家蝇抗药性相关羧酸酯酶MdαE7 G137D和乙酰胆碱酯酶V260L与F407Y突变的核酸检测方法

doi:10.11853/j.issn.1003.8280.2024.05.010

摘要/Abstract

摘要： 目的建立基于聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）策略,可用于家蝇抗药性相关羧酸酯酶MdαE7 G137D和乙酰胆碱酯酶（AChE）V260L与F407Y突变的分子检测方法。方法 2022年6—7月,在四川省内江市资中、威远、隆昌、市中和东兴5个县（市、区）,采用诱蝇笼或捕虫网采集家蝇成虫,用无水乙醇保存,带回实验室操作。根据家蝇羧酸酯酶MdαE7和AChE基因序列设计引物,以单只家蝇的基因组DNA为模板,进行家蝇MdαE7基因和AChE编码基因片段的PCR扩增,用限制性内切酶酶切PCR产物,根据酶切产物的电泳检测结果来区分家蝇个体的基因型。结果采用基因特异性引物,可扩增出长度分别为213、170和133 bp的PCR产物MdαE7-137,AChE-260和AChE-407。MdαE7-137经Bst XI酶切后,电泳检测显示26和187 bp 2条条带为137位点敏感纯合子（GG）,仅213 bp 1条条带为抗性纯合子（DD）,26、187和213 bp 3条条带为杂合子（G/D）。AChE-260经Sa1 I-HF^{^®}酶切后,电泳显示170 bp 1条条带的,为AChE 260位点敏感纯合子（VV）,显示26和144 bp 2条条带的,为抗性纯合子（LL）,显示26、144和170 bp 3 条条带的,为杂合子（V/L）。AChE-407经Eco RV-HF^{^®}酶切后,电泳条带存在133 bp 1条条带的,为AChE 407位点敏感纯合子（FF）,存在31和102 bp 2条条带的,为抗性纯合子（YY）,存在31、102和133 bp 3条条带的,为杂合子（F/Y）。结论建立的PCR-RFLP基因分型方法简便、准确性高,可分别用于快速检测野外家蝇种群中抗药性相关的羧酸酯酶MdαE7 G137D、AChE V260L和AchE F407Y突变的频率。

关键词: 家蝇, 羧酸酯酶MdαE7, 乙酰胆碱酯酶, 抗性突变, 检测方法

Abstract: Objective To establish a molecular method for detection of resistance-conferring mutations in carboxylesterase MdαE7 (G137D) and acetylcholinesterase (V260L and F407Y) of Musca domestica by adopting the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy. Methods Fly traps or capture nets were used to collect adult houseflies from June to July 2022 in five counties, county-level cities, or districts, i.e., Zizhong, Weiyuan, Longchang, Shizhong, and Dongxing in Neijiang City, Sichuan Province, China. The houseflies were preserved in absolute ethanol and brought back to the laboratory for processing. DNA fragments of M.domestica acetylcholinesterase-encoding gene and MdαE7 gene were amplified by polymerase chain reaction (PCR) using gene-specific primers and individual genomic DNA as the template. Gene fragments were amplified by polymerase chain reaction (PCR) using individual genomic DNA as the template. The PCR products were digested using specific restriction endonucleases. The genotype of individual houseflies was determined according to the restriction fragment length polymorphisms of digestion products via agarose gel electrophoresis. Results Using gene-specific primers, the PCR products, MdαE7-137 (213 bp), AChE-260 (170 bp), and AChE-407 (133 bp), were obtained. After MdαE7-137 was incubated with Bst XI, the wild homozygote (GG), resistant homozygote (DD) and heterozygote (G/D) could produce two bands ( 26 and 187 bp), a band (213 bp), and three bands (26, 187, and 213 bp), respectively. After AChE-260 was treated with Sal I-HF^{^®}, a band (170 bp), two bands (26 and 144 bp), and three bands (26, 144, and 170 bp) could be seen on gel for wild homozygote (VV), resistant homozygote (LL) and heterozygote (V/L) respectively. The presence of one band (133 bp), two bands (31 and 102 bp) and three bands (31, 102, and 133 bp) on the gel loaded with Eco RV-HF^{^®} digestion products of AChE-407 was scored as wild homozygote (FF), resistant homozygote (YY), and heterozygote (F/Y) respectively. Conclusions The established PCR-RFLP genotyping method is simple and highly accurate. This method can be used for rapid determination of the frequencies of the resistance-related mutations G137D in carboxylesterase MdαE7 and V260L and F407Y in AChE in field M.domestica populations.

Key words: Musca domestica, Carboxylesterase MdαE7, Acetylcholinesterase, Insecticide resistance mutation, Detection method

中图分类号:

R384.2

刘鹃, 刘鹏, 王雅伟, 余小梅, 黎雪梅, 邱星辉. 家蝇抗药性相关羧酸酯酶MdαE7 G137D和乙酰胆碱酯酶V260L与F407Y突变的核酸检测方法[J]. 中国媒介生物学及控制杂志, 2024, 35(5): 564-568.

LIU Juan, LIU Peng, WANG Ya-wei, YU Xiao-mei, LI Xue-mei, QIU Xing-hui. Nucleic acid-based method for detection of insecticide resistance-associated mutations in carboxylesterase MdαE7 （G137D） and acetylcholinesterase （V260L and F407Y） of Musca domestica[J]. Chinese Journal of Vector Biology and Control, 2024, 35(5): 564-568.

参考文献

[1] Nayduch D,Burrus RG. Flourishing in filth：House fly-microbe interactions across life history[J]. Ann Entomol Soc Am,2017,110（1）：6-18. DOI：10.1093/aesa/saw083.
[2] Sun XF,Li QL,Liu XW,et al. Harms of flies in livestock breeding farms and control methods[J]. J Tradit Chin Vet Med,2024,43（1）：82-86. DOI：10.13823/j.cnki.jtcvm.2023.124.（in Chinese）孙雪峰,李巧丽,刘希望,等. 养殖场的蝇类危害及防控方法[J]. 中兽医医药杂志,2024,43（1）：82-86. DOI：10.13823/j.cnki.jtcvm.2023.124.
[3] Wang QM,Li M,Pan J,et al. Diversity and frequencies of genetic mutations involved in insecticide resistance in field populations of the house fly （Musca domestica L.） from China[J]. Pestic Biochem Physiol,2012,102（2）：153-159. DOI：10.1016/j.pestbp.2011.12.007.
[4] Song BD,Zhang Y,Min Y,et al. Ecological surveillance result of flies and resistance of Musca domestica to commonly used insecticides in Nanjing,China,2022[J]. Chin J Vector Biol Control,2023,34（5）：637-641. DOI：10.11853/j.issn.1003.8280.2023.05.010.（in Chinese）宋丙栋,张艳,闵玥,等. 南京市2022年蝇类生态学监测及家蝇对常用杀虫剂的抗药性调查[J]. 中国媒介生物学及控制杂志,2023,34（5）：637-641. DOI：10.11853/j.issn.1003.8280.2023.05.010.
[5] Wu YY,Liu QM,Wang JN,et al. Resistance of Musca domestica to commonly used insecticides in Zhejiang Province,China,2021[J]. Chin J Vector Biol Control,2023,34（1）：26-30. DOI：10.11853/j.issn.1003.8280.2023.01.005.（in Chinese）吴瑜燕,刘钦梅,王金娜,等. 2021年浙江省家蝇对常见卫生杀虫剂的抗药性研究[J]. 中国媒介生物学及控制杂志,2023,34（1）：26-30. DOI：10.11853/j.issn.1003.8280.2023.01.005.
[6] Zhang Y,Li J,Ma Z,et al. Multiple mutations and overexpression of the MdαE7 carboxylesterase gene associated with male-linked malathion resistance in housefly,Musca domestica （Diptera：Muscidae）[J]. Sci Rep,2018,8（1）：224. DOI：10.1038/s41598-017-17325-x.
[7] Kozaki T,Brady SG,Scott JG. Frequencies and evolution of organophosphate insensitive acetylcholinesterase alleles in laboratory and field populations of the house fly,Musca domestica L.[J]. Pestic Biochem Physiol,2009,95（1）：6-11. DOI：10.1016/j.pestbp.2009.04.015.
[8] Kozaki T,Shono T,Tomita T,et al. Fenitroxon insensitive acetylcholinesterases of the housefly,Musca domestica associated with point mutations[J]. Insect Biochem Mol Biol,2001,31（10）：991-997. DOI：10.1016/S0965-1748（01）00047-9.
[9] Qiu XH,Pan J,Li M,et al. PCR-RFLP methods for detection of insecticide resistance-associated mutations in the house fly （Musca domestica）[J]. Pestic Biochem Physiol,2012,104（3）：201-205. DOI：10.1016/j.pestbp.2012.08.002.
[10] Rinkevich FD,Zhang L,Hamm RL,et al. Frequencies of the pyrethroid resistance alleles of Vssc1 and CYP6D1 in house flies from the eastern United States[J] Insect Mol Biol,2006,15（2）：157-167. DOI：10.1111/j.1365-2583.2006.00620.x.
[11] Hou YX,Liu ZW,Zhang YJ,et al. Resistance of Musca domestica to six insecticides commonly used in Hefei,China,2021[J]. Chin J Vector Biol Control,2023,34（1）：105-108. DOI：10.11853/j.issn.1003.8280.2023.01.019.（in Chinese）侯银续,刘志伟,张燕婕,等. 2021年合肥市家蝇对6种常用卫生杀虫剂的抗药性调查[J]. 中国媒介生物学及控制杂志,2023,34（1）：105-108. DOI：10.11853/j.issn.1003.8280.2023.01.019.
[12] Zhu CY,Zhao CC,Wang YG,et al. Establishment of an innovative and sustainable PCR technique for 1534 locus mutation of the knockdown resistance （kdr） gene in the dengue vector Aedes albopictus[J]. Parasit Vectors,2019,12（1）：603. DOI：10.1186/s13071-019-3829-5.
[13] Zhang L,Shi J,Shi XY,et al. Quantitative and qualitative changes of the carboxylesterase associated with beta-cypermethrin resistance in the housefly,Musca domestica （Diptera：Muscidae）[J]. Comp Biochem Physiol B Biochem Mol Biol,2010,156（1）：6-11. DOI：10.1016/j.cbpb.2010.01.011.
[14] Claudianos C,Russell RJ,Oakeshott JG. The same amino acid substitution in orthologous esterases confers organophosphate resistance on the house fly and a blowfly[J]. Insect Biochem Mol Biol,1999,29（8）：675-686. DOI：10.1016/S0965-1748（99）00035-1.
[15] Yang C,Pan J,Li M,et al. Occurrence and evolutionary origin of insecticide resistance-conferring mutations in carboxylesterase （MdαE7） in Chinese house fly populations from five provinces[J]. Acta Parasitol Med Entomol Sin,2023,30（2）：88-94. DOI：10.3969/j.issn.1005-0507.2023.02.006.（in Chinese）杨婵,潘婧,李梅,等. 家蝇羧酸酯酶MdαE7抗药性相关基因突变检测及其进化起源分析[J]. 寄生虫与医学昆虫学报,2023,30（2）：88-94. DOI：10.3969/j.issn.1005-0507.2023.02.006.
[16] Yang C,Zhu J,Pan J,et al. Evolutionary origins of insecticide resistance-conferring mutations in the acetylcholinesterase in Chinese house fly populations[J]. Acta Parasitol Med Entomol Sin,2023,30（1）：46-50. DOI：10.3969/j.issn.1005-0507.2023.01.008.（in Chinese）杨婵,朱江,潘婧,等. 家蝇抗药性相关乙酰胆碱酯酶基因突变的进化起源分析[J]. 寄生虫与医学昆虫学报,2023,30（1）：46-50. DOI：10.3969/j.issn.1005-0507.2023.01.008.