et al. Biological survey on the overwintering larvae of rice stem borer in Hubei Province[J]. Journal of Environmental Entomology,2018,40(5):1051-1057(in Chinese with English abstract).
[15] 周操,王召,杨洪,等. 贵州中部稻区大螟和二化螟发生规律初探[J]. 山地农业生物学报,2018,37(4):18-24,94. Zhou C,Wang Z,Yang H,et al. Occurrence of stem borers in rice field of central Guizhou[J]. Journal of Mountain Agriculture and Biology,2018,37(4):18-24,94(in Chinese with English abstract).
[16] 高忠文,陶岭梅,苏建亚,等. 防治稻纵卷叶螟高毒农药替代药剂的室内筛选[J]. 中国水稻科学,2008,22(6):631-636. Gao Z W,Tao L M,Su J Y,et al. Laboratory screening of alternatives to highly toxic insecticides against the rice leafroller,Cnaphalocrocis medinalis[J]. Chinese Journal of Rice Science,2008,22(6):631-636(in Chinese with English abstract).
[17] 周国辉,凌炎,龙丽萍. 不同杀虫剂对稻纵卷叶螟的毒效研究[J]. 中国农学通报,2012,28(6):202-206. Zhou G H,Ling Y,Long L P. Effects of different insecticides on Cnaphalocrocis medinalis Guenee[J]. Chinese Agricultural Science Bulletin,2012,28(6):202-206(in Chinese with English abstract).
[18] 常菊花,何月平. 二化螟对常用杀虫剂的抗性研究进展[J]. 长江大学学报(自然科学版),2016,13(9):4-6,21. Chang J H,He Y P. Progress in the study on resistance of the Chilo suppressalis to common insecticides[J]. Journal of Yangtze University(Natural Science Edition),2016,13(9):4-6,21(in Chinese).
[19] 姜海平,阚李斌,蔡超,等. 不同杀虫剂防治水稻大螟田间药效试验研究[J]. 农药科学与管理,2011,32(5):42-43. Jiang H P,Kan L B,Cai C,et al. Field efficacy of different insecticides against Sesamia inferens[J]. Pesticide Science and Administration,2011,32(5):42-43(in Chinese).
[20] 徐丽娜,周子燕,胡飞,等. 安徽省二化螟不同地理种群对主要杀虫剂的敏感性[J]. 安徽农业大学学报,2018,45(4):740-744. Xu L N,Zhou Z Y,Hu F,et al. The insecticide susceptibility of Chilo suppressalis in Anhui Province[J]. Journal of Anhui Agricultural University,2018,45(4):740-744(in Chinese with English abstract).
[21] 黄诚华,姚洪渭,叶恭银,等. 浙江省二化螟不同种群和大螟对三唑磷的敏感性研究[J]. 农药学学报,2005,7(4):323-328. Huang C H,Yao H W,Ye G Y,et al. Susceptibility of different populations of Chilo suppressalis and Sesamia inferens to triazophos in Zhejiang Province of China[J]. Chinese Journal of Pesticide Science,2005,7(4):323-328(in Chinese with English abstract).
[22] Li F,Han Z J. Mutations in acetylcholinesterase associated with insecticide resistance in the cotton aphid,Aphis gossypii Glover[J]. Insect Biochemistry and Molecular Biology,2004,34(4):397-405.
[23] Lee D W,Choi J Y,Kim W T,et al. Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella(L.)[J]. Biochem Biophys Res Commun,2007,353(3):591-597.
[24] 姜晓静. 二化螟对三唑磷的靶标抗性研究[D]. 南京:南京农业大学,2009. Jiang X J. Studies on target resistance of Chilo suppressalis(Walker)to triazophos[D]. Nanjing:Nanjing Agricultural University,2009(in Chinese with English abstract).
[25] 罗光华,张志春,韩光杰,等. 二化螟越冬种群特点及其对三唑磷靶标抗性突变频率分析[J]. 中国水稻科学,2012,26(4):481-486. Luo G H,Zhang Z C,Han G J,et al. Characteristics of overwintering populations of rice stem borers and mutation frequencies of resistance to triazophos[J]. Chinese Journal of Rice Science,2012,26(4):481-486(in Chinese with English abstract).
[26] Gawad C,Koh W,Quake S R. Single-cell genome sequencing:current state of the science[J]. Nature Reviews Genetics,2016,17(3):175-188.
[27] Castle P E,Porras C,Quint W G,et al. Comparison of two PCR-based human papillomavirus genotyping methods[J]. Journal of Clinical Microbiology,2008,46(10):3437-3445.
[28] Guo D H,Luo J P,Zhou Y N,et al. ACE:an efficient and sensitive tool to detect insecticide resistance-associated mutations in insect acetylcholinesterase from RNA-Seq data[J]. BMC Bioinformatics,2017,18(1):330.
[29] Mao Y,Tan F,Yan S G,et al. High-throughput genotyping of single-nucleotide polymorphisms in ace-1 gene of mosquitoes using MALDI-TOF mass spectrometry[J]. Insect Science,2013,20(2):167-174.
[30] 魏星. 褐飞虱烟碱型乙酰胆碱受体β1亚基氨基酸多态性分析及与吡虫啉抗性关系研究[D]. 南京:南京农业大学,2013. Wei X. Analysis of nAChR β1 subunit polymorphism and associate with imidacloprid resistance in the brown planthopper[D]. Nanjing:Nanjing Agricultural University,2013(in Chinese with English abstract).
[31] 董德臻,管利莎,童巧琼,等. 吡虫啉对褐飞虱DNA多态性的影响[J]. 核农学报,2013,27(5):584-590. Dong D Z,Guan L S,Tong Q Q,et al. Effects of imidacloprid on DNA polymorphism in the brown planthopper[J]. Journal of Nuclear Agricultural Sciences,2013,27(5):584-590(in Chinese with English abstract).
[32] 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 B:Biological Sciences,1998,353(1376):1685-1693.


 Gu Hainan.A PRELIMINARY STUDY ON THE NATURAL POPULATION DYNAMICS OF PINK RICE BORER,SESAMIA INFERENS WALKER[J].Journal of Nanjing Agricultural University,1987,10(6):31.[doi:10.7685/j.issn.1000-2030.1987.01.006]
 FENG Xiao-hui,LIU Bao-sheng,GUO Hui-fang,et al.Study on efficacy of several kinds of insecticides on the pink rice borer,Sesamia inferens(Walker),in laboratory and fields[J].Journal of Nanjing Agricultural University,2011,34(6):67.[doi:10.7685/j.issn.1000-2030.2011.05.013]
 JIN Junyan,ZHANG Yanan,YANG Ke,et al.Molecular identification and temporal expression of two general odorant-binding protein genes in the pink rice borer, Sesamia inferens(Walker)(Lepidoptera:Noctuidae)[J].Journal of Nanjing Agricultural University,2014,37(6):87.[doi:10.7685/j.issn.1000-2030.2014.01.015]
 ZHAO Jun,FU Wenxi,HAN Zhaojun.Resistance monitoring of Sesamia inferens(Walker)to seven insecticides and verification of related baseline data[J].Journal of Nanjing Agricultural University,2016,39(6):84.[doi:10.7685/jnau.201505021]


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