【作者】 陈志永；

【导师】 王进军；

【作者基本信息】 西南大学 ， 农药学， 2007， 硕士

【摘要】 本研究以桔全爪螨为对象，综合运用农药生物测定、抗性选育、生化分析、分子生物学等技术，在室内对其进行辛硫磷抗性选育的基础上，对产生抗性的品系进行抗药性生理生化机制研究，旨在延长有机磷类杀螨剂的使用时间，为该螨抗药性的治理提供有效的策略。此外，利用反转录多聚酶链式反应（RT-PCR）技术对桔全爪螨AChE cDNA片段进行扩增，并克隆测序，为获取桔全爪螨AChE基因全序列进而研究其变构AChE介导抗性的分子机制奠定基础。经过近两年的研究工作，获得了以下主要研究结果：1桔全爪螨对辛硫磷的抗性选育室内对桔全爪螨进行辛硫磷的抗性选育。随着抗性选育代数的增加，抗性指数逐渐增高。选育到第14代(F₁₄)，抗性指数达18.6倍。抗性选育过程中LD-p回归方程的斜率有变小的趋势，表明桔全爪螨群体中抗性个体不稳定，群体发生了异质性变化，抗性有进一步增强的潜力。2桔全爪螨辛硫磷抗性机理研究2．1桔全爪螨辛硫磷抗性与乙酰胆碱酯酶（AChE）的关系采用酶标仪技术对桔全爪螨辛硫磷敏感和抗性品系体内的AChE活性进行了测定，结果显示抗性品系和敏感品系体内的AChE比活力分别为4.3435和1.1399μmol／μg·mg／pro，差异达显著水平。而且抗性品系的Km值显著增大，Vm显著减小，表明该酶发生了质变，导致酶和底物的结合能力显著降低，桔全爪螨对辛硫磷存在着明显的靶标抗性。离体抑制作用测定结果表明，随着辛硫磷浓度升高，辛硫磷对桔全爪螨敏感和抗性品系AChE活性的抑制作用愈加明显，且对酶活性的抑制率与药剂浓度间存在明显的线性关系。2．2桔全爪螨辛硫磷抗性与羧酸酯酶（CarE）的关系对桔全爪螨辛硫磷敏感和抗性品系体内的CarE活性测定结果表明，敏感品系和抗性品系的CarE比活力分别为1.1399和4.3435μmol／μg·mg／pro，差异达显著水平。抗性品系的Km显著的高于抗性品系，说明该品系CarE对底物的亲和力下降。离体抑制作用测定结果表明，随着辛硫磷的浓度升高，辛硫磷对敏感品系和抗性品系体内CarE的抑制率均增加，且对敏感品系的抑制作用比抗性品系显。表明CarE活性的增强是桔全爪螨对辛硫磷产生抗药性的因素之一。2．3桔全爪螨对辛硫磷抗性与磷酸酯酶的关系对桔全爪螨辛硫磷敏感和抗性品系体内磷酸酯酶活性的测定结果表明，抗性品系和敏感品系酸性磷酸酯酶比活力分别为3.85105和5.5680μmol／mg·pro／30min，差异达显著水平；碱性磷酸酯酶的活性分别为3.0145和3.0968μmol／mg·pro／30min，差异不显著。表明桔全爪螨对辛硫磷抗性的形成与酸性磷酸酯酶活性增强有关，而与碱性磷酸酯酶的关联不大。2．4桔全爪螨对辛硫磷抗性与三种保护酶的关系对桔全爪螨辛硫磷敏感和抗性品系体内三种保护酶进行了比较。抗性品系和敏感品系体内超氧化物歧化酶SOD的比活力分别为0.1714和0.0810μnit／10mite／15min；过氧化酶POD的比活力分别为1.1367和0.8517mmol H₂O₂／mg．pro／min。抗性品系体内的SOD和POD比活力均显著高于敏感品系；抗性品系和敏感品系体内过氧化氢酶CAT的比活力分别为0.3098和0.3104μnit／mg．pro／min，差异不显著。说明桔全爪螨对辛硫磷的抗性与SOD和POD相关，而与CAT关联不大。2．5桔全爪螨对辛硫磷抗性与谷胱甘肽S-转移酶（GSTs）的关系对桔全爪螨辛硫磷敏感和抗性品系体内GSTs活性的测定结果表明，抗性品系和敏感品系体内GSTs的比活力分别为0.5570和0.3751μmol／min·mg／pro，差异达显著水平。以GSH为底物，抗性品系K_m值显著大于敏感品系，而以CDNB为底物时抗性品系K_m值显著小于敏感品系。说明与敏感品系相比抗性品系对底物GSH的亲和力减弱，而对CDNB的亲和力增强。表明GSTs与桔全爪螨对辛硫磷的抗性有一定的相关性。2．6桔全爪螨对辛硫磷抗性与多功能氧化酶（MFO）的关系比较桔全爪螨辛硫磷敏感和抗性品系体内MFO的酶学特性及辛硫磷和增效醚（PBO）对该酶的抑制作用，结果表明，抗性品系和敏感品系体内MFO的比活力分别为17.82和4.324（30min·mgpro），差异达显著水平。抗性品系的K_m显著高于敏感品系。辛硫磷对敏感品系MFO的抑制效果显著强于抗性品系，且MFO活性抑制率与药剂抑制浓度之间呈显著的线性关系。以PBO作用于桔全爪螨，敏感品系和抗性品系MFO的活力均受到抑制，但对抗性品系的抑制效果强于敏感品系。说明桔全爪螨对辛硫磷的抗性形成与MFO活力的增强相关。3桔全爪螨乙酰胆碱酯酶的cDNA片段的克隆采用RT-PCR技术克隆获得了桔全爪螨AChE的1个151bp的片段，推导的氨基酸序列为：EMWNPNTNISEDCLYLNIWVPQRLRIRHHGDKPPQERPKVPVLVWIYGGG。同源性比较结果表明该序列与其他生物AChE相应氨基酸序列的同源性较高，而且该序列还含有AChE多个保守的特征性序列，说明该片段编码桔全爪螨AChE的部分氨基酸序列。更多还原

【Abstract】 The resistance mechanisms of Panonychus cirri （McGregor） to phoxim were investigated on the basis of resistance selection of the mite in laboratory experiment with a series of techniques such as bioassay, resistance selection, biochemical analysis, and molecular biology. This research would help to completely understand the physiological and biochemical mechanisms of acaricides resistance of the mite in the future, and also possess great practical significance in the formulation of strategies for the delay and management of resistance. In addition, a fragment of acetylcholinesterase （ACHE） from P. cirri was amplified with degenerate primers by reverse transcriptase-polymerase chain reaction （RT-PCR） method and it provided some basic information for the further study about the complete sequence of the gene and the molecular mechanism of altered ACHE. After about two years’ work, the main research results are summarized as follows.1 Resistance selection ofP. citri to phoximThe results of resistance selection of P. citri to phoxim indicated that the resistance level gradually rose with the increase of selection time and the resistance factors at the fourteenth generation amounted to 18.6-fold. In the process of resistance selection, there was a decreased tendency about the slope （b） value of LD-p lines, suggesting that P. citri developed obvious resistance to phoxim and resistance would further develop if the selection continue.2 Resistance mechanisms ofP. citri to phoxim2.1 Relationship between AChE and the resistance of P. citri to phoximCompared to the susceptible strain, the activity and specific activity of AChE from the resistant strain increased significantly. The results also showed that ACHE in the resistant strain possessed a significantly less affinity to the substrate acetylthiocholine iodide （ATChI）. For the catalytic activity of AChE towards ATChI, the V_max value of the resistant strain decreased significantly compared to that from susceptible strain.The in vitro inhibition kinetics of phoxim on AChE activity showed that there was a strong linear relationship between inhibiting action and AChE activity from the two strains. The results suggested that there is target resistance in P. citri to phoxim.2.2 Relationship between Carboxylesterase （CarE） and the resistance of P. citri to phoximThe results showed that the susceptible and resistant strains differed significantly in the amount of protein per individual, and higher activity and specific activity of Care from the resistant strain were observed. For the catalytic activity of CarE towards the substrate, the K_m value of the resistant strain decreased significantly compared to that from susceptible strain.The inhibition of phoxim to CarE activity showed that CarE in the susceptible strain was more sensitive and it suggested that the change of Care activity in the resistant strain was an important reason for the resistance of P. citri to phoxim.2.3 Relationship between phosphatases （ACP,ALP） and the resistance of P. cirri to phoximThe activity and specific activity of ACP from the resistant strain were significantly higher than its susceptible counterpart. Although the activity and specific activity of ALP in the resistant strain were higher, no significant difference was observed.2.4 Relationship between SOD, POD, or CAT and the resistance of P. citri to phoximCompared to those of the susceptible strain, the specific activity of both SOD and POD from the resistant strain increased significantly while there was no significant difference of the specific activity of CAT between the two strains. These suggested that the resistance of P. citri to phoxim related to SOD and POD, and maybe have no relation with CAT.2.5 Relationship between GSTs and the resistance of P. citri to phoximThe GSTs activities of the resistant and susceptible strains were 0.55μmol/min.mg/pro and 0.3751μmol/min.mg/pro, respectively, and the difference was significant. The Km value of the resistant strain was significantly greater than that of the susceptible strain if GSH was used as substrate, while it was significantly smaller if CDNB was used as substrate. These suggested the affinity of GSTs from the resistant strain to GSH was less than that from the susceptible strain. All these implied that there was some relationship between the resistance of P. citri to phoxim and GSTs. 2.6 Relationship between MFO and the resistance of P. citriThe activities, specific activity and Km of MFO in the resistant strain were significantly higher than those in the susceptible strain, and the inhibition of phoxim to MFO was significantly greater in the susceptible strain. There was a significantly linear relationship between the inhibition rate of MFO activity and the inhibition concentration of phoxim. However, the inhibition of PBO to MFO in the resistant strain was significantly higher than that in the susceptible strain. The results suggested that the development of resistance to phoxim in P. citri might be related to the rise of the MFO activity.3 cDNA cloning of AChE in P. cirriA 151bp cDNA fragment of AChE gene in P. citri was amplified with the degenerate primers from the conserved peptide sequences of AChEs in insect and acari species by reverse transcription-polymerase chain reaction （RT-PCR） method, and the sequence analysis indicated there was a high degree of amino acid sequence homology between P. cirri and other species. In addition, it included several characteristic sequences of ACHE. Hence, it could be concluded that the fragment encoded the AChE in P. citri.更多还原