桔全爪螨对甲氰菊酯和阿维菌素的抗性及其酯酶基因的克隆与表达研究
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摘要
桔全爪螨Panonychus citri (McGregor)是一种对柑桔严重为害的世界性害螨。该螨的抗药性问题已引起国内外植保界和农药界的高度重视。本学位论文在国家公益性行业(柑桔)科研专项经费(Nyhyzx 07-057)、重庆市自然科学基金项目(CSTC,2009BA1042)和现代农业产业(柑桔)技术体系岗位科学家经费的支持下,较为系统地研究了桔全爪螨对两种主要杀螨剂甲氰菊酯和阿维菌素产生抗性的生态学原因及生化机理,并对其酯酶基因进行了克隆和表达,其主要研究结果如下：
1两种杀螨剂对桔全爪螨的亚致死效应研究
1.1甲氰菊酯亚致死浓度对桔全爪螨生长发育及繁殖的影响
用甲氰菊酯LC20的浓度处理桔全爪螨的若螨和成螨,观察其对当代和后代(F1、F2代)生长发育及繁殖的影响。结果表明,甲氰菊酯LC20浓度处理若螨后,当代若螨发育成为成螨的百分率(以下简称为羽化率)降低,雌成螨产卵量增加,但后代雌性比例下降。甲氰菊酯亚致死浓度处理若螨或成螨对F1和F2代的影响不完全相同,但两种处理基本相似。处理成螨和若螨后,F1、F2代的产卵前期显著缩短(P<0.05)。F1、F2代的后代雌性比例都增大,但F1代卵的孵化率和若螨羽化率降低,产卵量和成螨寿命增加,F2代却相反。利用生命表方法分析得知,甲氰菊酯LC20处理若螨和成螨后,其F1代的净增殖率、内禀增长率和周限增长率都增大,种群加倍时间缩短；F2代的净增殖率虽然减小,但由于世代历期缩短,内禀增长率和周限增长率仍然显著增大(P<0.05),种群加倍时间显著缩短(尸<0.05)。总体来看,甲氰菊酯LC20浓度可以促进桔全爪螨的增殖,其作用效应与用药时期(成螨期和若螨期)无关。
1.2阿维菌素亚致死浓度对桔全爪螨生长发育及繁殖的影响
在实验室条件下对阿维菌素亚致死浓度LC20对桔全爪螨生长发育和繁殖的影响进行了研究。结果表明,用阿维菌素处理若螨后,对当代若螨羽化率和后代的卵孵化率没有显著影响,但是,其F1、F2代产卵量减少,产卵前期和成螨寿命缩短,因此,世代历期也缩短。总体来看,在成螨期用药和若螨期用药对子代种群的影响不同,其后代的种群生命表参数在两种处理间具有显著差异,且这种差异在F2代更为明显。若螨期用药后对F1、F2代具有积极作用,如,内禀增长率、净增殖率、周限增长率均提高,种群加倍时间相应缩短。相反,成螨期用药后,其F1、F2代的内禀增长率、净增殖率、周限增长率均减小,种群加倍时间相应延长。
1.3两种杀螨剂对桔全爪螨体内主要解毒酶的诱导作用
甲氰菊酯和阿维菌素LC20浓度处理桔全爪螨成螨后,对体内三种主要解毒酶CarE、GSTs和MFO的诱导效应不同,其时间效应也不尽相同。甲氰菊酯处理后48 h内,CarE活性先上升后下降,又再次上升,波动范围很大,其比活力在12 h、36 h、48 h显著高于对照(P<0.05);对GSTs的诱导作用较为明显,在诱导高峰期的第36 h其比活力为对照的3.31倍,显著高于对照(P<0.05)；对桔全爪螨的多功能氧化酶O-脱甲基活力诱导作用相对较小。而阿维菌素处理桔全爪螨雌成螨后,CarE比活力在前12 h表现为上升趋势,12-24 h酶活力降低,以后趋于平缓；对GSTs的诱导作用也不如甲氰菊酯明显,仅在处理12 h和36 h后比活力有所提高；且阿维菌素LC20浓度处理对桔全爪螨体内微粒体MFO氧脱甲基活力具有一定的抑制作用,在处理24 h后抑制作用最为明显,与对照相比差异显著(P<0.05)。
2桔全爪螨抗药性选育、交互抗性及抗性风险评估
在实验室抗性品系选育基础上,应用数量遗传学中的域性状分析法,研究了桔全爪螨北碚种群对甲氰菊酯、阿维菌素两种杀螨剂的抗性现实遗传力,对两种药剂在不同致死率下抗性发展的速率进行了预测,并对这两种杀螨剂进行了交互抗性研究。结果表明,用甲氰菊酯、阿维菌素分别汰选16、11代后,桔全爪螨对两种药剂的抗性分别为29.92倍和3.80倍,抗性现实遗传力分别为0.1544和0.0475。在室内选择条件下,致死率为50%-90%时,要获得10倍抗性,甲氰菊酯仅需要16-8代,阿维菌素需要26-12代。在田间选择条件下,两种药剂都将需要更长的时间。并且,甲氰菊酯抗性品系(FeR)对哒螨灵、三氯杀螨醇和三唑锡产生了明显的交互抗性,阿维菌素抗性品系(AvR)对甲维盐产生了明显的交互抗性。这些结果表明,生物源农药阿维菌素的抗性风险明显低于菊酯类药剂甲氰菊酯,且甲氰菊酯的交互抗性谱明显大于阿维菌素。试验结果可为桔全爪螨抗性治理提供参考。
3桔全爪螨对两种杀螨剂的抗性的生化机理
3.1三种增效剂对两种杀螨剂的增效作用
甲氰菊酯、阿维菌素分别与三种增效剂(PBO、TPP、DEM,分别是多功能氧化酶、酯酶和谷胱甘肽S-转移酶的专一性抑制剂)组配对桔全爪螨敏感品系(SS)、甲氰菊酯抗性品系(FeR)、阿维菌素抗性品系(AvR)进行了毒力测定,并以抗性品系增效倍数与敏感品系增效倍数的比值作为增效作用的评定标准。结果显示,PBO、TPP、DEM三种增效剂对甲氰菊酯均有一定的增效作用,其相对增效倍数分别为3.18,2.90,1.68倍;而PBO、TPP、DEM对阿维菌素的增效作用相对较弱,其相对增效倍数分别为1.81、1.44、0.84倍。这表明桔全爪螨对甲氰菊酯的抗性与多功能氧化酶、羧酸酯酶、谷胱甘肽S-转移酶活性增强有关；对阿维菌素的抗性主要与多功能氧化酶和羧酸酯酶有关,而与谷胱甘肽S-转移酶关系不大。
3.2桔全爪螨不同品系羧酸酯酶(CarE)比较研究
测定CarE活性结果表明,与敏感品系相比,两抗性品系以α-NA和β-NA作为底物所测得的CarE活力均显著提高(P<0.05)；当以α-NA为底物时,甲氰菊酯抗性品系的Km值显著提高(P<0.05),阿维菌素抗性品系K_m值有所下降,两抗性品系V_(max)值均显著提高(P<0.05)；当以β-NA为底物时,两个抗性品系的Km值均有所下降,Vmax值均显著提高(P<0.05)。
通过体外离体抑制作用分析表明,在所选用的7种杀虫(螨)剂中,甲基对氧磷、马拉硫磷、丁硫克百威对桔全爪螨CarE有明显的抑制作用,高效氯氰菊酯、甲氰菊酯的抑制作用也较强,阿维菌素也具有一定的抑制作用,而哒螨灵抑制作用很低,仅在高浓度时表现出一定的抑制作用,在低浓度时有诱导桔全爪螨体内CarE活性的作用。且实验结果表明,7种杀虫(螨)剂对桔全爪螨不同品系CarE离体抑制作用差异很大,对甲氰菊酯抗性品系的抑制作用明显减小(马拉硫磷除外),I50最大。
以上结果表明,CarE参与了对甲氰菊酯和阿维菌素的代谢,在桔全爪螨对这两种杀螨剂抗性产生中起着十分重要的作用。
3.3桔全爪螨不同品系磷酸酯酶(ACP和ALP)比较研究
桔全爪螨磷酸酯酶测定结果表明,甲氰菊酯抗性品系的ACP比活力、ALP Vmax值显著高于敏感品系(尸<0.05)；阿维菌素抗性品系ACP和ALP比活力、米氏常数Km值和Vmax值与敏感品系相比均无显著差异(P>0.05)。这说明桔全爪螨磷酸酯酶与其对甲氰菊酯抗性的形成有一定关系,而与阿维菌素抗性的形成无关。
3.4桔全爪螨不同品系乙酰胆碱酯酶(AChE)比较研究
桔全爪螨AChE测定结果表明,三个品系中,以甲氰菊酯抗性品系的AChE活力、比活力以及最大反应速率Vmax值最大。与敏感品系相比,两抗性品系的AChE活力、比活力和Vmax值均显著高于敏感品系(P<0.05),且两抗性品系之间也存在显著差异(P<0.05)。米氏常数Km值在三个品系间无显著差异(P>0.05)。这说明AChE比活力提高是桔全爪螨对甲氰菊酯和阿维菌素产生抗性的原因之一
3.5桔全爪螨不同品系谷胱甘肽S-转移酶(GSTs)比较研究
桔全爪螨三个品系GSTs测定结果表明,甲氰菊酯抗性品系的GSTs活力和比活力显著提高(P<0.05)。当以CDNB为底物时,甲氰菊酯抗性品系的Km值和Vmax均高于敏感品系,分别是敏感品系的1.45倍和1.47倍；而以GSH为底物时,与敏感品系相比,甲氰菊酯抗性品系的Km和Vmax值均显著下降(P<0.05)。而阿维菌素抗性品系的GSTs活力、比活力以及分别以CDNB和GSH为底物的米氏常数Km值和最大反应速率Vmax值与敏感品系相比均无显著差异(P>0.05)。这些结果表明,谷胱甘肽S-转移酶与桔全爪螨对甲氰菊酯的抗性有关,与阿维菌素的抗性关系不大。
3.6桔全爪螨不同品系多功能氧化酶(MFO)比较研究
桔全爪螨甲氰菊酯抗性品系和阿维菌素抗性品系的MFO氧脱甲基比活力分别是敏感品系的1.36倍和1.31倍,均显著高于敏感品系(P<0.05)。其最大反应速率Vmax值在三个品系间无显著差异(P>0.05)。就米氏常数%、值来看,阿维菌素抗性品系的Km值显著大于敏感品系(P<0.05);甲氰菊酯抗性品系与敏感品系之间无显著差异(P>0.05)。说明桔全爪螨MFO在其阿维菌素抗性产生中具有重要作用,且与甲氰菊酯抗性的形成也有一定关系。
3.7桔全爪螨不同品系三磷酸腺苷酶(ATPase)比较研究
桔全爪螨不同品系Na+-K+-ATPase比活力从大到小分别是阿维菌素抗性品系(AvR)(112.98 nmol/mg/min)>敏感品系(SS) (102.48 nmol/mg/min)>甲氰菊酯抗性品系(FeR)(69.88 nmol/mg/min),甲氰菊酯抗性品系Na+-K+-ATPase比活力仅为敏感品系的0.68倍,与其它两个品系之间都有显著性差异(P<0.05),阿维菌素抗性品系与敏感品系之间无显著差异(P>0.05)。桔全爪螨甲氰菊酯抗性品系Na+-K+-ATPase的Km值和Vmax值显著高于其它两个品系(P<0.05),而阿维菌素抗性品系Na+-K+-ATPase的Vmax值也显著高于敏感品系(P<0.05)。就Mg2+-ATPase而言,三个品系的活力、比活力及Km值和Vmax值均无显著差异(P>0.05)
离体抑制作用表明,在甲氰菊酯6个浓度作用下,Na+-K+-ATPase的活力均受到了不同程度的抑制。其中,敏感品系的抑制率最大,其次为阿维菌素抗性品系,甲氰菊酯抗性品系的抑制率最低；甲氰菊酯抗性品系与其它两个品系之间差异达显著水平(P<0.05)。阿维菌素对桔全爪螨Na+-K+-ATPase的活力有一定的抑制作用,在实验浓度范围(0.0229-2290.7146μM)内抑制率在3.60%-12.76%,在较高浓度(如2290.7146μM)下,抗性品系与敏感品系间差异显著(P<0.05)。
这些结果表明,Na+-K+-ATPase活力降低及其敏感性下降是桔全爪螨对甲氰菊酯产生抗性的主要原因,推测Na+-K+-ATPase是甲氰菊酯的靶标酶。此外,Na+-K+-ATPase可能对阿维菌素抗性产生也有一定的作用,但其作用不同于对甲氰菊酯。
4桔全爪螨酯酶基因的克隆与表达研究
4.1桔全爪螨酯酶基因的克隆及序列分析
利用RT-PCR技术和RACE技术,成功克隆获得桔全爪螨1个酯酶(esterase)基因cDNA的全长序列,命名为PCE1, GenBank登录号为：GQ144324。该基因全长1892 bp,其中开放阅读框1653 bp,编码550个氨基酸组成的前体蛋白,N端19个氨基酸为信号肽,成熟蛋白的分子量为61.1 kDa,理论等电点为5.21。根据在线软件ScanProsite的算法原理,从该基因的氨基酸序列中均找到了羧酸酯酶(CarE)的2个保守结构域：1个是丝氨酸活性中心"FGGDPDQVTIFGESAG",另一个是保守的二硫键形成位点"EDCLTLNVITP"。另外C端还具有CarE内质网腔滞留信号"HEEL "。此外,ScanProsite分析表明,该基因PCE1还具有多个磷酸化位点、N端酰基化和N端糖基化位点。同源性分析表明,桔全爪螨酯酶基因PCE1与其它昆虫和蜱螨的酯酶基因之间同源性较低,但在活性中心处的序列则是高度保守的。使用MEGA 4.1软件,应用Neighbor Joining方法构建了系统发育树,结果表明克隆获得的桔全爪螨酯酶基因PCE1属于蜱螨酯酶族群。另外,应用网络蛋白模拟软件SWISS-MODEL对桔全爪螨酯酶基因PCE1的三维结构进行了模拟。
4.2桔全爪螨酯酶基因PCE1 mRNA表达
通过叶片带螨浸渍法明确了桔全爪螨对甲氰菊酯的敏感性,根据生测结果,用甲氰菊酯亚致死浓度LC50对桔全爪螨进行了活体诱导,采用实时定量PCR技术分析了上述克隆获得的酯酶基因PCE1经药剂诱导后其mRNA表达的时间动态(6、12、24、48 h)。结果表明,PCE1经甲氰菊酯诱导后,mRNA表达有明显的时间动态变化。在药剂处理初期,该酯酶基因mRNA表达水平下降,处理后6 h,其mRNA表达水平为对照的0.74倍,显著低于对照(P<0.05)以后逐渐上升,在12 h后上升为对照的1.17倍,显著高于对照(P<0.05)：此后再次下降,诱导处理24 h后为对照的1.05倍；此后mRNA水平又上升,48 h后为对照的1.36倍,此时的mRNA水平显著高于对照(P<0.05)。总的来看,甲氰菊酯LC50对桔全爪螨该酯酶基因PCE1是先抑制后诱导的作用,时间越长越趋于诱导作用。这说明该酯酶基因可能参与了对甲氰菊酯的代谢。
4.3桔全爪螨酯酶基因PCE1的异源表达
利用BamH、Xhol的双酶切以及DNA重组技术,成功构建了桔全爪螨酯酶基因PCE1基于pET-43.1a(+)的原核表达载体,应用SDS-PAGE检测到融合蛋白,表明构建的原核表达质粒可以在宿主菌中稳定、正确表达,为进一步研究该酯酶基因的性质和功能提供了有用的实验材料。
The citrus red mite, Panonychus citri (McGregor) (Acari:Tetranychidae), is an important pest mite in the world that devastates citrus trees and thus badly affects yield and quality of orange. To date, P. citri has developed severe resistance to most acaricides, which has resulted in extensive attentions and concerns of scientific researchers and administrative authority. In the current thesis, the effects of two acaricides (fenpropathrin and avermectin) on development and reproduction of P. citri and the biochemical mechanism of the mite resistance to the acaricides were systematically studied, and the molecular cloning and expression of esterase gene (PCE1) from the mite was also carried out. The study was supported by the Grants-in-Aid from the Ministry of Agriculture (nyhyzx07-057), Chongqing Natural Science Fund for Distinguished Young Scholars (CSTC, 2009BA1042) and the earmarked fund for Modern Agro-Industry (Citrus) Technology Research System of China. The main results are as follows:
1 Sublethal effects of two acaricides on P. citri
1.1 Effects of fenpropathrin with sublethal concentration on development and reproduction of P. citri
The effects of fenpropathrin with a sublethal concentration (the LC20 was chose as a representative) on the development and reproduction of P. citri were evaluated. The results showed that after the treatment of fenpropathrin at the sublethal dose, the emergence rate of the nymphs and sex ratio of offspring both decreased, while the number of eggs laid per female increased in F0 generation. The sublethal dose of fenpropathrin expressed different effects on the offspring (F1 and F2 generations) of the mite. However, there was no significant difference between two treatments (that is, exposed to sublethal concentration of fenpropathrin during the nymphal stages and the adult stages). After treatment with the sublethal dose of fenpropathrin, the pre-oviposition duration was significantly shortened (P< 0.05), and the sex ratio of offspring increased both in F1 and F2 generations. The hatching rate of eggs and the emergence rate of the nymphs decreased while the number of eggs laid per female and the adult longevity both increased in F1 generation, which were totally in verse in F2 generation. Furthermore, the net reproductive rate(Ro), intrinsic rate of increase (rm) and finite rate of increase (λ) values all increased, and the population doubling time (Dt) shortened in F1 generation; while Ro, Dt and the generation time (T) decreased, rm andλsignificantly increased in F2 generation. Generally, fenpropathrin with a sublethal concentration of LC20 facilitated reproduction of the mite population and there was no relationship between its effects and treatment period (adult and nymphal stages).
1.2 Effects of avermectin with sublethal concentration on development and reproduction of P. citri
The effects of avermectin with sublethal concentration on the development and reproduction of P. citri were evaluated and the results showed the sublethal dose of avermectin had no significant effect on the hatching rate of eggs and the emergence rate of the nymphs in F0 generation. However, after exposure to avermectin, progeny (F1 and F2 generations) produced fewer eggs, the pre-oviposition duration and the adult longevity shortened, resulting in a decrease in the mean generation time. The sublethal dose of avermectin expressed different effects on the offspring (F1 and F2 generations) of the mite and the effect of nymphal stage and adult stage treatments on the population life parameters of the offspring was more significant in F2 generation. After treatment during nymphal stages, higher rm, R0, andλvalues while lower Dt value were observed. However, the treatment during adult stages exhibited negative effects on population increase (i.e. lower rm, Ro, andλvalues while longer Dt).
1.3 Effects of fenpropathrin and avermectin with sublethal concentrations on activities of detoxifying enzymes in P. citri
After adult mites were treated applying fenpropathrin and avermectin with LC20 concentration, there were different induction effects on the activities of main detoxifying enzymes such as carboxylesterase (CarE), gutathione S-transferases (GSTs) and mixed-function oxidase (MFO) from the mite. After the treatment with fenpropathrin, the specific activity of CarE fluctuated a lot, increased firstly, then decreased and finally increased again, with the values at 12 h,36 h and 48 h significantly higher than the control (P< 0.05); the induction effects on GST activities were quite obvious and the specific activity at 36 h was 3.31 fold of control; there was no significant effects on MFO activity. For avermectin sublethal treatment, CarE specific activity ascended in the first 12 h, then decreased to a state value; the induction effect on GSTs was less significant than fenpropathrin treatment; the activities of O-demethylation of MFOs were distinctly inhibited by sublethal concentration of avermectin, especially after 24 h of the treatment, significant different from the control (P<0.05)
2 Resistance selection and risk assessment of resistance to fenpropathrin and avermectin in P. citri
After the further resistance selection in laboratory, the realized heritability of P. citri resistance to acaricides were conducted via threshold trait analysis, the resistance development ratio of the mite was forecast and the cross-resistance between the acaricides was also studied. After discontinuous selection with fenpropathrin and avermectin 16 and 11 times during 19 generations, resistance ratio of the mite to the acaricides amounted to 29.92 and 3.80 fold, respectively. The realized heritability of resistance to fenpropathrin and avermectin accounted for 0.1544 and 0.0475, respectively. Theoretically, in laboratory, to obtain a 10 fold resistance ratio requires 8 to 16 generations for fenpropathrin and 12 to 26 generations for avermectin under selective pressure of 50% -90% mortality for each selective generation. For field populations, more generations were required to obtain the same resistance levels. Bioassay revealed that the fenpropathrin resistant strain developed obvious cross-resistance to pyridaben, dicofol, azocyclotin and the avermectin resistant strain obtained cross-resistance to emamectin benzoate. The results of resistance risk assessment suggested fenpropathrin has higher resistance risk than avermectin in P. citri. The current results provide some information for the resistance management in P. citri.
3 Biochemical mechanism of P. citri resistance to two acaricides
3.1 Synergism effects of synergists on fenpropathrin and avermectin
The synergism of PBO, TPP and DEM all on fenpropathrin and avermectin respectively was test in susceptible and resistant strains of P. citri with the relative synergism ratio (the ratio between the synergism ratio in resistant and susceptible strains, SRFeR(AvR)/SRss) as the criterion. The results showed that PBO, TPP and DEM all possessed obvious synergism effects on fenpropathrin with the relative synergism ratio (SRFeR/SRss) of 3.18,2.90 and 1.68, respectively, the relative synergism ratios of PBO, TPP and DEM on avermectin was 1.81,1.44, and 0.84, respectively.
3.2 Comparison of carboxylesterase (CarE) in different strains of P. citri
Compared to their susceptible counterparts, the activities of CarE in FeR and AvR were significantly higher (P< 0.05). Kinetic parameters comparison showed that CarE from FeR expressed highest Km value to the substrate a-NA, while both two resistant strains exhibited lower Km values toβ-NA. For Vmax, the values in resistant strains were significantly higher.
In vitro inhibition study showed that paraoxon-methyl, carbosulfan and malathion expressed significant inhibitory effects on CarE in three strains of P. citri. Beta-cypermethrin and fenpropathrin also exhibited obvious inhibition effects, while avermectin expressed limited inhibition effect. For pyridaben, the inhibition effects were not obvious and some facilitated effects appeared at lower concentrations. The comparison analysis revealed that except malathion, CarE from SS strain were most sensitive (lowest I50 values) and the sensitivity of CarE from FeR was lowest (highest I50 values).
3.3 Comparison of acid phosphatase (ACP) and alkaline phosphatase (ALP) in different strains of P. citri
Compared to their susceptible counterparts, the specific activities of ACP and Vmax value of ALP from FeR expressed significantly higher than SS (P< 0.05), while the specific activities, the Vmax and Km values of of ACP and ALP had no significant difference between SS and AvR (P> 0.05). The results indicated that phosphatase might play some roles in fenpropathrin resistance of P. citri, but played little role in P. citri resistance to avermectin.
3.4 Comparison of acetylcholinesterase (AChE) in different strains of P. citri
Among three strains, the activity per mite and specific activity of AChE, as well as the Vmax value were highest in FeR. Compared to SS strain, these values in FeR and AvR were significantly higher(P< 0.05), and significant difference was observed between two resistant strains (P< 0.05). However, there was no significant difference of Km value among three strains (P> 0.05). The result indicated that increased activity of AChE plays important roles in P. citri resistant to fenpropathrin and avermectin.
3.5 Comparison of glutathione S-transferase (GSTs) in different strains of P. citri
The result revealed that the activity per mite and specific activity of GSTs in FeR were significantly higher than other two strains (SS and AvR) (P< 0.05). GSTs from FeR expressed highest Km and Vmax values to the substrate CDNB while exhibited lowest Km and Vmax values to GSH. There were significant difference between FeR and other two strains (P< 0.05) while no significant difference existed between AvR and SS strains (P> 0.05). The results indicated that GSTs was important in fenpropathrin resistance development, but played little role in P. citri resistance to avermectin.
3.6 Comparison of mixed function oxidases (MFO) in different strains of P. citri
Compared to SS strain, the specific activities of O-demethylation in FeR and AvR were significantly higher (1.36 and 1.31 fold of SS strain) (P< 0.05). For kinetic parameters, O-demethylation from AvR showed highest Km value while lowest Vmax value, and its Km value was significantly higher than that from SS (P< 0.05), indicating MFO might have changed in quality in AvR strain. However, there was no significant difference in the Km and Vmax values between FeR and SS strain (P> 0.05). These results indicated that MFO was essential in avermectin resistance development, and it played some roles in fenpropathrin resistance to P. citri.
3.7 Comparison of adenosine triphosphatase (ATPase) in different strains of P. citri
The biochemical characterization of ATPase comparison analysis showed that the specific activity of Na+-K+-ATPase from high to low was AvR> SS> FeR. The specific activity of Na+-K+-ATPase in FeR is 0.68-fold of SS, significantly lower than other two strains (P< 0.05). There was no significant difference between AvR and SS strain (P> 0.05). Further kinetic parameters comparison showed that Na+-K+-ATPase from FeR expressed significantly higher Km and Vmax values than other two strains(P< 0.05), and the Vmax value from AvR was also higher than SS strain (P< 0.05). For the activities per mite, the specific activities, the Km value and Vmax value of Mg2+-ATPase, no significant difference was observed among these strains of P. citri(P> 0.05).
In vitro inhibition of insecticides on Na+-K+-ATPase of P. citri showed that fenpropathrin expressed significant inhibitory effects under tested six concentrations. The sensitivity of Na+-K+-ATPase from SS was highest while from FeR was lowest, and significance existed between FeR and other two strains (P< 0.05). In addition, avermectin also exhibited some in vitro inhibition effect, and the inhibition rate on Na+-K+-ATPase of the three strains of P. citri ranged from 3.60%to 12.76% (under 0.0229-2290.7164μM) and significant difference existed between susceptible and resistant strains under higher concentration (i.e.2290.7146μM)(P< 0.05)
These results indicated that Na+-K+-ATPase played major role in fenpropathrin resistance of P. citri by its significantly lower activity and insensitivity to fenpropathrin and thus speculated that Na+-K+-ATPase is one of target enzymes of fenpropathrin. In addition, it may play some roles in avermectin resistance to P. citri.
4 Molecular cloning and expression of esterase gene in P. citri
4.1 Molecular cloning and sequence analysis of esterase gene in P. citri
The full length cDNA encoding esterase PCE1 (GenBank No. GQ144324) was cloned from P. citri by the method of reverse transcriptase PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The complete cDNA of this gene consists of 1892 bp with an open reading frame (ORF) of 1653 bp, encoding a protein of 550 amino acids residues with the putative signal peptide of 19 residues. The mature protein has a molecular weight of 61.1 kDa with an isoelectric point of 5.21. Based on the online software ScanProsite, two conserved regions of CarE were found:serine active site (FGGDPDQVTIFGESAG) and the conserved cysteine that compose disulfide bridge (EDCLTLNVITP). In addition, an endoplasmic reticulum targeting sequence (HEEL) as well as 14 phosphorylation sites,12 N-myristoylation sites and 4 N-glycosylation sites were also found. The sequence homology analysis showed that the esterase cloned from P. citri share low homology to other insect species, but the sequences is highly conserved in active site. Phylogenetic trees generated from the amino acid sequences revealed a closer relationship to other acaris than to insects. In addition, the PCE1 gene 3-D structure models were constructed by SWISS-MODEL.
4.2 mRNA expression level of the esterase gene PCE1 from P. citri
The adult female mites of P. citri were treated with LC50 concentration of fenpropathrin using leaf dip method and then the expression levels of PCE1 gene was determined. After treated for 6 h, 12 h,24 h and 48 h, the relative quantity of PCE1 was investigated applying real-time PCR, respectively. The results showed that the relative quantity of PCE1 from P. citri fluctuated significantly. At the beginning of fenpropatrin treatment, the relative quantity of PCE1 was inhibited (compared to the control group, PCE1 transcript was only 0.74 fold after 6 h treated). It increased gradually with the increase of time treated, then decreased, and then again increased. After 48 h treated, the relative quantity of PCE 1 is significantly higher than the control (just 1.36 fold compared with the control). In general, the transcript of PCE1 from P. citri treated was higher with longer treatment time.
4.3 Heterologous expression of the esterase gene PCE1 from P. citri
Applying the double digestion of BamHI and Xhol restriction enzymes with the DNA recombination technology, the expression vectors for PCE1 was constructed based on pET-43.1a (+) vector. Specific band appeared on the gel of SDS-PAGE. The current result provides useful base for furthermore exploration of the property and function of the esterase gene PCE1.
1两种杀螨剂对桔全爪螨的亚致死效应研究
1.1甲氰菊酯亚致死浓度对桔全爪螨生长发育及繁殖的影响
用甲氰菊酯LC20的浓度处理桔全爪螨的若螨和成螨,观察其对当代和后代(F1、F2代)生长发育及繁殖的影响。结果表明,甲氰菊酯LC20浓度处理若螨后,当代若螨发育成为成螨的百分率(以下简称为羽化率)降低,雌成螨产卵量增加,但后代雌性比例下降。甲氰菊酯亚致死浓度处理若螨或成螨对F1和F2代的影响不完全相同,但两种处理基本相似。处理成螨和若螨后,F1、F2代的产卵前期显著缩短(P<0.05)。F1、F2代的后代雌性比例都增大,但F1代卵的孵化率和若螨羽化率降低,产卵量和成螨寿命增加,F2代却相反。利用生命表方法分析得知,甲氰菊酯LC20处理若螨和成螨后,其F1代的净增殖率、内禀增长率和周限增长率都增大,种群加倍时间缩短；F2代的净增殖率虽然减小,但由于世代历期缩短,内禀增长率和周限增长率仍然显著增大(P<0.05),种群加倍时间显著缩短(尸<0.05)。总体来看,甲氰菊酯LC20浓度可以促进桔全爪螨的增殖,其作用效应与用药时期(成螨期和若螨期)无关。
1.2阿维菌素亚致死浓度对桔全爪螨生长发育及繁殖的影响
在实验室条件下对阿维菌素亚致死浓度LC20对桔全爪螨生长发育和繁殖的影响进行了研究。结果表明,用阿维菌素处理若螨后,对当代若螨羽化率和后代的卵孵化率没有显著影响,但是,其F1、F2代产卵量减少,产卵前期和成螨寿命缩短,因此,世代历期也缩短。总体来看,在成螨期用药和若螨期用药对子代种群的影响不同,其后代的种群生命表参数在两种处理间具有显著差异,且这种差异在F2代更为明显。若螨期用药后对F1、F2代具有积极作用,如,内禀增长率、净增殖率、周限增长率均提高,种群加倍时间相应缩短。相反,成螨期用药后,其F1、F2代的内禀增长率、净增殖率、周限增长率均减小,种群加倍时间相应延长。
1.3两种杀螨剂对桔全爪螨体内主要解毒酶的诱导作用
甲氰菊酯和阿维菌素LC20浓度处理桔全爪螨成螨后,对体内三种主要解毒酶CarE、GSTs和MFO的诱导效应不同,其时间效应也不尽相同。甲氰菊酯处理后48 h内,CarE活性先上升后下降,又再次上升,波动范围很大,其比活力在12 h、36 h、48 h显著高于对照(P<0.05);对GSTs的诱导作用较为明显,在诱导高峰期的第36 h其比活力为对照的3.31倍,显著高于对照(P<0.05)；对桔全爪螨的多功能氧化酶O-脱甲基活力诱导作用相对较小。而阿维菌素处理桔全爪螨雌成螨后,CarE比活力在前12 h表现为上升趋势,12-24 h酶活力降低,以后趋于平缓；对GSTs的诱导作用也不如甲氰菊酯明显,仅在处理12 h和36 h后比活力有所提高；且阿维菌素LC20浓度处理对桔全爪螨体内微粒体MFO氧脱甲基活力具有一定的抑制作用,在处理24 h后抑制作用最为明显,与对照相比差异显著(P<0.05)。
2桔全爪螨抗药性选育、交互抗性及抗性风险评估
在实验室抗性品系选育基础上,应用数量遗传学中的域性状分析法,研究了桔全爪螨北碚种群对甲氰菊酯、阿维菌素两种杀螨剂的抗性现实遗传力,对两种药剂在不同致死率下抗性发展的速率进行了预测,并对这两种杀螨剂进行了交互抗性研究。结果表明,用甲氰菊酯、阿维菌素分别汰选16、11代后,桔全爪螨对两种药剂的抗性分别为29.92倍和3.80倍,抗性现实遗传力分别为0.1544和0.0475。在室内选择条件下,致死率为50%-90%时,要获得10倍抗性,甲氰菊酯仅需要16-8代,阿维菌素需要26-12代。在田间选择条件下,两种药剂都将需要更长的时间。并且,甲氰菊酯抗性品系(FeR)对哒螨灵、三氯杀螨醇和三唑锡产生了明显的交互抗性,阿维菌素抗性品系(AvR)对甲维盐产生了明显的交互抗性。这些结果表明,生物源农药阿维菌素的抗性风险明显低于菊酯类药剂甲氰菊酯,且甲氰菊酯的交互抗性谱明显大于阿维菌素。试验结果可为桔全爪螨抗性治理提供参考。
3桔全爪螨对两种杀螨剂的抗性的生化机理
3.1三种增效剂对两种杀螨剂的增效作用
甲氰菊酯、阿维菌素分别与三种增效剂(PBO、TPP、DEM,分别是多功能氧化酶、酯酶和谷胱甘肽S-转移酶的专一性抑制剂)组配对桔全爪螨敏感品系(SS)、甲氰菊酯抗性品系(FeR)、阿维菌素抗性品系(AvR)进行了毒力测定,并以抗性品系增效倍数与敏感品系增效倍数的比值作为增效作用的评定标准。结果显示,PBO、TPP、DEM三种增效剂对甲氰菊酯均有一定的增效作用,其相对增效倍数分别为3.18,2.90,1.68倍;而PBO、TPP、DEM对阿维菌素的增效作用相对较弱,其相对增效倍数分别为1.81、1.44、0.84倍。这表明桔全爪螨对甲氰菊酯的抗性与多功能氧化酶、羧酸酯酶、谷胱甘肽S-转移酶活性增强有关；对阿维菌素的抗性主要与多功能氧化酶和羧酸酯酶有关,而与谷胱甘肽S-转移酶关系不大。
3.2桔全爪螨不同品系羧酸酯酶(CarE)比较研究
测定CarE活性结果表明,与敏感品系相比,两抗性品系以α-NA和β-NA作为底物所测得的CarE活力均显著提高(P<0.05)；当以α-NA为底物时,甲氰菊酯抗性品系的Km值显著提高(P<0.05),阿维菌素抗性品系K_m值有所下降,两抗性品系V_(max)值均显著提高(P<0.05)；当以β-NA为底物时,两个抗性品系的Km值均有所下降,Vmax值均显著提高(P<0.05)。
通过体外离体抑制作用分析表明,在所选用的7种杀虫(螨)剂中,甲基对氧磷、马拉硫磷、丁硫克百威对桔全爪螨CarE有明显的抑制作用,高效氯氰菊酯、甲氰菊酯的抑制作用也较强,阿维菌素也具有一定的抑制作用,而哒螨灵抑制作用很低,仅在高浓度时表现出一定的抑制作用,在低浓度时有诱导桔全爪螨体内CarE活性的作用。且实验结果表明,7种杀虫(螨)剂对桔全爪螨不同品系CarE离体抑制作用差异很大,对甲氰菊酯抗性品系的抑制作用明显减小(马拉硫磷除外),I50最大。
以上结果表明,CarE参与了对甲氰菊酯和阿维菌素的代谢,在桔全爪螨对这两种杀螨剂抗性产生中起着十分重要的作用。
3.3桔全爪螨不同品系磷酸酯酶(ACP和ALP)比较研究
桔全爪螨磷酸酯酶测定结果表明,甲氰菊酯抗性品系的ACP比活力、ALP Vmax值显著高于敏感品系(尸<0.05)；阿维菌素抗性品系ACP和ALP比活力、米氏常数Km值和Vmax值与敏感品系相比均无显著差异(P>0.05)。这说明桔全爪螨磷酸酯酶与其对甲氰菊酯抗性的形成有一定关系,而与阿维菌素抗性的形成无关。
3.4桔全爪螨不同品系乙酰胆碱酯酶(AChE)比较研究
桔全爪螨AChE测定结果表明,三个品系中,以甲氰菊酯抗性品系的AChE活力、比活力以及最大反应速率Vmax值最大。与敏感品系相比,两抗性品系的AChE活力、比活力和Vmax值均显著高于敏感品系(P<0.05),且两抗性品系之间也存在显著差异(P<0.05)。米氏常数Km值在三个品系间无显著差异(P>0.05)。这说明AChE比活力提高是桔全爪螨对甲氰菊酯和阿维菌素产生抗性的原因之一
3.5桔全爪螨不同品系谷胱甘肽S-转移酶(GSTs)比较研究
桔全爪螨三个品系GSTs测定结果表明,甲氰菊酯抗性品系的GSTs活力和比活力显著提高(P<0.05)。当以CDNB为底物时,甲氰菊酯抗性品系的Km值和Vmax均高于敏感品系,分别是敏感品系的1.45倍和1.47倍；而以GSH为底物时,与敏感品系相比,甲氰菊酯抗性品系的Km和Vmax值均显著下降(P<0.05)。而阿维菌素抗性品系的GSTs活力、比活力以及分别以CDNB和GSH为底物的米氏常数Km值和最大反应速率Vmax值与敏感品系相比均无显著差异(P>0.05)。这些结果表明,谷胱甘肽S-转移酶与桔全爪螨对甲氰菊酯的抗性有关,与阿维菌素的抗性关系不大。
3.6桔全爪螨不同品系多功能氧化酶(MFO)比较研究
桔全爪螨甲氰菊酯抗性品系和阿维菌素抗性品系的MFO氧脱甲基比活力分别是敏感品系的1.36倍和1.31倍,均显著高于敏感品系(P<0.05)。其最大反应速率Vmax值在三个品系间无显著差异(P>0.05)。就米氏常数%、值来看,阿维菌素抗性品系的Km值显著大于敏感品系(P<0.05);甲氰菊酯抗性品系与敏感品系之间无显著差异(P>0.05)。说明桔全爪螨MFO在其阿维菌素抗性产生中具有重要作用,且与甲氰菊酯抗性的形成也有一定关系。
3.7桔全爪螨不同品系三磷酸腺苷酶(ATPase)比较研究
桔全爪螨不同品系Na+-K+-ATPase比活力从大到小分别是阿维菌素抗性品系(AvR)(112.98 nmol/mg/min)>敏感品系(SS) (102.48 nmol/mg/min)>甲氰菊酯抗性品系(FeR)(69.88 nmol/mg/min),甲氰菊酯抗性品系Na+-K+-ATPase比活力仅为敏感品系的0.68倍,与其它两个品系之间都有显著性差异(P<0.05),阿维菌素抗性品系与敏感品系之间无显著差异(P>0.05)。桔全爪螨甲氰菊酯抗性品系Na+-K+-ATPase的Km值和Vmax值显著高于其它两个品系(P<0.05),而阿维菌素抗性品系Na+-K+-ATPase的Vmax值也显著高于敏感品系(P<0.05)。就Mg2+-ATPase而言,三个品系的活力、比活力及Km值和Vmax值均无显著差异(P>0.05)
离体抑制作用表明,在甲氰菊酯6个浓度作用下,Na+-K+-ATPase的活力均受到了不同程度的抑制。其中,敏感品系的抑制率最大,其次为阿维菌素抗性品系,甲氰菊酯抗性品系的抑制率最低；甲氰菊酯抗性品系与其它两个品系之间差异达显著水平(P<0.05)。阿维菌素对桔全爪螨Na+-K+-ATPase的活力有一定的抑制作用,在实验浓度范围(0.0229-2290.7146μM)内抑制率在3.60%-12.76%,在较高浓度(如2290.7146μM)下,抗性品系与敏感品系间差异显著(P<0.05)。
这些结果表明,Na+-K+-ATPase活力降低及其敏感性下降是桔全爪螨对甲氰菊酯产生抗性的主要原因,推测Na+-K+-ATPase是甲氰菊酯的靶标酶。此外,Na+-K+-ATPase可能对阿维菌素抗性产生也有一定的作用,但其作用不同于对甲氰菊酯。
4桔全爪螨酯酶基因的克隆与表达研究
4.1桔全爪螨酯酶基因的克隆及序列分析
利用RT-PCR技术和RACE技术,成功克隆获得桔全爪螨1个酯酶(esterase)基因cDNA的全长序列,命名为PCE1, GenBank登录号为：GQ144324。该基因全长1892 bp,其中开放阅读框1653 bp,编码550个氨基酸组成的前体蛋白,N端19个氨基酸为信号肽,成熟蛋白的分子量为61.1 kDa,理论等电点为5.21。根据在线软件ScanProsite的算法原理,从该基因的氨基酸序列中均找到了羧酸酯酶(CarE)的2个保守结构域：1个是丝氨酸活性中心"FGGDPDQVTIFGESAG",另一个是保守的二硫键形成位点"EDCLTLNVITP"。另外C端还具有CarE内质网腔滞留信号"HEEL "。此外,ScanProsite分析表明,该基因PCE1还具有多个磷酸化位点、N端酰基化和N端糖基化位点。同源性分析表明,桔全爪螨酯酶基因PCE1与其它昆虫和蜱螨的酯酶基因之间同源性较低,但在活性中心处的序列则是高度保守的。使用MEGA 4.1软件,应用Neighbor Joining方法构建了系统发育树,结果表明克隆获得的桔全爪螨酯酶基因PCE1属于蜱螨酯酶族群。另外,应用网络蛋白模拟软件SWISS-MODEL对桔全爪螨酯酶基因PCE1的三维结构进行了模拟。
4.2桔全爪螨酯酶基因PCE1 mRNA表达
通过叶片带螨浸渍法明确了桔全爪螨对甲氰菊酯的敏感性,根据生测结果,用甲氰菊酯亚致死浓度LC50对桔全爪螨进行了活体诱导,采用实时定量PCR技术分析了上述克隆获得的酯酶基因PCE1经药剂诱导后其mRNA表达的时间动态(6、12、24、48 h)。结果表明,PCE1经甲氰菊酯诱导后,mRNA表达有明显的时间动态变化。在药剂处理初期,该酯酶基因mRNA表达水平下降,处理后6 h,其mRNA表达水平为对照的0.74倍,显著低于对照(P<0.05)以后逐渐上升,在12 h后上升为对照的1.17倍,显著高于对照(P<0.05)：此后再次下降,诱导处理24 h后为对照的1.05倍；此后mRNA水平又上升,48 h后为对照的1.36倍,此时的mRNA水平显著高于对照(P<0.05)。总的来看,甲氰菊酯LC50对桔全爪螨该酯酶基因PCE1是先抑制后诱导的作用,时间越长越趋于诱导作用。这说明该酯酶基因可能参与了对甲氰菊酯的代谢。
4.3桔全爪螨酯酶基因PCE1的异源表达
利用BamH、Xhol的双酶切以及DNA重组技术,成功构建了桔全爪螨酯酶基因PCE1基于pET-43.1a(+)的原核表达载体,应用SDS-PAGE检测到融合蛋白,表明构建的原核表达质粒可以在宿主菌中稳定、正确表达,为进一步研究该酯酶基因的性质和功能提供了有用的实验材料。
The citrus red mite, Panonychus citri (McGregor) (Acari:Tetranychidae), is an important pest mite in the world that devastates citrus trees and thus badly affects yield and quality of orange. To date, P. citri has developed severe resistance to most acaricides, which has resulted in extensive attentions and concerns of scientific researchers and administrative authority. In the current thesis, the effects of two acaricides (fenpropathrin and avermectin) on development and reproduction of P. citri and the biochemical mechanism of the mite resistance to the acaricides were systematically studied, and the molecular cloning and expression of esterase gene (PCE1) from the mite was also carried out. The study was supported by the Grants-in-Aid from the Ministry of Agriculture (nyhyzx07-057), Chongqing Natural Science Fund for Distinguished Young Scholars (CSTC, 2009BA1042) and the earmarked fund for Modern Agro-Industry (Citrus) Technology Research System of China. The main results are as follows:
1 Sublethal effects of two acaricides on P. citri
1.1 Effects of fenpropathrin with sublethal concentration on development and reproduction of P. citri
The effects of fenpropathrin with a sublethal concentration (the LC20 was chose as a representative) on the development and reproduction of P. citri were evaluated. The results showed that after the treatment of fenpropathrin at the sublethal dose, the emergence rate of the nymphs and sex ratio of offspring both decreased, while the number of eggs laid per female increased in F0 generation. The sublethal dose of fenpropathrin expressed different effects on the offspring (F1 and F2 generations) of the mite. However, there was no significant difference between two treatments (that is, exposed to sublethal concentration of fenpropathrin during the nymphal stages and the adult stages). After treatment with the sublethal dose of fenpropathrin, the pre-oviposition duration was significantly shortened (P< 0.05), and the sex ratio of offspring increased both in F1 and F2 generations. The hatching rate of eggs and the emergence rate of the nymphs decreased while the number of eggs laid per female and the adult longevity both increased in F1 generation, which were totally in verse in F2 generation. Furthermore, the net reproductive rate(Ro), intrinsic rate of increase (rm) and finite rate of increase (λ) values all increased, and the population doubling time (Dt) shortened in F1 generation; while Ro, Dt and the generation time (T) decreased, rm andλsignificantly increased in F2 generation. Generally, fenpropathrin with a sublethal concentration of LC20 facilitated reproduction of the mite population and there was no relationship between its effects and treatment period (adult and nymphal stages).
1.2 Effects of avermectin with sublethal concentration on development and reproduction of P. citri
The effects of avermectin with sublethal concentration on the development and reproduction of P. citri were evaluated and the results showed the sublethal dose of avermectin had no significant effect on the hatching rate of eggs and the emergence rate of the nymphs in F0 generation. However, after exposure to avermectin, progeny (F1 and F2 generations) produced fewer eggs, the pre-oviposition duration and the adult longevity shortened, resulting in a decrease in the mean generation time. The sublethal dose of avermectin expressed different effects on the offspring (F1 and F2 generations) of the mite and the effect of nymphal stage and adult stage treatments on the population life parameters of the offspring was more significant in F2 generation. After treatment during nymphal stages, higher rm, R0, andλvalues while lower Dt value were observed. However, the treatment during adult stages exhibited negative effects on population increase (i.e. lower rm, Ro, andλvalues while longer Dt).
1.3 Effects of fenpropathrin and avermectin with sublethal concentrations on activities of detoxifying enzymes in P. citri
After adult mites were treated applying fenpropathrin and avermectin with LC20 concentration, there were different induction effects on the activities of main detoxifying enzymes such as carboxylesterase (CarE), gutathione S-transferases (GSTs) and mixed-function oxidase (MFO) from the mite. After the treatment with fenpropathrin, the specific activity of CarE fluctuated a lot, increased firstly, then decreased and finally increased again, with the values at 12 h,36 h and 48 h significantly higher than the control (P< 0.05); the induction effects on GST activities were quite obvious and the specific activity at 36 h was 3.31 fold of control; there was no significant effects on MFO activity. For avermectin sublethal treatment, CarE specific activity ascended in the first 12 h, then decreased to a state value; the induction effect on GSTs was less significant than fenpropathrin treatment; the activities of O-demethylation of MFOs were distinctly inhibited by sublethal concentration of avermectin, especially after 24 h of the treatment, significant different from the control (P<0.05)
2 Resistance selection and risk assessment of resistance to fenpropathrin and avermectin in P. citri
After the further resistance selection in laboratory, the realized heritability of P. citri resistance to acaricides were conducted via threshold trait analysis, the resistance development ratio of the mite was forecast and the cross-resistance between the acaricides was also studied. After discontinuous selection with fenpropathrin and avermectin 16 and 11 times during 19 generations, resistance ratio of the mite to the acaricides amounted to 29.92 and 3.80 fold, respectively. The realized heritability of resistance to fenpropathrin and avermectin accounted for 0.1544 and 0.0475, respectively. Theoretically, in laboratory, to obtain a 10 fold resistance ratio requires 8 to 16 generations for fenpropathrin and 12 to 26 generations for avermectin under selective pressure of 50% -90% mortality for each selective generation. For field populations, more generations were required to obtain the same resistance levels. Bioassay revealed that the fenpropathrin resistant strain developed obvious cross-resistance to pyridaben, dicofol, azocyclotin and the avermectin resistant strain obtained cross-resistance to emamectin benzoate. The results of resistance risk assessment suggested fenpropathrin has higher resistance risk than avermectin in P. citri. The current results provide some information for the resistance management in P. citri.
3 Biochemical mechanism of P. citri resistance to two acaricides
3.1 Synergism effects of synergists on fenpropathrin and avermectin
The synergism of PBO, TPP and DEM all on fenpropathrin and avermectin respectively was test in susceptible and resistant strains of P. citri with the relative synergism ratio (the ratio between the synergism ratio in resistant and susceptible strains, SRFeR(AvR)/SRss) as the criterion. The results showed that PBO, TPP and DEM all possessed obvious synergism effects on fenpropathrin with the relative synergism ratio (SRFeR/SRss) of 3.18,2.90 and 1.68, respectively, the relative synergism ratios of PBO, TPP and DEM on avermectin was 1.81,1.44, and 0.84, respectively.
3.2 Comparison of carboxylesterase (CarE) in different strains of P. citri
Compared to their susceptible counterparts, the activities of CarE in FeR and AvR were significantly higher (P< 0.05). Kinetic parameters comparison showed that CarE from FeR expressed highest Km value to the substrate a-NA, while both two resistant strains exhibited lower Km values toβ-NA. For Vmax, the values in resistant strains were significantly higher.
In vitro inhibition study showed that paraoxon-methyl, carbosulfan and malathion expressed significant inhibitory effects on CarE in three strains of P. citri. Beta-cypermethrin and fenpropathrin also exhibited obvious inhibition effects, while avermectin expressed limited inhibition effect. For pyridaben, the inhibition effects were not obvious and some facilitated effects appeared at lower concentrations. The comparison analysis revealed that except malathion, CarE from SS strain were most sensitive (lowest I50 values) and the sensitivity of CarE from FeR was lowest (highest I50 values).
3.3 Comparison of acid phosphatase (ACP) and alkaline phosphatase (ALP) in different strains of P. citri
Compared to their susceptible counterparts, the specific activities of ACP and Vmax value of ALP from FeR expressed significantly higher than SS (P< 0.05), while the specific activities, the Vmax and Km values of of ACP and ALP had no significant difference between SS and AvR (P> 0.05). The results indicated that phosphatase might play some roles in fenpropathrin resistance of P. citri, but played little role in P. citri resistance to avermectin.
3.4 Comparison of acetylcholinesterase (AChE) in different strains of P. citri
Among three strains, the activity per mite and specific activity of AChE, as well as the Vmax value were highest in FeR. Compared to SS strain, these values in FeR and AvR were significantly higher(P< 0.05), and significant difference was observed between two resistant strains (P< 0.05). However, there was no significant difference of Km value among three strains (P> 0.05). The result indicated that increased activity of AChE plays important roles in P. citri resistant to fenpropathrin and avermectin.
3.5 Comparison of glutathione S-transferase (GSTs) in different strains of P. citri
The result revealed that the activity per mite and specific activity of GSTs in FeR were significantly higher than other two strains (SS and AvR) (P< 0.05). GSTs from FeR expressed highest Km and Vmax values to the substrate CDNB while exhibited lowest Km and Vmax values to GSH. There were significant difference between FeR and other two strains (P< 0.05) while no significant difference existed between AvR and SS strains (P> 0.05). The results indicated that GSTs was important in fenpropathrin resistance development, but played little role in P. citri resistance to avermectin.
3.6 Comparison of mixed function oxidases (MFO) in different strains of P. citri
Compared to SS strain, the specific activities of O-demethylation in FeR and AvR were significantly higher (1.36 and 1.31 fold of SS strain) (P< 0.05). For kinetic parameters, O-demethylation from AvR showed highest Km value while lowest Vmax value, and its Km value was significantly higher than that from SS (P< 0.05), indicating MFO might have changed in quality in AvR strain. However, there was no significant difference in the Km and Vmax values between FeR and SS strain (P> 0.05). These results indicated that MFO was essential in avermectin resistance development, and it played some roles in fenpropathrin resistance to P. citri.
3.7 Comparison of adenosine triphosphatase (ATPase) in different strains of P. citri
The biochemical characterization of ATPase comparison analysis showed that the specific activity of Na+-K+-ATPase from high to low was AvR> SS> FeR. The specific activity of Na+-K+-ATPase in FeR is 0.68-fold of SS, significantly lower than other two strains (P< 0.05). There was no significant difference between AvR and SS strain (P> 0.05). Further kinetic parameters comparison showed that Na+-K+-ATPase from FeR expressed significantly higher Km and Vmax values than other two strains(P< 0.05), and the Vmax value from AvR was also higher than SS strain (P< 0.05). For the activities per mite, the specific activities, the Km value and Vmax value of Mg2+-ATPase, no significant difference was observed among these strains of P. citri(P> 0.05).
In vitro inhibition of insecticides on Na+-K+-ATPase of P. citri showed that fenpropathrin expressed significant inhibitory effects under tested six concentrations. The sensitivity of Na+-K+-ATPase from SS was highest while from FeR was lowest, and significance existed between FeR and other two strains (P< 0.05). In addition, avermectin also exhibited some in vitro inhibition effect, and the inhibition rate on Na+-K+-ATPase of the three strains of P. citri ranged from 3.60%to 12.76% (under 0.0229-2290.7164μM) and significant difference existed between susceptible and resistant strains under higher concentration (i.e.2290.7146μM)(P< 0.05)
These results indicated that Na+-K+-ATPase played major role in fenpropathrin resistance of P. citri by its significantly lower activity and insensitivity to fenpropathrin and thus speculated that Na+-K+-ATPase is one of target enzymes of fenpropathrin. In addition, it may play some roles in avermectin resistance to P. citri.
4 Molecular cloning and expression of esterase gene in P. citri
4.1 Molecular cloning and sequence analysis of esterase gene in P. citri
The full length cDNA encoding esterase PCE1 (GenBank No. GQ144324) was cloned from P. citri by the method of reverse transcriptase PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The complete cDNA of this gene consists of 1892 bp with an open reading frame (ORF) of 1653 bp, encoding a protein of 550 amino acids residues with the putative signal peptide of 19 residues. The mature protein has a molecular weight of 61.1 kDa with an isoelectric point of 5.21. Based on the online software ScanProsite, two conserved regions of CarE were found:serine active site (FGGDPDQVTIFGESAG) and the conserved cysteine that compose disulfide bridge (EDCLTLNVITP). In addition, an endoplasmic reticulum targeting sequence (HEEL) as well as 14 phosphorylation sites,12 N-myristoylation sites and 4 N-glycosylation sites were also found. The sequence homology analysis showed that the esterase cloned from P. citri share low homology to other insect species, but the sequences is highly conserved in active site. Phylogenetic trees generated from the amino acid sequences revealed a closer relationship to other acaris than to insects. In addition, the PCE1 gene 3-D structure models were constructed by SWISS-MODEL.
4.2 mRNA expression level of the esterase gene PCE1 from P. citri
The adult female mites of P. citri were treated with LC50 concentration of fenpropathrin using leaf dip method and then the expression levels of PCE1 gene was determined. After treated for 6 h, 12 h,24 h and 48 h, the relative quantity of PCE1 was investigated applying real-time PCR, respectively. The results showed that the relative quantity of PCE1 from P. citri fluctuated significantly. At the beginning of fenpropatrin treatment, the relative quantity of PCE1 was inhibited (compared to the control group, PCE1 transcript was only 0.74 fold after 6 h treated). It increased gradually with the increase of time treated, then decreased, and then again increased. After 48 h treated, the relative quantity of PCE 1 is significantly higher than the control (just 1.36 fold compared with the control). In general, the transcript of PCE1 from P. citri treated was higher with longer treatment time.
4.3 Heterologous expression of the esterase gene PCE1 from P. citri
Applying the double digestion of BamHI and Xhol restriction enzymes with the DNA recombination technology, the expression vectors for PCE1 was constructed based on pET-43.1a (+) vector. Specific band appeared on the gel of SDS-PAGE. The current result provides useful base for furthermore exploration of the property and function of the esterase gene PCE1.
引文
1.http://www.ensam.inra.fr/CBGP/spmweb/index.php.
2. 冉春.2007.桔全爪螨田间种群对杀螨剂的敏感性及其生化机理研究.博士学位论文重庆：西南大学
3. 范志金,李永强,刘秀峰,于维强.2004.农业害螨的抗药性.现代农药3：1-4
4. 胡冠芳.1998.国内叶螨类抗药性及治理对策研究概况.甘肃科学学报10：92-96
5. 袁明龙,刘永华,张昆,高玉珍,王进军.2009.桔全爪螨抗药性研究进展.粮食安全与植保科技创新,中国农业科学技术出版社.343-350.
6. Stumpf N, Nauen R.2001. Cross-resistance, inheritance, and biochemistry of mitochondrial electron transport inhibitor-acaricide resistance in Tetranychus urticae (Acari:Tetranychidae). Journal of Economic Entomology 94:1577-1583
7. Solomon MG, Easterbrook MA, Fitzgerald JD.1993. Mite-management programmes based on organophosphate-resistant Typhlodromus pyri in UK apple orchards. Crop Protection 12: 249-254
8. 盂昭礼,董瑞瑞.1989.苹果红蜘蛛抗药性研究.农药28：3-6
9. 黄汉杰,黄媛云.1993.尼索朗与阿波罗等杀螨剂对桔全爪螨的交护抗药性.江西柑桔科技4：30-30
10. 孟和生,王开运,姜兴印.2000.桔全爪螨对甲氰菊酯的抗性选育及交互抗性的研究.植物保护26：7-9
11. 陈洋.2006.柑桔全爪螨Panonychus citri (Mcgregor)抗药性监测及其抗性机理的初步研究.硕士学位论文重庆：西南大学
12. 袁明龙,冉春,李勇,王进军.2008.取食不同柑桔种质资源对桔全爪螨药剂敏感性及酯酶同工酶的影响.植物保护学报35：187-188
13. Takeyama K, Mori N, Osakabe M.2006. Effect of cytochrome P450 inhibitor, piperonyl butoxide, on survival of Panonychus citri (McGregor)(Acari: Tetranychidae) on citrus leaves. Applied Entomology and Zoology 41:487-491
14. Osakabe M, Goka K, Toda S, Shintaku T, Amano H.2005. Significance of habitat type for the genetic population structure of Panonychus citri (Acari:Tetranychidae). Experimental and Applied Acarology 36:25-40
15. 刘孝纯,李巧丝.1998.杀虫剂对朱砂叶螨扩散行为及繁殖力的影响.植物保护学报25：156-160
16. 黄振东,严得胜,陈道茂.2002.噻菌铜对桔全爪螨繁殖影响的研究.中国南方果树31：14-15
17. Jones VP, Parrella MP.1984. The sublethal effects of selected insecticides on life table parameters of Panonychus citri (Acari:Tetranychidae). The Canadian Entomologist 116: 1033-1040
18. Yamamoto A, Yoneda H, Hatano R, Asada M.1995. Influence of hexythiazox resistance on life history parameters in the citrus red mite, Panonychus citri (McGregor). Pesticide Science 2: 521-527
1 9. 夏冰,石泰.2002.杀虫剂亚致死剂量对小菜蛾羧酸酯酶的影响.农药学学报4：23-27
20. 曾春祥,王进军.2007.吡虫啉亚致死剂量处理对桃蚜乙酰胆碱酯酶的时间与剂量效应.植
物保护33：50-54
21. 赵善欢.2000.植物化学保护(第三版)北京：中国农业出版社：49-55
22. 曹传旺.2007.羧酸酯酶引起的棉蚜对氧化乐果和溴氰菊酯抗性分子机制.博士学位论文北京：中国农业大学
23. 唐振华.1993.昆虫抗药性北京：中国农业出版社：302-335
24. 孟和生.2002.桔全爪螨抗药性监测及抗性机理.硕士学位论文山东：山东农业大学
25. Chen ZY, Ran C, Zhang L, Dou W, Wang JJ.2009. Susceptibility and esterase activity in citrus red mite Panonychus citri (McGregor)(Acari:Tetranychidae) after selection with phoxim. International Journal of Acarology 35:33-40
26. 陈达荣,钟捷英.1990.桔全爪螨体内羧酸酯酶活性与对有机磷抗药性的关系.福建农学院学报19：312-325
27. 陈年春,彭云良.1996.柑桔全爪螨抗药性监测及主导抗性机制的初步研究.农药35：15-18
28. 盂和生,王开运.2002.桔全爪螨的抗药性选育及其解毒酶活力变化.昆虫学报45：58-62
29. 陈洋,冉春,熊琳,干进军.2006.不同柑桔种质资源对桔全爪螨酶活性及其对药剂敏感性的影响.西南农业学报19：434-437
30. Gotoh T, Ishikawa Y, Takayama K, Suzuki M.1991. Differences in esterase zymograms between non-diapausing and diapausing individuals of the hawthorn spider mite, Tetranychus viennensis Zacher (Acari:Tetranychidae). Applied Entomology and Zoology 26:153-155
3 1. 豆威,王进军,张淑莲,陈志杰,张锋,王琦,董志平,甘耀进,董立,马继芳.2005.昆虫谷胱甘肽S-转移酶研究进展.农业生物灾害预防与控制研究.26：35-37
32. 孟和生,王开运.2000.桔全爪螨对哒螨灵抗性的选育及其生化机理.农药学学报2：30-34
33. 陈志永.2007.桔全爪螨Panonychus citri (McGregor)对辛硫磷的抗性及AChE cDNA片段克隆.硕士学位论文重庆：西南大学
34. 唐振华,吴士雄.2000.昆虫抗药性的遗传与进化：上海：上海科学技术文献出版社
35. Anazawa Y, Tomita T, Aiki Y, Kozaki T, Kono Y.2003. Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant two-spotted spider mite, Tetranychus urticae. Insect Biochemistry and Molecular Biology 33:509-514
36. Kwon DH, Clark JM, Lee SH.2009. Cloning of a sodium channel gene and identification of mutations putatively associated with fenpropathrin resistance in Tetranychus urticae. Pesticide Biochemistry and Physiology: doi:10.1016/j.pestbp.2009.1007.1009
37. 赵卫东,王开运,姜兴印,仪美芹.2003.二斑叶螨对阿维菌素,哒螨灵和甲氰菊酯的抗性选育及其解毒酶活力变化.昆虫学报46：788-792
38. 何林,赵志模.2002.朱砂叶螨对三种杀螨剂的抗性选育与抗性风险评估.昆虫学报45：688-692
39. Yamamoto A, Yoneda H, Hatano R, Asada M.1995. Field selection experiments with hexythiazox in the citrus red mite, Panonychus citri (McGregor). Journal of Pesticide Science (Japan) 20:307-315
40. Yamamoto A, Yoneda H, Hatano R, Asada M.1995. Laboratory selections of populations in the citrus red mite, Panonychus citri (McGregor), with hexythiazox and their cross resistance spectrum. Pesticide Science 20:493-501
41. 林祥文,沈晋良.2001.棉铃虫对辛硫磷抗性的风险评估与预报.昆虫学报44：462-468
42. Via S.1986. Genetic covariance between oviposition preference and larval performance in an insect herbivore. Evolution 40:778-785
43. Yamamoto A, Yoneda H, Hatano R, Asada M.1996. Realized heritability estimates of hexythiazox resistance in the citrus red mite, Panonychus citri (McGregor). Journal of Pesticide Science (Japan) 21:43-47
44. Inoue K.1991. Genetic analysis of acaricide resistance in citrus red mite, Panonychus citri McGregor. Japan Agricultural Research Quarterly 25:3-9
45. Yamamoto A, Yoneda H, Hatano R, Asada M.1995. Genetic analysis of hexythiazox resistance in the citrus red mite, Panonychus citri (McGregor). Journal of Pesticide Science (Japan) 20: 513-519
46. Georghiou GP, Saito T.1983. Pest resistance to pesticides. Plenum Press New York
47. 黄国洋.1999.浙江省主要害虫抗药性发展现状及治理策略综述.农药科学与管理20：31-34
48. 孟和生,王开运.2000.桔全瓜螨对常用杀螨剂的抗药性测定.农药39：26-28
49. Yamamoto A, Yoneda H, Hatano R, Asada M.1996. Stability of hexythiazox resistance in the citrus red mite, Panonychus citri (McGregor) under laboratory and field conditions. Pesticide Science 21:37-42
50. Devonshire AL, Moores GD.1982. A carboxylesterase with broad substrate specificity causes organophosphorus, carbamate and pyrethroid resistance in peach-potato aphids (Myzus persicae). Pesticide Biochemistry and Physiology 18:235-246
51. 滕霞,孙曼霁.2003.羧酸酯酶研究进展.生命科学15：31-35
52. 宋宏图.2007.野桑蚕羧酸酯酶基因的克隆及其在大肠杆菌中的表达.硕士学位论文苏州：苏州大学
53. Wheelock CE, Shan G, Ottea J.2005. Overview of carboxylesterases and their role in the metabolism of insecticides. Journal of Pesticide Science 30:75-83
54. 郭晶,高菊芳,唐振华.2007.羧酸酯酶及其在含酯类化合物代谢中的作用.农药46：365-368
55. Wheelock CE, Colvin ME, Uemura I, Olmstead MM, Sanborn JR, Nakagawa Y, Jones AD, Hammock BD.2002. Use of ab initio calculations to predict the biological potency of carboxylesterase inhibitors. Journal of Medical Chemistry 45:5576-5593
56. Huang H, Stok JE, Stoutamire DW, Gee SJ, Hammock BD.2005. Development of optically pure pyrethroid-like fluorescent substrates for carboxylesterases. Chemical Research in Toxicology 18:516-527
57. 姚洪渭,蒋彩英.2001.白背飞虱羧酸酯酶与乙酰胆碱酯酶的体躯与亚细胞分布特征.浙江大学学报：农业与生命科学版27：5-10
58. 李腾武,高希武.1999.小菜蛾不同亚细胞层羧酸酯酶的性质研究.农药学学报1：47-53
59. Motoyama N, Kao LR, Lin PT, Dauterman WC.1984. Dual role of esterases in insecticide resistance in the green rice leafhopper. Pesticide Biochemistry and Physiology 21:139-147
60. Ebenezer 00, Kazuhiro K, Michio H, Chisato H.1994. Some properties of carboxylesterase from Aphis gossypii glover (Homoptera:Aphididae). Applied Entomology and Zoology 29: 47-53
61. Chen WL, Sun CN.1994. Purification and characterization of carboxylesterases of a rice brown planthopper, Nilaparvata lugens. Insect Biochemistry and Molecular Biology 24:347-355
62. 唐培安.2009.3种书虱酯酶基因的克隆及其mRNA表达水平研究.博十学位论文.重庆：西南大学
63. 王东,李兵,管京敏,赵华强,陈玉华,沈卫德.2008.棉铃虫羧酸酯酶基因的克隆,序列分 析及组织表达.昆虫学报51：979-985
64. 唐培安,邓永学,王进军.2007.甲酸乙酯对米象乙酰胆碱酯酶和羧酸酯酶的影响.植物保护33：44-47
65. 高希武,梁同庭.1993.阿特拉津和敌敌畏对棉铃虫和家蝇羧酸酯酶以及谷胱甘肽S-转移酶的诱导作用.昆虫学报36：167-171
66. 黄诚华,姚洪渭,叶恭银,程家安.2006.氟虫腈亚致死剂量处理对二化螟和大螟幼虫体内解毒酶系活力的影响.中国水稻科学20：447-450
67. 李晓涛,黄青春,唐振华.2006.氟虫腈对二化螟生长发育的影响及对解毒酶的诱导效应.农药学学报 8：250-254
68. Gao G-T Chen KP, Yao Q, Chen HQ, Wang LI, Xu JP, Zhao Y, Wang YJ.2007. A Study on the Activity of Carboxylesterase and the Differential Expression of Its Gene in the Midguts of Bombyx mori Resistant to BmDNV-Z. Agricultural Sciences in China 6:1018-1026
69. 张帆.2009.棉花防御与烟粉虱反防御的交互作用.硕十学位论文新疆：新疆农业大学
70. 薛传华.2008.朱砂叶螨酯酶基因克隆及其mRNA表达研究.硕士学位论文重庆：西南大学
71. 何林,谭仕禄,曹小芳,赵志模,邓新平,王进军.2003.朱砂叶螨的抗药性选育及其解毒酶活性研究.农药学学报5：23-29
72. Hughes PB, Raftos DA.2009. Genetics of an esterase associated with resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina (Wiedemann)(Diptera: Calliphoridae). Bulletin of Entomological Research 75:535-544
73. Guerrero FD, Nene VM.2008. Gene structure and expression of a pyrethroid-metabolizing esterase, CzEst9, from a pyrethroid resistant Mexican population of Rhipicephalus (Boophilus) microplus (Acari:Ixodidae). Journal of Medical Entomology 45:677-695
74. Newcomb RD, Campbell PM, Ollis DL, Cheah E, Russell RJ, Oakeshott JG.1997. A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly. Proceedings of the National Academy of Sciences of the United States of America 94:7464
75. Devonshire AL, Sawicki RM.1979. Insecticide-resistant Myzus persicae as an example of evolution by gene duplication. Nature 280:140-141
76. Field LM, Devonshire AL, Forde BG.1988. Molecular evidence that insecticide resistance in peach potato aphids (Myzus persicae Sulz) results from amplification of an esterase gene. Biochemical Journal 251:309-312
77. 乔传令,Hemingway J,李瑄.2003.有机磷抗性致倦库蚊种群中酯酶基因扩增的定量分析.昆虫学报 46：11-17
78. 郭惠琳,高希武.2005.棉蚜抗氧化乐果品系的羧酸酯酶基因突变.昆虫学报 48：194-202
79. Cao CW, Zhang J, Gao XW, Liang P, Guo HL.2008. Overexpression of carboxylesterase gene associated with organophosphorous insecticide resistance in cotton aphids, Aphis gossypii (Glover). Pesticide Biochemistry and Physiology 90:175-180
80. Baffi MA, de Souza GRL, de Sousa CS, Ceron CR, Bonetti AM.2008. Esterase enzymes involved in pyrethroid and organophosphate resistance in a Brazilian population of Riphicephallus (Boophilus) microplus (Acari, Ixodidae). Molecular and Biochemical Parasitology 160:70-73
81. Campbell PM, Newcomb RD, Russell RJ, Oakeshott JG.1998. Two different amino acid substitutions in the ali-esterase, E3, confer alternative types of organophosphorus insecticide resistance in the sheep blowfly, Lucilia cuprina. Insect Biochemistry and Molecular Biology 28: 139-150
82. Zhu YC, Dowdy AK, Baker JE. 1999. Differential mRNA expression levels and gene sequences of a putative carboxylesterase-like enzyme from two strains of the parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae). Insect Biochemistry and Molecular Biology 29: 417-425
83. Claudianos C, Russell RJ, Oakeshott JG.1999. The same amino acid substitution in orthologous esterases confers organophosphate resistance on the house fly and a blowfly. Insect Biochemistry and Molecular Biology 29:675-686
84. Pan YO, Guo HL, Gao XW.2009. Carboxylesterase activity, cDNA sequence, and gene expression in malathion susceptible and resistant strains of the cotton aphid, Aphis gossypii. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology 152: 266-270
85. 高贵田,陈克平,姚勤,王林玲,陈慧卿.2006.家蚕羧酸酯酶基因克隆及差异表达,.昆虫学报49：930-937
86. Hernandez R, He H, Chen AC, Waghela SD, Wayne Ivie G, George JE, Gale Wagner G.2000. Identification of a point mutation in an esterase gene in different populations of the southern cattle tick, Boophilus microplus. Insect Biochemistry and Molecular Biology 30: 969-977
87. Hernandez R, Guerrero F, George J, Wagner G.2002. Allele frequency and gene expression of a putative carboxylesterase-encoding gene in a pyrethroid resistant strain of the tick Boophilus microplus. Insect Biochemistry and Molecular Biology 32:1009-1016
88. Baffi MA, de Souza GRL, Vieira CU, de Sousa CS, Gourlart LR, Bonetti AM.2007. Identification of point mutations in a putative carboxylesterase and their association with acaricide resistance in Rhipicephalus (Boophilus) microplus (Acari:Ixodidae). Veterinary parasitology 148:301-309
89. 曹小芳,何林,赵志模,邓新平,王进军.2004.朱砂叶螨不同抗性品系酯酶同工酶研究.蛛形学报13：95-102
90. 何林,谭仕禄,曹小芳,赵志模,邓新平,王进军.2003.朱砂叶螨的抗药性选育及其解毒酶活性研究.农药学学报5：23-29
91. Ran C, Chen Y, Wang JJ.2009. Susceptibility and carboxylesterase activity of five field populations of Panonychus citri (McGregor)(Acari:Tetranychidae) to four acaricides. International Journal of Acarology 35:115-121
92. Chen AC, He H, Temeyer KB, Jones S, Green P, Barker SC.2009. A survey of Rhipicephalus microplus populations for mutations associated with pyrethroid resistance. Journal of Economic Entomology 102:373-380
93. 冉春,陈洋,袁明龙,王进军,刘浩强,姚廷山.2008.桔全爪螨田间种群对杀螨剂的敏感性.植物保护学报35：537-540
94. 洪源范,洪青,沈雨佳,李顺鹏.2007.甲氰菊酯降解菌Sphingomonas sp.JQL4-5对污染土壤的生物修复.环境科学 28 1121-1125
95. 孟和生.2002.两种生物方法对杀螨剂毒力测定结果的影响比较.植物保护28：49-51
96. 丁岩钦.1994.昆虫数学生态学.北京：科学出版社：153-169
97. 沈慧敏,张新虎.2002.三氟氯氰菊酯和甲氰菊酯对二点叶螨生命活力及繁殖的影响.植物保护学报29：183-188
98. 李定旭,田娟,沈佐锐.2006.不同药剂对山楂叶螨的亚致死效应.植物保护学报33：
187-192
99. 高宗仁,李巧丝,刘孝纯,等.1991.杀虫剂对朱砂叶螨某些生物学特性的影响.植物保护学报18：283-287
100. Gerson U, Cohen E.1989. Resurgences of spider mites (Acari:Tetranychidae) induced by synthetic pyrethroids. Experimental and Applied Acarology 6: 29-46
101. 陈道茂,陈卫民.1990.二种拟除虫菊酯对桔全爪螨繁殖的影响.植物保护学报17：279-282
102. Luckey TD.1968. Insecticide hormoligosis. Journal of Economic Entomology 61:7-12
103. 高君晓,梁沛,宋敦伦,高希武.2008.高效氯氰菊酯亚致死剂量对大豆蚜实验种群的影响.植物保护学报35：379-380
104. Kolar L, Erzen, NK, Hogerwerf L, van Gestel CAM.2008. Toxicity of abamectin and doramectin to soil invertebrates. Environmental Pollution 151:182-189
105. Flores AE, Landeros J, Badii MH.2000. Evaluation of population parameters of Tetranychus urticae Koch (Acari:Prostigmata: Tetranyciudae) exposed to avermectin. Southwestern Entomologist 25:287-293
106. Landeros J, Mora N, Badii M, Cerda PA, Flores AE. 2002. Effect of sublethal concentrations of avermectin on population parameter of Tetranychus urticae (Acari:Tetranychidae) on strawberry. Southwestern Entomologist 27:283-289
107. Marcic D, Sekulic D.1998. Effects of acaricide resistance on life history parameters of Tetranychus urticae Koch (Acari:Tetranychidae). Pesticides 13:13-46
108. 唐振华,毕强.2003.杀虫剂作用的分子行为：上海：上海远东出版社.100-250.
109. Rumpf S, Hetzel F, Frampton C.1997. Lacewings (Neuroptera:Hemerobiidae and Chrysopidae) and integrated pest management: enzyme activity as biomarker of sublethal insecticide exposure. Journal of Economic Entomology 90:102-108
110. Bradford MM.1976. A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye release. Anal Biochem 72:248-254
111. Clark AG, Dick GL, Smith JN.1984. Kinetic studies on a glutathione S-transferase from the larvae of Costelytra zealandica. Biochemical Journal 217:51-58
112. Habig WH, Pabst MJ, Jakoby WB.1974. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. The Journal of Biological Chemistry 249:7130
113. Kim YJ, Lee SH, Lee SW, Ahn YJ.2004. Fenpyroximate resistance in Tetranychus urticae Acari: Tetranychidae:cross-resistance and biochemical resistance mechanisms. Pest Management Science 60:1001-1006
114. 尹显慧,吴青君,李学锋,张友军,徐宝云.2008.多杀菌素亚致死浓度对小菜蛾解毒酶系活力的影响.农药学学报10：28-34
115. 梁沛,夏冰,石泰,高希武.2003.阿维菌素和高效氯氰菊酯亚致死剂量对小菜蛾谷胱甘肽S-转移酶的影响.中国农业大学学报8：65-68
116. 魏波,郝赤,李会仙.2006.杀虫剂亚致死剂量对棉铃虫靶标酶的影响.现代农药5：35-38
117. 高希武,董向丽.1997.棉铃虫的谷胱甘肽S-转移酶(GSTs)：杀虫药剂和植物次生性物质的诱导与GSTs对杀虫药剂的代谢.昆虫学报40：122-127
118. 魏辉,李洪山,戴华国,王娟.2006.多杀菌素对小菜蛾体内多功能氧化酶和保幼激素酯酶活力的影响.农药学学报8：239-244
119. 茹李军,范贤林.1997.棉铃虫对拟除虫菊酯类杀虫剂抗性遗传力的分析.植物保护学报24：356-360
120. Tabashnik BE.1992. Resistance risk assessment: realized heritability of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae), tobacco budworm (Lepidoptera: Noctuidae), and Colorado potato beetle (Coleoptera: Chrysomelidae). Journal of Economic Entomology 85:1551-1559
121. 林荣寿,邓新平.1994.四川省柑桔害螨抗药性监测与综合治理.西南农业学报7：75-81
122. Cochran DG.1989. Monitoring for insecticide resistance in field-collected strains of the German cockroach (Dictyoptera: Blattellidae). Journal of Economic Entomology 82:336-341
123. Firko MJ, HAyes JL.1990. Quantification of larval resistance to cypermethrin in tobacco budworm (Lepidoptera: Noctuidae) and the effects of larval weight. Journal of Economic Entomology 83:1222-1228
124. Bloch G, Wool D.1994. Methidathion resistance in the sweetpotato whitefly (Aleyrodidae: Homoptera) in Israel: selection, heritability, and correlated changes of esterase activity. Journal of Economic Entomology 87:1147-1156
125. 韩启发,庄佩君,唐振华.1995.抗杀螟硫磷二化螟的抗性遗传力研究.昆虫学报38：402-406
126. 兰亦全,赵士熙,吴刚.2006.甜菜夜蛾对三种拟除虫菊酯类杀虫剂的抗性遗传力及风险评估.应用生态学报17：468-471
127. 贾变桃,沈晋良,刘叙杆.2007.甜菜夜蛾对虫酰肼的抗性选育,风险评估及交互抗性.昆虫学报50：1116-1121
128. 牛洪涛,罗万春,宗建平,魏书娟,王海迎,潘志孝.2008.小菜蛾对丁烯氟虫腈的抗性遗传力及风险评估.植物保护学报35：165-168
129. 莫建初,唐振华.1997.数量遗传学在害虫种群抗性进化研究中的应用.昆虫知识34：183-186
130. 唐振华.2000.我国昆虫抗药性研究的现状及展望.昆虫知识37：97-103
131. 许雄山,韩召军.1999.增效醚对棉铃虫的毒理学研究.农药学学报 1：42-46
132. He L, Xue CH, Wang JJ, Li M, Lu WC Zhao ZM.2009. Resistance selection and biochemical mechanism of resistance to two Acaricides in Tetranychus cinnabarinus (Boiduval). Pesticide Biochemistry and Physiology 93:47-52
133. 何林.2003.朱砂叶螨抗药性机理及抗性适合度研究.博士学位论文重庆：西南大学
134. 范志金,陈年春.1996.截形叶螨抗药性主导机制的研究.植物保护学报23：175-180
135. 王利华,吴益东.2008.kdr突变和解毒代谢在B型烟粉虱对高效氯氰菊酯抗性中的作用.昆虫学报51：277-283
136. 孙耘芹,冯国蕾,袁家珪,祝平,龚坤元.1987.棉蚜对有机磷杀虫剂抗性的生化机理.昆虫学报30：13-20
137. 冉春,陈志永,陈洋,王进军.2007.桔全爪螨田间种群对双甲脒的敏感性及其两种酶的生化特性.中国农学通报23：284-288
138. 陈巧云,姜家良,林国芳,刘维德.1980.淡色库蚊对敌百虫抗性的研究——水解酶同敌百虫抗性关系.昆虫学报23：350-357
139. Senthil Nathan S, Kalaivani K, Chung PG.2005. The effects of azadirachtin and nucleopolyhedrovirus on midgut enzymatic profile of Spodoptera litura Fab.(Lepidoptera: Noctuidae). Pesticide Biochemistry and Physiology 83:46-57
140. Ren Y, Wei XP, Wu S, Dou W, Wang JJ.2008. Comparison of acetylcholinesterase from three field populations of Liposcelis paeta Pearman (Psocoptera: Liposcelididae):Implications of insecticide resistance. Pesticide Biochemistry and Physiology 90:196-202
141. Ellman GL, Courtney KD.1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical pharmacology 7:88-90
142. Wilkinson GN.1961. Statistical estimations in enzyme kinetics. Biochemical Journal 80:324
143. Chai YX, Liu GY, Wang JJ.2007. Toxicological and biochemical characterizations of AChE in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae). Pesticide Biochemistry and Physiology 88:197-202
144. 王新国,王怀位,甄天民,江洪涛,孙传红,赵玉强,程鹏,王海防.2005.淡色库蚊三种抗性品系抗性发展与乙酰胆碱酯酶相关性的研究.中国媒介生物学及控制杂志 16：264-266
145. 杨帆,帅霞.2008.桃蚜高效氯氰菊酯抗药性与乙酰胆碱酯酶的关系.植物保护34：60-62
146. Rauch N, Nauen R.2004. Characterization and molecular cloning of a glutathione S-transferase from the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae). Insect Biochemistry and Molecular Biology 34:321-329
147. 豆威.2009.书虱谷胱甘肽S-转移酶的纯化及生化毒理学特性研究.博十学位论文重庆：西南大学
148. 孙耘芹,袁家圭.1990.家蝇对拟除虫菊酯农药的抗性机制.昆虫学报33：265-273
149. 韩启发,庄佩君.1995.二化螟对杀螟硫磷产生抗性的机理.昆虫学报38：266-271
150. 冷欣大,唐振华,王荫长.1996.杀虫药剂分子毒理学及昆虫抗药性.北京：农业出版社47-89
151. 邱立红,李学锋.1999.抗溴氰菊酯家蝇在不同用药方式下的敏感性变化及其机制.昆虫学报42：248-256
152. 刘永杰,沈晋良.2003.甜菜夜蛾抗氯氟氰菊酯品系相对适合度,抗性生化机理及抗性遗传方式.昆虫学报46：567-572
153. 沈同,王镜岩.1991.生物化学(第二版).北京：高等教育出版社：103-107
1 54. 李宏.1996.ATPase的研究进展.生物学杂志 13：9-12
155. 祁鸣,薛京伦.1986.Na+,K+-ATP酶的特性研究及其应用.生物化学与生物物理进展13：22-26
156. 兰亦全,赵十熙.2004.甜菜夜蛾抗药性监测及机理.福建农林大学学报：自然科学版33：26-29
157. 冯北元,徐慕禹.1981.大鼠脑突触体Na+-K+-ATPase活力的微量测定方法.生物化学与生物物理学进展2：48-49
158. 张钧,邓蜀李,刘扬,黄叔怀.2003.运动对大鼠心肌线粒体功能的影响.北京体育大学学报26：198-200
159. 程伟霞.2007.嗜卷书虱和嗜虫书虱比较毒理学研究.博士学位论文重庆：西南大学
160. Feng GL, Marion JR., Clark JM.1992. Suppression of pyrethroid-dependent neurotransmitter release from synaptosomes of knockdown-resistant house flies under pulsed-depolarization conditions during continuous perfusion. Pesticide Biochemistry and Physiology 42:64-77
161. Sarkar PK.2002. A quick assay for Na+-K+-ATPase specific activity. Zeitschrift fur Naturforschung 57c:562-564
1 62. 何运转,李梅,冯国蕾,王荫长.1999.拟除虫菊酯对家蝇Na+-K+-ATPase抑制作用的研究.昆虫学报1：19-24
163. 蒋志胜,尚稚珍,王晓博,杨淑华.2000.美洲大蠊Na+-K+-ATPase作为筛选靶标的初步研究.农药学学报2：28-32
164. Li HP, Feng T, Liang P, Shi XY, Gao XW, Jiang H.2006. Effect of temperature on toxicity of pyrethroids and endosulfan, activity of mitochondrial Na+,K+-ATPase and Ca2+,Mg2*-ATPase in Chilo suppressalis (Walker)(Lepidoptera: Pyralidae). Pesticide Biochemistry and Physiology 86: 151-156
165. 伦才智,李艳伟,刘永杰,沈晋良,束怀瑞.2006.抗性甜菜夜蛾Na+-K+-ATP酶,Ca2+-ATP酶和Ca2+-Mg2+-ATP酶对高效氯氟氰菊酯的敏感性.农药学学报8：335-338
166. 蒋志胜,颜增光,何佳,杜育哲,尚稚珍.2003.典型光活化毒素α-三噻吩对棉铃虫和亚洲玉米螟Na+-K+-ATPase的抑制作用.农药学学报5：47-52
167. 田雨,冷欣夫.1999.溴氰菊酯对不同品系家蝇脑突触体膜蛋白磷酸化及ATP酶活性的影响.昆虫学报42：113-119
168. 李海平,冯涛,陶岭梅,刘学,姜辉,林荣华,梁沛,高希武.2006.九种常用杀虫剂对二化螟线粒体ATPase活力的抑制作用.昆虫学报49：254-259
169. 曾晓P.1995.杀虫剂对家蝇线粒体ATP酶活性影响的研究.中国媒介生物学及控制杂志6：253-256
170. 郭志波,马志卿,冯俊涛,张兴.2008.松油烯4-醇及其酯类衍生物对家蝇Na+,K+-ATPase活性的抑制作用.中国农业科学41：3110-3115
171. 马志卿,栾正春,张兴.2009.松油烯-4-醇对淡色库蚊的熏蒸毒力及对其Na+,K+-ATP酶的抑制作用.农药学学报11：230-234
172. 马志卿,冯俊涛,黄石周,张兴.2004.松油烯-4-醇对家蝇体内几种酶系的影响.植物保护学报31：283-288
173. 蒋志胜,白淑萍,尚稚珍.2002.具保幼激素活性的昆虫生长调节剂对亚洲玉米螟Na+-K+-ATPase的影响初探.南开大学学报(自然科学版)35：76-79
174. 宋宏图.2007.野桑蚕羧酸酯酶基因的克隆及在大肠杆菌中的表达.硕士学位论文江苏：江苏大学
175. 李飞.2003.棉蚜的杀虫剂神经靶标分子生物学研究.博士学位论文南京：南京农业大学
176. Field LM, Devonshire AL.1998. Evidence that the E4 and FE4 esterase genes responsible for insecticide resistance in the aphid Myzus persicae (Sulzer) are part of a gene family. Biochemical Journal 330:169
177. Vaughan C, Nemeth NM, Cary J, Temple S.2005. Response of a Scarlet Macaw Ara macao population to conservation practices in Costa Rica. Bird Conservation International 15:119-130
178. Vontas JG, Small GJ, Hemingway J.2000. Comparison of esterase gene amplification, gene expression and esterase activity in insecticide susceptible and resistant strains of the brown planthopper, Nilaparvata lugens (Stal). Insect Molecular Biology 9: 655-660
179. 曲明静,许新军,韩召军,陈茂华.2007.酯酶基因扩增及突变与昆虫抗药性.昆虫知识44：29-31
180. 唐振华.1993.昆虫抗药性中的酯酶基因扩增研究进展.昆虫知识30：53-56
181. Jones CD, Yeung C, Zehnder JL.2003. Comprenensive validation of a realtime quantitative bcrabl assay for clinical laboratory use. American Journal of Clinical Pathology 120:42-48
182. Livak KJ, Schmittgen TD.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods 25:402-408
183. Cao CW, Zhang J, Gao XW, Liang P, Guo HL.2008. Overexpression of carboxylesterase gene associated with organophosphorous insecticide resistance in cotton aphids, Aphis gossypii (Glover). Pesticide Biochemistry and Physiology 90:175-180
184. Furlong J, Meighan M, Conner J, Murray J, Clements, JB.1992. Methods for Improved Protein Expression Using Pet Vectors. Nucleic Acids Reseach 20:4668-4673
2. 冉春.2007.桔全爪螨田间种群对杀螨剂的敏感性及其生化机理研究.博士学位论文重庆：西南大学
3. 范志金,李永强,刘秀峰,于维强.2004.农业害螨的抗药性.现代农药3：1-4
4. 胡冠芳.1998.国内叶螨类抗药性及治理对策研究概况.甘肃科学学报10：92-96
5. 袁明龙,刘永华,张昆,高玉珍,王进军.2009.桔全爪螨抗药性研究进展.粮食安全与植保科技创新,中国农业科学技术出版社.343-350.
6. Stumpf N, Nauen R.2001. Cross-resistance, inheritance, and biochemistry of mitochondrial electron transport inhibitor-acaricide resistance in Tetranychus urticae (Acari:Tetranychidae). Journal of Economic Entomology 94:1577-1583
7. Solomon MG, Easterbrook MA, Fitzgerald JD.1993. Mite-management programmes based on organophosphate-resistant Typhlodromus pyri in UK apple orchards. Crop Protection 12: 249-254
8. 盂昭礼,董瑞瑞.1989.苹果红蜘蛛抗药性研究.农药28：3-6
9. 黄汉杰,黄媛云.1993.尼索朗与阿波罗等杀螨剂对桔全爪螨的交护抗药性.江西柑桔科技4：30-30
10. 孟和生,王开运,姜兴印.2000.桔全爪螨对甲氰菊酯的抗性选育及交互抗性的研究.植物保护26：7-9
11. 陈洋.2006.柑桔全爪螨Panonychus citri (Mcgregor)抗药性监测及其抗性机理的初步研究.硕士学位论文重庆：西南大学
12. 袁明龙,冉春,李勇,王进军.2008.取食不同柑桔种质资源对桔全爪螨药剂敏感性及酯酶同工酶的影响.植物保护学报35：187-188
13. Takeyama K, Mori N, Osakabe M.2006. Effect of cytochrome P450 inhibitor, piperonyl butoxide, on survival of Panonychus citri (McGregor)(Acari: Tetranychidae) on citrus leaves. Applied Entomology and Zoology 41:487-491
14. Osakabe M, Goka K, Toda S, Shintaku T, Amano H.2005. Significance of habitat type for the genetic population structure of Panonychus citri (Acari:Tetranychidae). Experimental and Applied Acarology 36:25-40
15. 刘孝纯,李巧丝.1998.杀虫剂对朱砂叶螨扩散行为及繁殖力的影响.植物保护学报25：156-160
16. 黄振东,严得胜,陈道茂.2002.噻菌铜对桔全爪螨繁殖影响的研究.中国南方果树31：14-15
17. Jones VP, Parrella MP.1984. The sublethal effects of selected insecticides on life table parameters of Panonychus citri (Acari:Tetranychidae). The Canadian Entomologist 116: 1033-1040
18. Yamamoto A, Yoneda H, Hatano R, Asada M.1995. Influence of hexythiazox resistance on life history parameters in the citrus red mite, Panonychus citri (McGregor). Pesticide Science 2: 521-527
1 9. 夏冰,石泰.2002.杀虫剂亚致死剂量对小菜蛾羧酸酯酶的影响.农药学学报4：23-27
20. 曾春祥,王进军.2007.吡虫啉亚致死剂量处理对桃蚜乙酰胆碱酯酶的时间与剂量效应.植
物保护33：50-54
21. 赵善欢.2000.植物化学保护(第三版)北京：中国农业出版社：49-55
22. 曹传旺.2007.羧酸酯酶引起的棉蚜对氧化乐果和溴氰菊酯抗性分子机制.博士学位论文北京：中国农业大学
23. 唐振华.1993.昆虫抗药性北京：中国农业出版社：302-335
24. 孟和生.2002.桔全爪螨抗药性监测及抗性机理.硕士学位论文山东：山东农业大学
25. Chen ZY, Ran C, Zhang L, Dou W, Wang JJ.2009. Susceptibility and esterase activity in citrus red mite Panonychus citri (McGregor)(Acari:Tetranychidae) after selection with phoxim. International Journal of Acarology 35:33-40
26. 陈达荣,钟捷英.1990.桔全爪螨体内羧酸酯酶活性与对有机磷抗药性的关系.福建农学院学报19：312-325
27. 陈年春,彭云良.1996.柑桔全爪螨抗药性监测及主导抗性机制的初步研究.农药35：15-18
28. 盂和生,王开运.2002.桔全爪螨的抗药性选育及其解毒酶活力变化.昆虫学报45：58-62
29. 陈洋,冉春,熊琳,干进军.2006.不同柑桔种质资源对桔全爪螨酶活性及其对药剂敏感性的影响.西南农业学报19：434-437
30. Gotoh T, Ishikawa Y, Takayama K, Suzuki M.1991. Differences in esterase zymograms between non-diapausing and diapausing individuals of the hawthorn spider mite, Tetranychus viennensis Zacher (Acari:Tetranychidae). Applied Entomology and Zoology 26:153-155
3 1. 豆威,王进军,张淑莲,陈志杰,张锋,王琦,董志平,甘耀进,董立,马继芳.2005.昆虫谷胱甘肽S-转移酶研究进展.农业生物灾害预防与控制研究.26：35-37
32. 孟和生,王开运.2000.桔全爪螨对哒螨灵抗性的选育及其生化机理.农药学学报2：30-34
33. 陈志永.2007.桔全爪螨Panonychus citri (McGregor)对辛硫磷的抗性及AChE cDNA片段克隆.硕士学位论文重庆：西南大学
34. 唐振华,吴士雄.2000.昆虫抗药性的遗传与进化：上海：上海科学技术文献出版社
35. Anazawa Y, Tomita T, Aiki Y, Kozaki T, Kono Y.2003. Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant two-spotted spider mite, Tetranychus urticae. Insect Biochemistry and Molecular Biology 33:509-514
36. Kwon DH, Clark JM, Lee SH.2009. Cloning of a sodium channel gene and identification of mutations putatively associated with fenpropathrin resistance in Tetranychus urticae. Pesticide Biochemistry and Physiology: doi:10.1016/j.pestbp.2009.1007.1009
37. 赵卫东,王开运,姜兴印,仪美芹.2003.二斑叶螨对阿维菌素,哒螨灵和甲氰菊酯的抗性选育及其解毒酶活力变化.昆虫学报46：788-792
38. 何林,赵志模.2002.朱砂叶螨对三种杀螨剂的抗性选育与抗性风险评估.昆虫学报45：688-692
39. Yamamoto A, Yoneda H, Hatano R, Asada M.1995. Field selection experiments with hexythiazox in the citrus red mite, Panonychus citri (McGregor). Journal of Pesticide Science (Japan) 20:307-315
40. Yamamoto A, Yoneda H, Hatano R, Asada M.1995. Laboratory selections of populations in the citrus red mite, Panonychus citri (McGregor), with hexythiazox and their cross resistance spectrum. Pesticide Science 20:493-501
41. 林祥文,沈晋良.2001.棉铃虫对辛硫磷抗性的风险评估与预报.昆虫学报44：462-468
42. Via S.1986. Genetic covariance between oviposition preference and larval performance in an insect herbivore. Evolution 40:778-785
43. Yamamoto A, Yoneda H, Hatano R, Asada M.1996. Realized heritability estimates of hexythiazox resistance in the citrus red mite, Panonychus citri (McGregor). Journal of Pesticide Science (Japan) 21:43-47
44. Inoue K.1991. Genetic analysis of acaricide resistance in citrus red mite, Panonychus citri McGregor. Japan Agricultural Research Quarterly 25:3-9
45. Yamamoto A, Yoneda H, Hatano R, Asada M.1995. Genetic analysis of hexythiazox resistance in the citrus red mite, Panonychus citri (McGregor). Journal of Pesticide Science (Japan) 20: 513-519
46. Georghiou GP, Saito T.1983. Pest resistance to pesticides. Plenum Press New York
47. 黄国洋.1999.浙江省主要害虫抗药性发展现状及治理策略综述.农药科学与管理20：31-34
48. 孟和生,王开运.2000.桔全瓜螨对常用杀螨剂的抗药性测定.农药39：26-28
49. Yamamoto A, Yoneda H, Hatano R, Asada M.1996. Stability of hexythiazox resistance in the citrus red mite, Panonychus citri (McGregor) under laboratory and field conditions. Pesticide Science 21:37-42
50. Devonshire AL, Moores GD.1982. A carboxylesterase with broad substrate specificity causes organophosphorus, carbamate and pyrethroid resistance in peach-potato aphids (Myzus persicae). Pesticide Biochemistry and Physiology 18:235-246
51. 滕霞,孙曼霁.2003.羧酸酯酶研究进展.生命科学15：31-35
52. 宋宏图.2007.野桑蚕羧酸酯酶基因的克隆及其在大肠杆菌中的表达.硕士学位论文苏州：苏州大学
53. Wheelock CE, Shan G, Ottea J.2005. Overview of carboxylesterases and their role in the metabolism of insecticides. Journal of Pesticide Science 30:75-83
54. 郭晶,高菊芳,唐振华.2007.羧酸酯酶及其在含酯类化合物代谢中的作用.农药46：365-368
55. Wheelock CE, Colvin ME, Uemura I, Olmstead MM, Sanborn JR, Nakagawa Y, Jones AD, Hammock BD.2002. Use of ab initio calculations to predict the biological potency of carboxylesterase inhibitors. Journal of Medical Chemistry 45:5576-5593
56. Huang H, Stok JE, Stoutamire DW, Gee SJ, Hammock BD.2005. Development of optically pure pyrethroid-like fluorescent substrates for carboxylesterases. Chemical Research in Toxicology 18:516-527
57. 姚洪渭,蒋彩英.2001.白背飞虱羧酸酯酶与乙酰胆碱酯酶的体躯与亚细胞分布特征.浙江大学学报：农业与生命科学版27：5-10
58. 李腾武,高希武.1999.小菜蛾不同亚细胞层羧酸酯酶的性质研究.农药学学报1：47-53
59. Motoyama N, Kao LR, Lin PT, Dauterman WC.1984. Dual role of esterases in insecticide resistance in the green rice leafhopper. Pesticide Biochemistry and Physiology 21:139-147
60. Ebenezer 00, Kazuhiro K, Michio H, Chisato H.1994. Some properties of carboxylesterase from Aphis gossypii glover (Homoptera:Aphididae). Applied Entomology and Zoology 29: 47-53
61. Chen WL, Sun CN.1994. Purification and characterization of carboxylesterases of a rice brown planthopper, Nilaparvata lugens. Insect Biochemistry and Molecular Biology 24:347-355
62. 唐培安.2009.3种书虱酯酶基因的克隆及其mRNA表达水平研究.博十学位论文.重庆：西南大学
63. 王东,李兵,管京敏,赵华强,陈玉华,沈卫德.2008.棉铃虫羧酸酯酶基因的克隆,序列分 析及组织表达.昆虫学报51：979-985
64. 唐培安,邓永学,王进军.2007.甲酸乙酯对米象乙酰胆碱酯酶和羧酸酯酶的影响.植物保护33：44-47
65. 高希武,梁同庭.1993.阿特拉津和敌敌畏对棉铃虫和家蝇羧酸酯酶以及谷胱甘肽S-转移酶的诱导作用.昆虫学报36：167-171
66. 黄诚华,姚洪渭,叶恭银,程家安.2006.氟虫腈亚致死剂量处理对二化螟和大螟幼虫体内解毒酶系活力的影响.中国水稻科学20：447-450
67. 李晓涛,黄青春,唐振华.2006.氟虫腈对二化螟生长发育的影响及对解毒酶的诱导效应.农药学学报 8：250-254
68. Gao G-T Chen KP, Yao Q, Chen HQ, Wang LI, Xu JP, Zhao Y, Wang YJ.2007. A Study on the Activity of Carboxylesterase and the Differential Expression of Its Gene in the Midguts of Bombyx mori Resistant to BmDNV-Z. Agricultural Sciences in China 6:1018-1026
69. 张帆.2009.棉花防御与烟粉虱反防御的交互作用.硕十学位论文新疆：新疆农业大学
70. 薛传华.2008.朱砂叶螨酯酶基因克隆及其mRNA表达研究.硕士学位论文重庆：西南大学
71. 何林,谭仕禄,曹小芳,赵志模,邓新平,王进军.2003.朱砂叶螨的抗药性选育及其解毒酶活性研究.农药学学报5：23-29
72. Hughes PB, Raftos DA.2009. Genetics of an esterase associated with resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina (Wiedemann)(Diptera: Calliphoridae). Bulletin of Entomological Research 75:535-544
73. Guerrero FD, Nene VM.2008. Gene structure and expression of a pyrethroid-metabolizing esterase, CzEst9, from a pyrethroid resistant Mexican population of Rhipicephalus (Boophilus) microplus (Acari:Ixodidae). Journal of Medical Entomology 45:677-695
74. Newcomb RD, Campbell PM, Ollis DL, Cheah E, Russell RJ, Oakeshott JG.1997. A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly. Proceedings of the National Academy of Sciences of the United States of America 94:7464
75. Devonshire AL, Sawicki RM.1979. Insecticide-resistant Myzus persicae as an example of evolution by gene duplication. Nature 280:140-141
76. Field LM, Devonshire AL, Forde BG.1988. Molecular evidence that insecticide resistance in peach potato aphids (Myzus persicae Sulz) results from amplification of an esterase gene. Biochemical Journal 251:309-312
77. 乔传令,Hemingway J,李瑄.2003.有机磷抗性致倦库蚊种群中酯酶基因扩增的定量分析.昆虫学报 46：11-17
78. 郭惠琳,高希武.2005.棉蚜抗氧化乐果品系的羧酸酯酶基因突变.昆虫学报 48：194-202
79. Cao CW, Zhang J, Gao XW, Liang P, Guo HL.2008. Overexpression of carboxylesterase gene associated with organophosphorous insecticide resistance in cotton aphids, Aphis gossypii (Glover). Pesticide Biochemistry and Physiology 90:175-180
80. Baffi MA, de Souza GRL, de Sousa CS, Ceron CR, Bonetti AM.2008. Esterase enzymes involved in pyrethroid and organophosphate resistance in a Brazilian population of Riphicephallus (Boophilus) microplus (Acari, Ixodidae). Molecular and Biochemical Parasitology 160:70-73
81. Campbell PM, Newcomb RD, Russell RJ, Oakeshott JG.1998. Two different amino acid substitutions in the ali-esterase, E3, confer alternative types of organophosphorus insecticide resistance in the sheep blowfly, Lucilia cuprina. Insect Biochemistry and Molecular Biology 28: 139-150
82. Zhu YC, Dowdy AK, Baker JE. 1999. Differential mRNA expression levels and gene sequences of a putative carboxylesterase-like enzyme from two strains of the parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae). Insect Biochemistry and Molecular Biology 29: 417-425
83. Claudianos C, Russell RJ, Oakeshott JG.1999. The same amino acid substitution in orthologous esterases confers organophosphate resistance on the house fly and a blowfly. Insect Biochemistry and Molecular Biology 29:675-686
84. Pan YO, Guo HL, Gao XW.2009. Carboxylesterase activity, cDNA sequence, and gene expression in malathion susceptible and resistant strains of the cotton aphid, Aphis gossypii. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology 152: 266-270
85. 高贵田,陈克平,姚勤,王林玲,陈慧卿.2006.家蚕羧酸酯酶基因克隆及差异表达,.昆虫学报49：930-937
86. Hernandez R, He H, Chen AC, Waghela SD, Wayne Ivie G, George JE, Gale Wagner G.2000. Identification of a point mutation in an esterase gene in different populations of the southern cattle tick, Boophilus microplus. Insect Biochemistry and Molecular Biology 30: 969-977
87. Hernandez R, Guerrero F, George J, Wagner G.2002. Allele frequency and gene expression of a putative carboxylesterase-encoding gene in a pyrethroid resistant strain of the tick Boophilus microplus. Insect Biochemistry and Molecular Biology 32:1009-1016
88. Baffi MA, de Souza GRL, Vieira CU, de Sousa CS, Gourlart LR, Bonetti AM.2007. Identification of point mutations in a putative carboxylesterase and their association with acaricide resistance in Rhipicephalus (Boophilus) microplus (Acari:Ixodidae). Veterinary parasitology 148:301-309
89. 曹小芳,何林,赵志模,邓新平,王进军.2004.朱砂叶螨不同抗性品系酯酶同工酶研究.蛛形学报13：95-102
90. 何林,谭仕禄,曹小芳,赵志模,邓新平,王进军.2003.朱砂叶螨的抗药性选育及其解毒酶活性研究.农药学学报5：23-29
91. Ran C, Chen Y, Wang JJ.2009. Susceptibility and carboxylesterase activity of five field populations of Panonychus citri (McGregor)(Acari:Tetranychidae) to four acaricides. International Journal of Acarology 35:115-121
92. Chen AC, He H, Temeyer KB, Jones S, Green P, Barker SC.2009. A survey of Rhipicephalus microplus populations for mutations associated with pyrethroid resistance. Journal of Economic Entomology 102:373-380
93. 冉春,陈洋,袁明龙,王进军,刘浩强,姚廷山.2008.桔全爪螨田间种群对杀螨剂的敏感性.植物保护学报35：537-540
94. 洪源范,洪青,沈雨佳,李顺鹏.2007.甲氰菊酯降解菌Sphingomonas sp.JQL4-5对污染土壤的生物修复.环境科学 28 1121-1125
95. 孟和生.2002.两种生物方法对杀螨剂毒力测定结果的影响比较.植物保护28：49-51
96. 丁岩钦.1994.昆虫数学生态学.北京：科学出版社：153-169
97. 沈慧敏,张新虎.2002.三氟氯氰菊酯和甲氰菊酯对二点叶螨生命活力及繁殖的影响.植物保护学报29：183-188
98. 李定旭,田娟,沈佐锐.2006.不同药剂对山楂叶螨的亚致死效应.植物保护学报33：
187-192
99. 高宗仁,李巧丝,刘孝纯,等.1991.杀虫剂对朱砂叶螨某些生物学特性的影响.植物保护学报18：283-287
100. Gerson U, Cohen E.1989. Resurgences of spider mites (Acari:Tetranychidae) induced by synthetic pyrethroids. Experimental and Applied Acarology 6: 29-46
101. 陈道茂,陈卫民.1990.二种拟除虫菊酯对桔全爪螨繁殖的影响.植物保护学报17：279-282
102. Luckey TD.1968. Insecticide hormoligosis. Journal of Economic Entomology 61:7-12
103. 高君晓,梁沛,宋敦伦,高希武.2008.高效氯氰菊酯亚致死剂量对大豆蚜实验种群的影响.植物保护学报35：379-380
104. Kolar L, Erzen, NK, Hogerwerf L, van Gestel CAM.2008. Toxicity of abamectin and doramectin to soil invertebrates. Environmental Pollution 151:182-189
105. Flores AE, Landeros J, Badii MH.2000. Evaluation of population parameters of Tetranychus urticae Koch (Acari:Prostigmata: Tetranyciudae) exposed to avermectin. Southwestern Entomologist 25:287-293
106. Landeros J, Mora N, Badii M, Cerda PA, Flores AE. 2002. Effect of sublethal concentrations of avermectin on population parameter of Tetranychus urticae (Acari:Tetranychidae) on strawberry. Southwestern Entomologist 27:283-289
107. Marcic D, Sekulic D.1998. Effects of acaricide resistance on life history parameters of Tetranychus urticae Koch (Acari:Tetranychidae). Pesticides 13:13-46
108. 唐振华,毕强.2003.杀虫剂作用的分子行为：上海：上海远东出版社.100-250.
109. Rumpf S, Hetzel F, Frampton C.1997. Lacewings (Neuroptera:Hemerobiidae and Chrysopidae) and integrated pest management: enzyme activity as biomarker of sublethal insecticide exposure. Journal of Economic Entomology 90:102-108
110. Bradford MM.1976. A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye release. Anal Biochem 72:248-254
111. Clark AG, Dick GL, Smith JN.1984. Kinetic studies on a glutathione S-transferase from the larvae of Costelytra zealandica. Biochemical Journal 217:51-58
112. Habig WH, Pabst MJ, Jakoby WB.1974. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. The Journal of Biological Chemistry 249:7130
113. Kim YJ, Lee SH, Lee SW, Ahn YJ.2004. Fenpyroximate resistance in Tetranychus urticae Acari: Tetranychidae:cross-resistance and biochemical resistance mechanisms. Pest Management Science 60:1001-1006
114. 尹显慧,吴青君,李学锋,张友军,徐宝云.2008.多杀菌素亚致死浓度对小菜蛾解毒酶系活力的影响.农药学学报10：28-34
115. 梁沛,夏冰,石泰,高希武.2003.阿维菌素和高效氯氰菊酯亚致死剂量对小菜蛾谷胱甘肽S-转移酶的影响.中国农业大学学报8：65-68
116. 魏波,郝赤,李会仙.2006.杀虫剂亚致死剂量对棉铃虫靶标酶的影响.现代农药5：35-38
117. 高希武,董向丽.1997.棉铃虫的谷胱甘肽S-转移酶(GSTs)：杀虫药剂和植物次生性物质的诱导与GSTs对杀虫药剂的代谢.昆虫学报40：122-127
118. 魏辉,李洪山,戴华国,王娟.2006.多杀菌素对小菜蛾体内多功能氧化酶和保幼激素酯酶活力的影响.农药学学报8：239-244
119. 茹李军,范贤林.1997.棉铃虫对拟除虫菊酯类杀虫剂抗性遗传力的分析.植物保护学报24：356-360
120. Tabashnik BE.1992. Resistance risk assessment: realized heritability of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae), tobacco budworm (Lepidoptera: Noctuidae), and Colorado potato beetle (Coleoptera: Chrysomelidae). Journal of Economic Entomology 85:1551-1559
121. 林荣寿,邓新平.1994.四川省柑桔害螨抗药性监测与综合治理.西南农业学报7：75-81
122. Cochran DG.1989. Monitoring for insecticide resistance in field-collected strains of the German cockroach (Dictyoptera: Blattellidae). Journal of Economic Entomology 82:336-341
123. Firko MJ, HAyes JL.1990. Quantification of larval resistance to cypermethrin in tobacco budworm (Lepidoptera: Noctuidae) and the effects of larval weight. Journal of Economic Entomology 83:1222-1228
124. Bloch G, Wool D.1994. Methidathion resistance in the sweetpotato whitefly (Aleyrodidae: Homoptera) in Israel: selection, heritability, and correlated changes of esterase activity. Journal of Economic Entomology 87:1147-1156
125. 韩启发,庄佩君,唐振华.1995.抗杀螟硫磷二化螟的抗性遗传力研究.昆虫学报38：402-406
126. 兰亦全,赵士熙,吴刚.2006.甜菜夜蛾对三种拟除虫菊酯类杀虫剂的抗性遗传力及风险评估.应用生态学报17：468-471
127. 贾变桃,沈晋良,刘叙杆.2007.甜菜夜蛾对虫酰肼的抗性选育,风险评估及交互抗性.昆虫学报50：1116-1121
128. 牛洪涛,罗万春,宗建平,魏书娟,王海迎,潘志孝.2008.小菜蛾对丁烯氟虫腈的抗性遗传力及风险评估.植物保护学报35：165-168
129. 莫建初,唐振华.1997.数量遗传学在害虫种群抗性进化研究中的应用.昆虫知识34：183-186
130. 唐振华.2000.我国昆虫抗药性研究的现状及展望.昆虫知识37：97-103
131. 许雄山,韩召军.1999.增效醚对棉铃虫的毒理学研究.农药学学报 1：42-46
132. He L, Xue CH, Wang JJ, Li M, Lu WC Zhao ZM.2009. Resistance selection and biochemical mechanism of resistance to two Acaricides in Tetranychus cinnabarinus (Boiduval). Pesticide Biochemistry and Physiology 93:47-52
133. 何林.2003.朱砂叶螨抗药性机理及抗性适合度研究.博士学位论文重庆：西南大学
134. 范志金,陈年春.1996.截形叶螨抗药性主导机制的研究.植物保护学报23：175-180
135. 王利华,吴益东.2008.kdr突变和解毒代谢在B型烟粉虱对高效氯氰菊酯抗性中的作用.昆虫学报51：277-283
136. 孙耘芹,冯国蕾,袁家珪,祝平,龚坤元.1987.棉蚜对有机磷杀虫剂抗性的生化机理.昆虫学报30：13-20
137. 冉春,陈志永,陈洋,王进军.2007.桔全爪螨田间种群对双甲脒的敏感性及其两种酶的生化特性.中国农学通报23：284-288
138. 陈巧云,姜家良,林国芳,刘维德.1980.淡色库蚊对敌百虫抗性的研究——水解酶同敌百虫抗性关系.昆虫学报23：350-357
139. Senthil Nathan S, Kalaivani K, Chung PG.2005. The effects of azadirachtin and nucleopolyhedrovirus on midgut enzymatic profile of Spodoptera litura Fab.(Lepidoptera: Noctuidae). Pesticide Biochemistry and Physiology 83:46-57
140. Ren Y, Wei XP, Wu S, Dou W, Wang JJ.2008. Comparison of acetylcholinesterase from three field populations of Liposcelis paeta Pearman (Psocoptera: Liposcelididae):Implications of insecticide resistance. Pesticide Biochemistry and Physiology 90:196-202
141. Ellman GL, Courtney KD.1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical pharmacology 7:88-90
142. Wilkinson GN.1961. Statistical estimations in enzyme kinetics. Biochemical Journal 80:324
143. Chai YX, Liu GY, Wang JJ.2007. Toxicological and biochemical characterizations of AChE in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae). Pesticide Biochemistry and Physiology 88:197-202
144. 王新国,王怀位,甄天民,江洪涛,孙传红,赵玉强,程鹏,王海防.2005.淡色库蚊三种抗性品系抗性发展与乙酰胆碱酯酶相关性的研究.中国媒介生物学及控制杂志 16：264-266
145. 杨帆,帅霞.2008.桃蚜高效氯氰菊酯抗药性与乙酰胆碱酯酶的关系.植物保护34：60-62
146. Rauch N, Nauen R.2004. Characterization and molecular cloning of a glutathione S-transferase from the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae). Insect Biochemistry and Molecular Biology 34:321-329
147. 豆威.2009.书虱谷胱甘肽S-转移酶的纯化及生化毒理学特性研究.博十学位论文重庆：西南大学
148. 孙耘芹,袁家圭.1990.家蝇对拟除虫菊酯农药的抗性机制.昆虫学报33：265-273
149. 韩启发,庄佩君.1995.二化螟对杀螟硫磷产生抗性的机理.昆虫学报38：266-271
150. 冷欣大,唐振华,王荫长.1996.杀虫药剂分子毒理学及昆虫抗药性.北京：农业出版社47-89
151. 邱立红,李学锋.1999.抗溴氰菊酯家蝇在不同用药方式下的敏感性变化及其机制.昆虫学报42：248-256
152. 刘永杰,沈晋良.2003.甜菜夜蛾抗氯氟氰菊酯品系相对适合度,抗性生化机理及抗性遗传方式.昆虫学报46：567-572
153. 沈同,王镜岩.1991.生物化学(第二版).北京：高等教育出版社：103-107
1 54. 李宏.1996.ATPase的研究进展.生物学杂志 13：9-12
155. 祁鸣,薛京伦.1986.Na+,K+-ATP酶的特性研究及其应用.生物化学与生物物理进展13：22-26
156. 兰亦全,赵十熙.2004.甜菜夜蛾抗药性监测及机理.福建农林大学学报：自然科学版33：26-29
157. 冯北元,徐慕禹.1981.大鼠脑突触体Na+-K+-ATPase活力的微量测定方法.生物化学与生物物理学进展2：48-49
158. 张钧,邓蜀李,刘扬,黄叔怀.2003.运动对大鼠心肌线粒体功能的影响.北京体育大学学报26：198-200
159. 程伟霞.2007.嗜卷书虱和嗜虫书虱比较毒理学研究.博士学位论文重庆：西南大学
160. Feng GL, Marion JR., Clark JM.1992. Suppression of pyrethroid-dependent neurotransmitter release from synaptosomes of knockdown-resistant house flies under pulsed-depolarization conditions during continuous perfusion. Pesticide Biochemistry and Physiology 42:64-77
161. Sarkar PK.2002. A quick assay for Na+-K+-ATPase specific activity. Zeitschrift fur Naturforschung 57c:562-564
1 62. 何运转,李梅,冯国蕾,王荫长.1999.拟除虫菊酯对家蝇Na+-K+-ATPase抑制作用的研究.昆虫学报1：19-24
163. 蒋志胜,尚稚珍,王晓博,杨淑华.2000.美洲大蠊Na+-K+-ATPase作为筛选靶标的初步研究.农药学学报2：28-32
164. Li HP, Feng T, Liang P, Shi XY, Gao XW, Jiang H.2006. Effect of temperature on toxicity of pyrethroids and endosulfan, activity of mitochondrial Na+,K+-ATPase and Ca2+,Mg2*-ATPase in Chilo suppressalis (Walker)(Lepidoptera: Pyralidae). Pesticide Biochemistry and Physiology 86: 151-156
165. 伦才智,李艳伟,刘永杰,沈晋良,束怀瑞.2006.抗性甜菜夜蛾Na+-K+-ATP酶,Ca2+-ATP酶和Ca2+-Mg2+-ATP酶对高效氯氟氰菊酯的敏感性.农药学学报8：335-338
166. 蒋志胜,颜增光,何佳,杜育哲,尚稚珍.2003.典型光活化毒素α-三噻吩对棉铃虫和亚洲玉米螟Na+-K+-ATPase的抑制作用.农药学学报5：47-52
167. 田雨,冷欣夫.1999.溴氰菊酯对不同品系家蝇脑突触体膜蛋白磷酸化及ATP酶活性的影响.昆虫学报42：113-119
168. 李海平,冯涛,陶岭梅,刘学,姜辉,林荣华,梁沛,高希武.2006.九种常用杀虫剂对二化螟线粒体ATPase活力的抑制作用.昆虫学报49：254-259
169. 曾晓P.1995.杀虫剂对家蝇线粒体ATP酶活性影响的研究.中国媒介生物学及控制杂志6：253-256
170. 郭志波,马志卿,冯俊涛,张兴.2008.松油烯4-醇及其酯类衍生物对家蝇Na+,K+-ATPase活性的抑制作用.中国农业科学41：3110-3115
171. 马志卿,栾正春,张兴.2009.松油烯-4-醇对淡色库蚊的熏蒸毒力及对其Na+,K+-ATP酶的抑制作用.农药学学报11：230-234
172. 马志卿,冯俊涛,黄石周,张兴.2004.松油烯-4-醇对家蝇体内几种酶系的影响.植物保护学报31：283-288
173. 蒋志胜,白淑萍,尚稚珍.2002.具保幼激素活性的昆虫生长调节剂对亚洲玉米螟Na+-K+-ATPase的影响初探.南开大学学报(自然科学版)35：76-79
174. 宋宏图.2007.野桑蚕羧酸酯酶基因的克隆及在大肠杆菌中的表达.硕士学位论文江苏：江苏大学
175. 李飞.2003.棉蚜的杀虫剂神经靶标分子生物学研究.博士学位论文南京：南京农业大学
176. Field LM, Devonshire AL.1998. Evidence that the E4 and FE4 esterase genes responsible for insecticide resistance in the aphid Myzus persicae (Sulzer) are part of a gene family. Biochemical Journal 330:169
177. Vaughan C, Nemeth NM, Cary J, Temple S.2005. Response of a Scarlet Macaw Ara macao population to conservation practices in Costa Rica. Bird Conservation International 15:119-130
178. Vontas JG, Small GJ, Hemingway J.2000. Comparison of esterase gene amplification, gene expression and esterase activity in insecticide susceptible and resistant strains of the brown planthopper, Nilaparvata lugens (Stal). Insect Molecular Biology 9: 655-660
179. 曲明静,许新军,韩召军,陈茂华.2007.酯酶基因扩增及突变与昆虫抗药性.昆虫知识44：29-31
180. 唐振华.1993.昆虫抗药性中的酯酶基因扩增研究进展.昆虫知识30：53-56
181. Jones CD, Yeung C, Zehnder JL.2003. Comprenensive validation of a realtime quantitative bcrabl assay for clinical laboratory use. American Journal of Clinical Pathology 120:42-48
182. Livak KJ, Schmittgen TD.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods 25:402-408
183. Cao CW, Zhang J, Gao XW, Liang P, Guo HL.2008. Overexpression of carboxylesterase gene associated with organophosphorous insecticide resistance in cotton aphids, Aphis gossypii (Glover). Pesticide Biochemistry and Physiology 90:175-180
184. Furlong J, Meighan M, Conner J, Murray J, Clements, JB.1992. Methods for Improved Protein Expression Using Pet Vectors. Nucleic Acids Reseach 20:4668-4673