甜菜夜蛾黄素单加氧酶的原核表达及其对杀虫剂的代谢作用  

Prokaryotic expressions of Spodoptera exigua flavin-dependent monooxygenases and the role in insecticide metabolism

作　　者：汤雨洁 田祥瑞 胡波 黄河 薛圆 苏建亚 TANG Yujie;TIAN Xiangrui;HU Bo;HUANG He;XUE Yuan;SU Jianya(College of Plant Protection/Key Laboratory of Integrated Management of Crop Disease and Pests,Ministry of Education,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[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

出　　处：《南京农业大学学报》2019年第4期672-681,共10页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31272063)

摘　　要：[目的]采用原核表达技术对甜菜夜蛾的黄素单加氧酶(FMO)进行体外表达,获得具有活性的FMO酶,在此基础上开展对杀虫剂的代谢功能研究。[方法]通过将甜菜夜蛾FMO基因的开放阅读框构建到pET32a原核表达载体中,在大肠杆菌中表达目的蛋白,利用该载体的His标签使用镍柱对表达蛋白进行纯化,再通过咪唑梯度洗脱得到纯化的重组FMO酶蛋白。采用还原型辅酶Ⅱ(NADPH)法测定FMO酶的活性以及对杀虫剂的氧化活性,并用HPLC法分析FMO酶催化杀虫剂降解的能力,最后用液质联用技术鉴定代谢产物。[结果]通过原核表达技术成功得到可溶性的SeFMO2与SeFMO3酶,这2种酶对FMO的模式底物甲巯咪唑与苄达明均具有氧化活性,SeFMO2与SeFMO3对甲巯咪唑S-氧化的代谢动力学参数Km值分别为37.59与3.00μmol·L^-1,对苄达明N-氧化的Km值分别为165.98与17.71μmol·L^-1。对模式底物的S-氧化活性与N-氧化活性均是SeFMO3高于SeFMO2,2种酶的S-氧化活性均高于N-氧化活性。SeFMO2和SeFMO3对毒死蜱、硫双威以及溴虫腈具有一定的代谢活性,HPLC分析表明这2种FMO酶均能催化这3种杀虫剂的氧化降解,SeFMO2对毒死蜱、溴虫腈和硫双威的降解率依次是11.8%、11.5%和27.8%,SeFMO3对它们的降解率分别是28.8%、23.1%和30.2%,其中对硫双威的降解作用较强。对硫双威代谢产物的鉴定表明:FMO主要催化硫双威的硫醚氧化,形成硫双威的砜化合物。[结论]甜菜夜蛾黄素单加氧酶对外源物具有S-氧化活性与N-氧化活性,对某些杀虫剂具有氧化代谢能力,是昆虫的重要解毒酶系之一。[Objectives] The prokaryotic expression was used to express flavin-dependent monooxygenase of Spodoptera exigua ( SeFMO )to obtain the soluble and active proteins of SeFMO,and then the metabolic function of SeFMO on insecticides was analyzed.[Methods] The open read frame of SeFMO was constructed into the vector pET32a to express the SeFMO proteins in Escherichia coli . The expressed protein was purified using Ni-NTA gel affinity chromatography and imidazole gradient washes. The enzymatic activities were evaluated by NADPH method,and the oxidative activities of SeFMO on insecticides were also determined. The breakdown rates of insecticides by SeFMO were also quantified by HPLC analysis,and finally the breakdown products of insecticides by SeFMO were identified by HPLC-MS/MS technology.[Results] The soluble and active SeFMO2 and SeFMO3 enzymes were obtained successfully;SeFMO2 and SeFMO3 had the oxidative activities on the model substrate of FMO:methimazole and benzydamin. The metabolically kinetic parameter K m values for S -oxidation of SeFMO2 and SeFMO3 on methimazole were 37.59 and 3.00 μmol · L^-1 ,respectively. The K m values for N -oxidation of two enzymes on benzydamin were 165.98 and 17.71 μmol · L^-1 ,respectively. Both S - and N -oxidation the metabolic activities of SeFMO3 were higher than those of SeFMO2,and S -oxidation activity was higher than N -oxidation activity for both enzymes. Two SeFMO were also found to have the oxidative activities against insecticides,such as chlorpyrifos,thiodicarb and chlorfenapyr. HPLC analysis demonstrated that SeFMO2 and SeFMO3 catalyzed the oxidative turnover of these three insecticides,after reaction with SeFMO2 the content of chlorpyrifos,chlorfenapyr and thiodicarb were reduced by 11.8%,11.5% and 27.8%,respectively;SeFMO3 reduced these insecticides for 28.8%,23.1% and 30.2%,respectively. Among three analyzed insecticides the clearance of thiodicarb by SeFMO was the most obvious. Finally,the metabolic products of thiodicarb by SeFMO were identified,the oxidative

关 键 词：黄素单加氧酶 原核表达 酶活性 杀虫剂代谢 甜菜夜蛾 

分 类 号：Q965.9[生物学—昆虫学]