淡色库蚊抗吡丙醚种群与敏感种群谷胱甘肽硫转移酶的生物化学性质
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摘要
目的比较淡色库蚊抗吡丙醚种群和敏感种群谷胱甘肽硫转移酶(GSTs)的生物化学性质,初步探讨其生化抗性机制。方法以室内连续筛选的淡色库蚊抗吡丙醚种群及淡色库蚊敏感种群为研究对象,参照Habig等(1976)的方法测定GSTs活性,采用t检验进行样本均数差别的统计学分析。结果淡色库蚊GSTs的最适底物为1-氯-2,4-二硝基苯(CDNB)。CDNB为底物时,抗吡丙醚种群和敏感种群GSTs的酶活性分别为3.626×10-4和2.737×10-4nmol/mg protein·min。淡色库蚊GSTs水解活性随底物浓度〔CDNB和DCNB(1,2-二氯-4-硝基苯)〕的增加而升高;在一定浓度范围内,抗性种群GSTs对这2种底物的水解活性略高于敏感种群。CDNB为底物时,抗性种群GSTs的米氏常数(Km)与最大反应速度(Vmax)分别为8.01 mmol/L和4.87×102μmol/min·mg,敏感种群的Km与Vmax分别为1.11 mmol/L和3.87×102μmol/min·mg,抗性种群的Km、Vmax与敏感种群差异有统计学意义(ta=11.415,tb=6.411,均P<0.05)。DCNB为底物时,抗性种群的Km与Vmax与敏感品系差异无统计学意义(tc=0.134,td=1.280,均P>0.05)。结论 GSTs在淡色库蚊对吡丙醚抗性形成过程中可能起一定解毒代谢作用。
Objective To preliminarily explore the resistance mechanism of pyriproxyfen-resistant Culex pipiens pallens by comparing the biochemical characteristics of glutathione s-transferases(GSTs) between resistant and susceptible populations of Cx. pipiens pallens. Methods The pyriproxyfen-resistant and susceptible populations of Cx. pipiens pallens were chosen through indoor successive selection as the research objects. And the activities of GSTs were determined according to the method reported by Habig et al.(1976). Statistical analysis was thus performed using the t-test for sample mean comparison.Results The optimal substrate of GSTs from Cx. pipiens pallens was 1-chloro-2, 4-dinitrobenzene(CDNB). When CDNB was used as the substrate, the activities of GSTs in the resistant and susceptible populations were 3.626×10-4 and 2.737×10-4 nmol/mg protein·min, respectively. The hydrolytic activity of Cx. pipiens pallens GSTs increased with the elevation of substrate concentrations〔i.e., CDNB and DCNB(1, 2-dichloro-4-nitrobenzene)〕. In a certain concentration range, the hydrolytic activity of GSTs in the resistant population was slightly higher than that in the susceptible population towards both substrates. When CDNB was used as the substrate, the Michaelis constant(Km) and maximum reaction rate(Vmax) of GSTs in the resistant population were 8.01 mmol/L and 4.87×102μmol/min·mg, respectively, while those in the susceptible population were 1.11 mmol/L and 3.87×102μmol/min·mg, respectively. There were significant differences between these two populations regarding Km and Vmax(ta= 11.415, tb= 6.411, all P<0.05). When DCNB was used as the substrate,there was no significant difference between the two populations regarding Km and Vmax of GSTs(tc=0.134, td=1.280, all P>0.05). Conclusion GSTs may play an antidotal and metabolic role in the formation of pyriproxyfen resistance in Cx.pipiens pallens.
Objective To preliminarily explore the resistance mechanism of pyriproxyfen-resistant Culex pipiens pallens by comparing the biochemical characteristics of glutathione s-transferases(GSTs) between resistant and susceptible populations of Cx. pipiens pallens. Methods The pyriproxyfen-resistant and susceptible populations of Cx. pipiens pallens were chosen through indoor successive selection as the research objects. And the activities of GSTs were determined according to the method reported by Habig et al.(1976). Statistical analysis was thus performed using the t-test for sample mean comparison.Results The optimal substrate of GSTs from Cx. pipiens pallens was 1-chloro-2, 4-dinitrobenzene(CDNB). When CDNB was used as the substrate, the activities of GSTs in the resistant and susceptible populations were 3.626×10-4 and 2.737×10-4 nmol/mg protein·min, respectively. The hydrolytic activity of Cx. pipiens pallens GSTs increased with the elevation of substrate concentrations〔i.e., CDNB and DCNB(1, 2-dichloro-4-nitrobenzene)〕. In a certain concentration range, the hydrolytic activity of GSTs in the resistant population was slightly higher than that in the susceptible population towards both substrates. When CDNB was used as the substrate, the Michaelis constant(Km) and maximum reaction rate(Vmax) of GSTs in the resistant population were 8.01 mmol/L and 4.87×102μmol/min·mg, respectively, while those in the susceptible population were 1.11 mmol/L and 3.87×102μmol/min·mg, respectively. There were significant differences between these two populations regarding Km and Vmax(ta= 11.415, tb= 6.411, all P<0.05). When DCNB was used as the substrate,there was no significant difference between the two populations regarding Km and Vmax of GSTs(tc=0.134, td=1.280, all P>0.05). Conclusion GSTs may play an antidotal and metabolic role in the formation of pyriproxyfen resistance in Cx.pipiens pallens.
引文
[1]刘洪霞,冷培恩,徐仁权,等.上海地区蚊虫对常用杀虫剂的抗性及防治对策[J].中华卫生杀虫药械,2009,15(2):112-115.
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[9] Habig WH,Jacoby WB. Assay for differentiation of glutathioneS-transferase[M]//Jacoby WB. Methods in Enzymology. New York:Academic Press,1981∶398-405.
[10]刘洪霞,徐劲秋,刘曜,等.淡色库蚊吡丙醚抗性品系和敏感品系羧酸酯酶生化性质差异研究[J].中国媒介生物学及控制杂志,2016,27(2):103-106. DOI:10.11853/j.issn.1003.8280.2016.02.003.
[11] Ishaaya I,Kontsedalov S,Horowitz AR. Biorational insecticides:mechanism and cross-resistance[J]. Arch Insect Biochem Physiol,2005,58(4):192-199. DOI:10.1002/arch.20042.
[12] Michaelakis A,Porichi AE,Koliopoulos G. Activity of pyriproxyfen,an insect growth regulator,on Culex pipiens(Diptera:Culicidae)[J]. Hellenic Plant Prot J,2009,2(1):41-46.
[13]刘洪霞,冷培恩,徐劲秋,等.淡色库蚊对吡丙醚的抗性选育及抗性风险评估[J].上海预防医学,2015,27(9):533-536.DOI:10.19428/j.cnki.sjpm.2015.09.004.
[14]刘洪霞,冷培恩,徐劲秋.抗吡丙醚淡色库蚊对5种常用杀虫剂的交互抗性[J].中国媒介生物学及控制杂志,2015,26(3):252-253. DOI:10.11853/j.issn.1003.4692.2015.03.008.
[2]孟凤霞,靳建超,陈云,等.我国淡色库蚊/致倦库蚊对常用化学杀虫剂的抗药性[J].中国媒介生物学及控制杂志,2011,22(6):517-520,528.
[3] Cui F,Raymond M,Qiao CL. Insecticide resistance in vector mosquitoes in China[J]. Pest Manag Sci,2006,62(11):1013-1022. DOI:10.1002/ps.1288.
[4]王永明,辛正,刘慧媛,等. 5%吡丙醚微乳剂阻止蝇幼虫化蛹及羽化实验观察[J].预防医学论坛,2006,12(5):566-568.DOI:10.3969/j.issn.1672-9153.2006.05.031.
[5]徐仁权,刘洪霞,冷培恩,等. 0.5%吡丙醚颗粒剂对白纹伊蚊控制效果的研究[J].中国媒介生物学及控制杂志,2010,21(4):297-299.
[6] Shah RM,Abbas N,Shad SA,et al. Selection,resistance risk assessment,and reversion toward susceptibility of pyriproxyfen in Musca domestica L.[J]. Parasitol Res,2015,114(2):487-494. DOI:10.1007/s00436-014-4206-0.
[7]师灿南,开文龙,刘起勇,等.景洪市登革热媒介伊蚊对吡丙醚的敏感性及其作用特点[J].中国媒介生物学及控制杂志,2017,28(2):108-112. DOI:10.11853/j.issn.1003.8280.2017.02.003.
[8]唐振华,吴士雄.昆虫抗药性的遗传与进化[M].上海:上海科学技术文献出版社,1999:103.
[9] Habig WH,Jacoby WB. Assay for differentiation of glutathioneS-transferase[M]//Jacoby WB. Methods in Enzymology. New York:Academic Press,1981∶398-405.
[10]刘洪霞,徐劲秋,刘曜,等.淡色库蚊吡丙醚抗性品系和敏感品系羧酸酯酶生化性质差异研究[J].中国媒介生物学及控制杂志,2016,27(2):103-106. DOI:10.11853/j.issn.1003.8280.2016.02.003.
[11] Ishaaya I,Kontsedalov S,Horowitz AR. Biorational insecticides:mechanism and cross-resistance[J]. Arch Insect Biochem Physiol,2005,58(4):192-199. DOI:10.1002/arch.20042.
[12] Michaelakis A,Porichi AE,Koliopoulos G. Activity of pyriproxyfen,an insect growth regulator,on Culex pipiens(Diptera:Culicidae)[J]. Hellenic Plant Prot J,2009,2(1):41-46.
[13]刘洪霞,冷培恩,徐劲秋,等.淡色库蚊对吡丙醚的抗性选育及抗性风险评估[J].上海预防医学,2015,27(9):533-536.DOI:10.19428/j.cnki.sjpm.2015.09.004.
[14]刘洪霞,冷培恩,徐劲秋.抗吡丙醚淡色库蚊对5种常用杀虫剂的交互抗性[J].中国媒介生物学及控制杂志,2015,26(3):252-253. DOI:10.11853/j.issn.1003.4692.2015.03.008.