【作者】 何恒果；

【导师】 赵志模； 王进军；

【作者基本信息】 西南大学 ， 农业昆虫与害虫防治， 2010， 博士

【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。更多还原

【Abstract】 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. citri1.1 Effects of fenpropathrin with sublethal concentration on development and reproduction of P. citriThe 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. citriThe 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. citriAfter 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. citriAfter 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 acaricides3.1 Synergism effects of synergists on fenpropathrin and avermectinThe 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. citriCompared 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. citriCompared 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. citriAmong 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. citriThe 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. citriCompared 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. citri4.1 Molecular cloning and sequence analysis of esterase gene in P. citriThe 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. citriThe 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. citriApplying 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.更多还原