【作者】 张金振；

【导师】 沈晋良；

【作者基本信息】 南京农业大学 ， 农药学， 2004， 硕士

【摘要】 确定了酶标动力学法测定乙酰胆碱酯酶活性的最佳反应体系为：pH为7.4（0.02mol／L）的磷酸缓冲液，0.4mmol／L的DTNB和2mmol／L的ATChI。酶终点法测定AChE的最佳反应体系为：0.15mmol／L的DTNB乙醇溶液，6mmol／L的ATChI，2头．ml^-1的酶液，反应时间10min。 采用药膜法比较了Org、JS、NJ等3个不同家蝇种群分别对甲胺磷、二嗪农、久效磷、灭多威、呋喃丹、甲基对硫磷等6种杀虫剂的敏感性，结果表明Org品种对除二嗪农以外的5种杀虫剂均比JS敏感，而NJ种群最不敏感；同时采用酶动力学法比较了这3种不同家蝇种群头部乙酰胆碱酯酶（AChE）对甲胺磷和灭多威的敏感性，结果发现Org品种的头部AChE对灭多威和甲胺磷最为敏感，其次是JS，最不敏感的是NJ种群。这说明家蝇对有机磷和氨基甲酸酯类杀虫剂的敏感性与其头部AChE对这两种药剂的敏感性具有相关性，因此，通过选育对这两类药剂更加敏感的家蝇品系可以提高其AChE对药剂的敏感性。由于Org种群相对其它种群更加敏感，故以该种群的家蝇为亲本能相对快速选育出更加敏感的家蝇品系，从而用作提取敏感AChE的酶源。 以Org家蝇种群为亲本，进行单对选育4代后发现敏感单对家蝇后代的毒力与其原始亲本种群（Org）相比，其LC₅₀虽然有所下降，但其置信区间重叠；测定甲胺磷和灭多威对选育的家蝇品系及原始品系乙酰胆碱酯酶的抑制中浓度(IC₅₀)发现单对选育并没有使家蝇头部乙酰胆碱酯酶对药剂的敏感性有显著提高，说明Org家蝇种群已经对甲胺磷和灭多威这两种药剂非常敏感，通过单对选育很难使其敏感性发生显著变化。 家蝇羽化后，其头部AChE对药剂的敏感性随羽化后天数的不同而变化，且对不同药剂敏感性变化趋势不同。但家蝇羽化后第3d其头部的AChE对甲胺磷和灭多威两种药剂敏感性畏高，其IC₅₀分别为3.001±0.031 μg／ml和0.319±0.003 μg／ml。在家蝇羽化第3d，不同部位AChE的活性及对不同药剂的敏感性差异较大，但以头部AChE活性最高，且对甲胺磷和灭多威均最为敏感。因此以羽化3d家蝇头部AChE作为酶源最佳。 不同药剂对乙酰胆碱酯酶的抑制率受抑制时间和温度的影响程度不同。灭多威对乙酰胆碱酯酶的抑制率受时间的影响不大，在抑制30min后，抑制率才开始明显下降；应用家蝇乙酞胆碱酷酶进行农药残留快速检测技术研究而甲胺磷对乙酞胆碱醋酶的抑制率则随着抑制时间延长而增加。灭多威对乙酞胆碱醋酶的抑制率随温度的上升而显著降低，甲胺磷对酶的抑制率在25℃一35℃时，随温度的增加而增加，在35℃时达到最大值，而后开始下降。由于在农药残留测定时要求快速且检测限低，常用高毒农药又以有机磷类为主，因此选用的抑制时间为巧min，温度为35℃。 在相同测定条件下，加入相同浓度不同体积的农药对AChE的抑制程度也有一定的影响，在体积较小时影响不大，但当体积相差较大时，如o.lml和4ml，两者对AChE的抑制率相差在10%以上。考虑到实际检测时样本提取液的体积，建议加药量为3ml。 速测仪和酶终点法1测定市售酶粉（BuchE）与自制粗酶液（AChE）对杀虫剂的敏感性和检出限的结果并不一致。研究发现，速测仪测定市售酶粉对甲胺磷和灭多威的敏感性大于自制粗酶液;甲胺磷对自制粗酶液检出限小于其对市售酶粉，灭多威对自制粗酶液的检出限仅为市售酶粉的1 .06倍。而酶终点法测得甲胺磷对市售酶粉的检出限低于自制粗酶液;灭多威对市售酶粉的敏感性低于自制粗酶液，检出限高于自制粗酶液。 采用3种方法测定了家蝇头部乙酞胆碱醋酶对水溶液和青菜中杀虫剂残留的检出限。从结果来看，酶动力学法最为理想，测得甲胺磷和灭多威在水溶液的检出限分别为0.502件g/ml和0.040林g/ml，在青菜中分别为7.452抖g/ml和0.151协g/ml。其中青菜中甲胺磷的检出限是水溶液中的9.29倍，灭多威是水溶液中的378倍。 将家蝇乙酞胆碱醋酶制成冻干粉在4℃下保存130d后，测得酶活性为1625士0.031（O D.mg一1·min一l），比初提取的粗酶液活性下降了12.9%，与保存第ld的酶活性相比，下降了8.6%。更多还原

【Abstract】 To detection the activity of acetylcholinesterase of housefly (Musca domestica), two methods were used, when the kinetic method was used, the optimum condition was: 0.02mol/L phosphate buffer with pH value of 7.4, 0.4 mmol/L 5,5’ -dithiobis-(2-nitrobenzoic acid)(DTNB), 2mmol/L Achl. And when the terminal condition was: 0.02mol/L phosphate buffer with pH value of 7.4, 0.15mmol/LDTNB ehyl alchol solution, 6mmol/L AChI, 2 head/ml enzyme solution and react for 1 Omin.The sensitivity of three Musca domestica populations (Org, JS and NJ) to four organophosphrous and two carbamate insecticides were determined by glass vial bioassay method. The results showed that among the three populations, the laboratory population Org was most sensitive to all the insecticides tested except diazion, the NJ population was most insensitive. The sensitivity of AChE to methamidophos and methomyl were also tested, the results indicated that the AChE of Org was also more sensitive to the two insecticides than those of other two populations. These results demonstrated that the sensitivity of Musca domestica to organophosphates and carbamates determined by traditional bioassay method were closely correlated with the sensitivity of their AChE. Therefore, the sensitivity of AChE could be improved by breeding more sensitive housefly in laboratory. The preparation of susceptible housefly maybe speed up by selecting the parents from Org population.The susceptible population was selected 4 generations in this study. Comparing to the origin parent housefly, the LC50 values of the offspring to methamidophos and methomyl decreased, but the confidence limits overlapped. The IC50 of methamidophos and methomyl to AChE in the selected strains and original parent houseflies were also conducted. The results showed that the sensitivity of AChE weren’ t improved significantly by single pair selection method, which is consistent to the bioassay results, that suggested that the Org population was very sensitive to methamidophos and methomyl, and it was difficult to get more sensitive offsprings by the traditional selection of single pair selection method.The sensitivities of AChE in different old housefly were also studied, the resultsshowed that the sensitivity of AChE to methamidophos and methomyl was most sensitive in the third day after emergence. The IC50 of methamidophos and methomyl to the AChE was 3.001 +0.03 lug/ml and 0.319+0.003ug/ml respectively. We also found that the specific activity of AChE in the head part was higher than those in other parts of the housefly. So AChE in the head part of 3-days old of housefly was used for pesticides residue determination.The inhibition rate of pesticides to AChE were determined under different temperature and reaction time. The results showed that the inhibition rate of methomyl to AChE was changed evidently with the inhibition time increased and it began to decrease after inhibition for 30s. While the inhibition rate of methamidophos increased all the time. The inhibition rate of methomyl decreased as the temperature going up, but the inhibition rate of methamidophos increased at the range of 25C to 35C, and reached the highest at 35C. Because the rapid and lower limit were required for detection the pesticide residue, and the organophosphate were maily used toxic pesticides, so the inhibition time and temperature were defined as 15 min and 35C respectively.The study revealed that the inhibition rate increased as the volume of pesticides increased, and when the volume was 0.4ml, the inhibition rate was ten percent more than that of 0.1 ml.The sensitivity and detection limit of commercial enzyme powdery and extracted crude liquid enzyme were compared by the rapid detection instrument and terminal method. Commercial enzyme powdery were more sensitive to methamidophos and methomyl than crude liquid enzyme, comparing with the commercial enzyme powdery, the detection limit of crude liquid to methamidophos was higher, to Methomyl, which was only 1.06 fold when the rapid detection instrument更多还原