摘要
昆虫羧酸酯酶(carboxylesterases,COEs)在昆虫降解杀虫剂和代谢植物次生物质的过程中起重要作用。克隆了家蚕中肠特异表达的4个羧酸酯酶α-家族基因Bmae2、Bmae17、Bmae18和Bmae27。序列分析表明Bmae2和Bmae17编码的蛋白质均具有酯酶活性必需的催化残基Ser、Glu、His以及α-酯酶/β-酯酶家族的特征基序Gly-x-Ser-x-Gly,而Bmae18与Bmae27编码蛋白质的Ser残基分别突变为Asp和Gly残基,推测Bmae2和Bmae17具有酯酶的水解活性。4龄起蚕分别添食4种浓度的槲皮素(quercetin)后,通过半定量RT-PCR和定量PCR检测4个COEs基因在各处理组家蚕中肠中的表达均有显著上调,最大诱导表达量分别为对照组的6.90、2.21、1.30、8.78倍。分别用亚致死剂量的有机磷农药辛硫磷、敌敌畏和毒死蜱处理5龄第2天幼虫,检测中肠COEs活性均显著增强。通过半定量RT-PCR和定量PCR检测发现:Bmae17和Bmae27在低浓度辛硫磷处理的3 d内均上调表达,与酶活性检测结果基本一致,推测这2个基因可能主要参与了对辛硫磷的解毒作用;敌敌畏处理组家蚕只在处理第3天时中肠中的Bmae17、Bmae18和Bmae27的表达呈现诱导上调,在处理后的前2 d呈现出表达被抑制或与对照组无显著差异的现象,毒死蜱处理组也出现了类似现象,与酶活性检测结果不完全一致,推测与在中肠表达的其它COEs参与了对这2种农药的解毒作用有关。研究结果为进一步深入研究家蚕中肠COEs基因的解毒机制奠定了一定基础。
Insect carboxylesterases play important roles in detoxification of insecticides and plant secondary metabolites.Four midgut specific COE genes(Bmae2,Bmae17,Bmae18 and Bmae27) were cloned from silkworm(Bombyx mori).Sequence analysis indicated that these COE genes belonged to α-esterase family.Bmae2 and Bmae17 contained the catalytic residues Ser,Glu and His which are essential to esterase activity,and the characteristic motif Gly-x-Ser-x-Gly of α/β-esterase family.However,the Ser residue of Bmae18 and Bmae27 has been mutated to Asp and Gly,respectively.It was suggested that Bmae2 and Bmae17 might contain the hydrolysis activity of esterase.After the 4th instar newly exuviated larvae were fed with quercetin at four concentrations,the expressions of all four BmCOEs were significantly up-regulated.The maximum induced expression of Bmae2,Bmae17,Bmae18 and Bmae27 was 6.90,2.21,1.30 and 8.78 times to the control,respectively.After day 2 larvae of the 5th instar were fed with sub-lethal doses of phoxim,dichlorvos and chlorpyrifos,COE activities were all significantly increased in the midgut.Semi-quantitative RT-PCR and qPCR results indicated that the expressions of Bmae17 and Bmae27 were significantly up-regulated within 3 d of treatment by low concentration phoxim,being in general agreement with the analytical result of enzymatic activity.This suggested that Bmae17 and Bmae27 might play important roles in detoxification of phoxim.Only a certain dosage of dichlorvos could induce up-regulation of Bmae17,Bmae18 and Bmae27 expressions on the third day of treatment,and inhibition or no significant difference was found in the first and second days of treatment.Such patterns were also observed in chlorpyrifos-treated groups,which were inconsistent with the analytical result of enzymatic activity.It is inferred that some other COE genes expressed in the midgut might be involved in the detoxification of dichlorvos and chlorpyrifos.The data presented in this study provide useful information for further studies on detoxificatio
出处
《蚕业科学》
CAS
CSCD
北大核心
2013年第2期247-256,共10页
ACTA SERICOLOGICA SINICA
基金
重庆市科委自然科学基金项目(No.cstc2012jjA00010)
中央高校基本科研业务费项目(No.CDJZR10290002)
关键词
家蚕
羧酸酯酶
基因克隆
有机磷杀虫剂
槲皮素
诱导
Bombyx mori
Carboxylesterases
Gene cloning
Organophosphorus Insecticide
Quercetin
Induction
