摘要
棉蚜(Aphis gossypii Glover)是世界性分布的重大农业害虫，生产上主要依赖于化学防治，这导致了棉蚜对多种杀虫剂产生了不同程度的抗药性。解毒代谢酶活力增强和杀虫剂靶标敏感性下降是棉蚜抗药性形成的重要机制，其中乙酰胆碱酯酶敏感性下降是棉蚜对有机磷和氨基甲酸酯类杀虫剂产生抗性的重要机制，而羧酸酯酶也在棉蚜对有机磷类杀虫剂的抗性中扮演着重要角色。因此，本文就抗性和敏感品系棉蚜乙酰胆碱酯酶和酯酶进行了一系列的比较研究。
1 棉蚜乙酰胆碱酯酶粗酶液稳定性研究
在抗药性研究中，经常利用粗酶液研究乙酰胆碱酯酶的生理毒理学特性。但棉蚜乙酰胆碱酯酶极易失活，因此对棉蚜乙酰胆碱酯酶粗酶液的稳定性进行研究，有助于获得可靠的结果和结论。本研究通过比较不同温度、以及加入甘油和牛血清白蛋白对酶活力的影响，分析了影响棉蚜乙酰胆碱酯酶粗酶液稳定性的重要因子。
研究结果显示，在4种不同温度下(-20℃、4℃、15℃、25℃)，随着环境温度的升高，棉蚜乙酰胆碱酯酶活性丧失速度加快，-20℃保存时，乙酰胆碱酯酶最为稳定，酶活力可以维持较长的时间。而在25℃下，乙酰胆碱酯酶最不稳定，活性丧失很快。一定浓度的甘油可提高乙酰胆碱酯酶在低温保存时的稳定性，但加入稳定剂牛血清白蛋白，对粗酶液中酶的稳定性并不能产生显著效果。由此认为，研究棉蚜乙酰胆碱酯酶的粗酶液，应保存在-20℃以下，并应加入一定量的甘油，以稳定酶活性，延长保存时间。
此外，本研究还发现，无论如何处理，酶液制备后的一段时间内，酶活力均呈现下降趋势，且不同品系棉蚜制备的乙酰胆碱酯酶粗酶液，其活力随时间的变化情况明显不同。因此建议，进行棉蚜乙酰胆碱酯酶活力的比较研究时，除需在-20℃下长期保存以外，还需要控制酶液保存时间的一致性。
2 不同抗性品系棉蚜的乙酰胆碱酯酶和羧酸酯酶比较
采用改进的棉蚜饲养方法，建立了不同抗性品系的棉蚜。通过测定来自不同地区(南京、北京和安阳)的抗性品系棉蚜乙酰胆碱酯酶对3种药剂(甲胺磷、乙酰甲胺磷和抗蚜威)的敏感性(I_(50)和K_i)，发现安阳品系棉蚜乙酰胆碱酯酶对杀虫剂的敏感性显著下降，而南京、北京品系则接近于敏感品系，但这3个抗性品系的全酯酶和羧
抗性和敏感性棉蚜的乙酞胆碱酷酶和酷酶的比较研究
酸醋酶的活性均显著高于敏感品系。分析认为，低抗水平的南京、北京品系只存在代
谢杭性，还未产生靶标抗性，而高杭的安阳品系，代谢抗性和靶标抗性均存在，导致
了高水平抗性的形成。
利用3种杀虫剂对不同品系棉蚜乙酞胆碱醋酶所做的抑制曲线，其结果与与和龙
的测定结果完全一致，但并没有发现因2种(或抗、感)乙酸胆碱醋酶的存在而出现
的“平台”。由此认为，利用抑制毒理曲线的“平台”作为鉴别有无2种酶的存在，
并不完全可靠。
此外，以a一乙酸蔡醋为底物时，棉蚜欺酸醋酶的活性显著高于以日一乙酸蔡醋
为底物时的活性，但不同品系没有发现顺序差异，说明棉蚜发酸醋酶对底物的空间构
型可能很敏感。
3不同品系棉蚜乙酞胆碱醋酶和梭酸醋酶亚细胞分布特征
通过差速离心法分析杭性品系和敏感品系棉蚜乙酞胆碱醋酶和数酸醋酶的亚细胞
分布，结果显示杭性品系和敏感品系棉蚜的乙酞胆碱醋酶和发酸醋酶的亚细胞分布均
存在显著差异。未用Tr 1 tonX一100处理时，抗性品系乙酞胆碱醋酶活性主要分布于细
胞核、细胞碎片及线粒体层中，而敏感品系乙酸胆碱醋酶活性主要分布于微粒体层中，
用Tr 1 tonx一100处理后，杭性品系和敏感品系乙酞胆碱醋酶均主要集中到上清液中。
由于微粒体可能包含了合成和分泌该酶的内质网和囊泡，而细胞核、细胞碎片及线粒
体的酶活性更能体现酶的膜结合状态，因此认为杭性品系棉蚜的乙酞胆碱醋酶主要以
膜结合蛋白形式存在，而敏感品系的主要以游离状态存在。
抗性品系欺酸醋酶活性主要分布在细胞核、细胞碎片及线粒体层中，敏感品系狡
酸醋酶活性则主要集中在上清液中，并且杭性品系各亚细胞组分的活性均显著高于敏
感品系。
显然，杭性品系和敏感品系的乙酞胆碱醋酶和致酸醋酶不仅存在着杭性水平的差
异，还存在着亚细胞分布上的差异，杭性品系的乙酸胆碱醋酶和戎酸醋酶具有显著的
亲膜特性。这种亲膜特性是否与抗药性直接相关，尚需要做进一步研究，但这一研究
结果表明，做抗、感棉蚜酶活力比较研究时，必须加入Tr 1 tonx一100等表面活性剂，
才能保证酶提取的一致性。
上述研究成果对于今后准确分析棉蚜乙酞胆碱醋酶和发酸醋酶的活力和特性，以
及对于不同抗性水平棉蚜的治理措施的设计，均具有重要的意义。
As a w orldwide-distributed p est, c otton a phid, Aphis g ossypii G lover, a ttracts 1 ots o f attention. The rapid development of resistance to insecticides has brought difficulty in the field control of this important pest. Acetylcholinesterase (AChE) is a target for organophosphate (OP) and carbamate insecticides. Insecticides resistance due to the insensitivity of acetylcholinesterase to the inhibition by these two groups of insecticides has been found in many insects including cotton aphid. Carboxylesterase (CarE) also plays an important role in organophosphate resistance of cotton aphid. In this paper, comparing studies on AChE and CarE from resistant and susceptible strains have been performed, including their stability, toxicology and sub-cellular distribution.
1 Study of stability of crude acetylcholinesterase from cotton aphid
The crude preparation of AChE is usually used in pest resistance study. However, AChE is not stable and easy to breakdown. Studies on the factors affecting the stability of the enzyme would be helpful for getting correct results in the further researches. So, series of experiments were carried out to demonstrate if and how temperature, glycerol and bovine serum albumin (BSA) affect the stability of the enzyme.
The results showed that the lower temperature when crude AChE preparation was stored at, the more stable it was. Glycerol could improve its stability effectively when AChE was stored at -20C. But BSA couldn't significantly improve the stability of the enzyme in crude preparations when stored at higher temperature. It was suggested that crude AChE preparations should be stored at -20C and with some glycerol.
AChE was also found having the tendency to lose its activity gradually short after it was extracted, and the stability of the crude AChE prepared from different strains, resistant and susceptible, were apparently different. So, the enzyme preparations stored for similar time should be used for comparing study of their activity.
2 Comparison of acetylcholinesterase and carboxylesterase from different aphid strains
With improved caging method of Li fei (2001), four laboratory strains of cotton aphid were set up. A susceptible strain 171B was introduced from Rothamsted Experiment Strains, UK. Three resistant strains were bred from the aphids collected from different districts in China, Nanjing, Beijing and Anyang. With the AChE preparations from these 4 strains, the
I50 and Ki of two OP and one carbamate insecticides were tested. Compared with that from 171B, the AChE from Anyang strain was found apparently insensitive to all the insecticides tested. But the sensitivity of AChE from Nanjing and Beijing strains were similar as that from 171B. However, the esterase (total esterase and carboxylesterase) activity increased dramatically in all the three strains. So, it could be deduced that pesticide resistance in Nanjing and Beijing strains only attributed to increased pesticide sequestration or detoxification by esterase, while the resistance in Anyang strain attributed to both increased esterase activity and reduced sensitivity of AChE.
The inhibition curve test was also carried out with the same insecticides and cotton aphid strains. The results showed the similar insensitivity of the AChE from different strains. But no flat on the curves was observed as usually expected when different enzymes present. So, it was thought it is not reliable to identify different enzymes with the flat on inhibition curves.
3 Subcellular distribution of acetylcholinesterase and carboxylesterase from different aphids strains
With differential centrifugation, subcellular distribution of AChE and CarE from susceptible strain (171B) and resistance strain (Anyang strain) were studied. Results showed that both AChE and CarE from susceptible strain and resistance strain were differently sub-cellular distributed. AChE activities in resistant strain were mainly distributed in cell nucleus, cell debris and mitochondrion. While AChE activities in susceptible strain were mainly
引文
1． 陈安良，冯俊涛，张兴．陕西棉蚜抗药性发展动态研究．西北农业大学学报，2000，28(1)：48-51．
2． 曹干．甘蔗不同基因型2种光合酶的热稳定性．福建农学院学报，1990，19(1)：19-22
3． 陈明，张新瑞，胡冠芳，等．20％丁硫克百威乳油防治棉田蚜虫试验研究[J]．农药，1996，35(4)：33-34．
4． 程桂林，刘润玺．新疆，山东棉蚜抗药性对比．农药，1997，36(11)：6-9．
5． 冯国蕾，李梅，赵章武，等．酶标板法监测棉蚜乙酰胆碱酯酶对杀虫剂的不敏感性．昆虫学报，2000，43(增刊)：32-37．
6． 冯俊涛，王兴林，杨崇珍，等．陕西棉花苗蚜对几种杀虫剂的抗药性测定[J]．西北农业大学学报，1996，24(3)：63-67．
7． 高希武，王政国，郑炳宗，等．1990．六种常用杀虫剂对八种蚜虫的选择性毒性．昆虫学报，33(3)：274-279．
8． 高希武，郑炳宗，梁同庭，等．杀虫剂混用或加增效剂对瓜—棉蚜增效作用及机制的研究．植物保护学报，1989，16(4)：273-278．
9． 高希武，郑润勇，宁世民，等．棉蚜不同品系羧酸酯酶的酶标仪动力学测定研究．中国农业大学学报，1997，2(5)：59-63．
10．高希武，周序国，王荣京等．棉铃虫乙酰胆碱酯酶(AChE)的体躯分布及部分纯化．昆虫学报，1998，41(增)：19-25．
11．韩招久．二化螟对杀虫单和甲胺磷的抗性机理及神经靶标乙酰胆碱受体的基因克隆．南京农业大学博士论文，2002．
12．韩召军，任晓霞，李飞，等．棉铃虫和棉蚜对有机磷靶标抗性的分子机理．第二届全国植物农药暨第六届药剂毒理学术会议论文集，2001，380-381．
13．韩召军，王荫长，Graham Moores，等．中国昆虫学会第六次全国代表大会暨学术讨论会论文摘要集，1997，10．
14．韩召军，张明，王荫长．五种蚜虫羧酸酯酶的活力和同工酶谱变异的初步研究．南京农业大学学报，1987，4(增刊)：31-35．
15．洪波，陈安良，冯俊涛，等．我国棉蚜抗药性研究现状．西北农业学报，2000，9(1)：118-122．
16．李飞，韩召军，吴智锋，王荫长．我国棉蚜抗药性研究进展．棉花学报，2001，Vol 13(2)：121-124
17． 李飞，韩召军，吴智锋．取食不同寄主棉蚜羧酸酯酶和乙酰胆碱酯酶特性的比较．南京农业大学学报，2002，25(2)：57-60．
18．李飞，韩召军．棉蚜2个乙酰胆碱酯酶cDNA片段的基因克隆与序列分析．动物学研究，2002，23(5)：444-448．
19．李飞，韩召军．棉蚜杀虫剂神经靶标的分子生物学研究．第六届全国昆虫学大会．2002，会议论文．
20．李飞，韩召军．棉蚜饲养技术—笼罩法．昆虫知识，2001，Vol38(3)：225-227．
21．李飞，韩召军．棉蚜羧酸酯酶同工酶cDNA片段的基因克隆和序列分析．棉花学报，2002，14(6)：336-339．
22．李莲，罗长才．饲用酶制剂中木聚糖酶酶学性质的研究．科技动态，2002，12：23-26．
23．李士根，刘永春，甄天民．蚊虫抗药性机制研究进展．中国媒介生物学及控制杂志，2001，12(1)：76-78．
24．李腾武，高希武，郑炳宗，等．小菜蛾不同亚细胞层羧酸酯酶的性质研究．农药学学报，1999，1(2)：47-53．
25．李卫芬，孙建义，顾赛红，等．里氏木酶β-葡聚糖酶稳定性研究．西南农业大学学报，2001，23(2)：97-99，
26．李显春，王荫长．昆虫抗药性靶标不敏感机制的研究进展．昆虫学报，1998，41(4)：417-425．
27．林忠莲，张立力．磷化氢对谷蠹和玉米象成虫体内乙酰胆碱酯酶的影响．郑州工程学院学报，2001，22(4)：35-41．
28．刘德明，杨秀芬，王树礼，等．辽宁棉区棉蚜抗药性监测与治理抗性研究．昆虫知识，1994，31(2)：81-85．
29．刘环宇，林森，梅德胜．酶在精细有机化工中的应用综述．江西化工，2003，9(3)：1-4．
30．刘泽文，韩召军，王荫长．褐飞虱抗有机磷品系的交互抗性及适合度研究．南京农业大学学报，2001，24(4)：37-40．
31．刘泽文，韩召军，张玲春，等．稻飞虱饲养与抗药性筛选的方法研究．中国水稻科学，2002，16(2)：167-170．
32．刘泽文，韩召军，张玲春．褐飞虱抗甲胺磷品系的交互抗性和抗性生化机制．昆虫学报，2002，45(4)：447-452．
33．罗万春，凌冰，岳留强，等．新疆棉蚜抗药性研究．植物保护学报，1 990，17(3)：283-288
34．罗万春，凌冰，岳留强，等．新疆棉蚜抗药性研究[J]．植物保护学报，1990，17(3)：283-288．
35．潘江球，刘坚，李思东，等．Polyacrylamide对菠萝蛋白酶活力的稳定作用．热带作物学报，2002，23(4)：29-31．
36．彭宇．二化螟对甲胺磷的抗性及AchE的研究．博士后研究工作报告，2000，62～70．
37．任晓霞，韩召军，王荫长．棉铃虫乙酰胆碱酯酶cDNA片段的克隆和序列分析．动物学报，2002，48(1)：121-124．
38．任晓霞，韩召军，王荫长．对久效磷抗性品系的选育及其乙酰胆碱酯酶的研究．南京农业大学学报，2001，24(1)：47-50．
39．任晓霞．棉铃虫对有机磷杀虫剂靶标抗性机制的研究．南京农业大学博士论文，2002．
40．沈晋良，韩召军，尤子平．南方棉蚜抗药性监测．南京农业大学学报．1987，4(增)：1-12．
41．沈晋良，吴益东．棉铃虫抗药性及其治理．北京：中国农业出版社，1997．
42．施明安，袁建忠，唐振华．乙酰胆碱酯酶的晶体结构及功能位点．农药学学报，2000，2(3)：1-7．
43．石键，崔光先，郑港庆．河北棉区棉蚜对内吸磷抗药性的调查．昆虫知识，1965，2(6)：329-330．
44．孙鲁娟，高希武，郑炳宗．棉蚜抗氧化乐果品系及敏感品系羧酸酯酶性质的比较．昆虫学报，2002，45(6)：724-727．
45．孙耘芹，冯国蕾，袁家圭，等．棉蚜对有机磷杀虫剂抗性的生化机理．昆虫学报，1987，30(1)：13-19．
46．孙中涛，闫艳春．蚊虫抗药性及酯酶基因扩增研究进展．山东农业大学学报，2001，32(3)：381-385．
47．唐振华，吴士雄．昆虫抗药性的遗传与进化．上海：上海科学技术文献出版社2000．
48．唐振华．昆虫抗药性及其治理．北京：农业出版社 1993．
49．王建军．小菜蛾靶标抗性研究．南京农业大学博士论文，2001．
50．王金信，刘峰，慕卫，等．几类农药防治棉蚜的药效评价．农药，1997，36(1)：8-9，15．
51．王开运，姜兴印，仪美芹．山东省主要棉花害虫的防治现状与综合防治．农药，1997，36(2)：6-9．
52．王兴林，冯俊涛，杨崇珍，等．陕西棉蚜抗药性监测．西北农业学报，1997，6(3)：57-60．
53．王旭，高希武，郑炳宗，等．有机磷药剂对棉铃虫羧酸酯酶的抑制作用．昆虫学
报，1998，41(增刊)：12-18．
54．王义平，吴鸿．昆虫抗药性的分子机理研究进展．中国森林病虫，2002，21(5)：34-36．
55．王荫长，韩召军．我国农业害虫抗药性发生概况．昆虫知识，1991，28(2)：120-121．
56．吴益东，沈晋良，谭福杰，等．山东省阳谷县棉铃虫抗药性监测．南京农业大学学报，1995，18(3)：48～53．
57．夏敬源．我国棉蚜抗药性现状与治理对策．中国棉花，1992，5：2．
58．肖海军，贺筱蓉．固定化酶及其应用研究进展．生物学通报，2001，36(7)：9-10．
59．谢佳燕，何凤琴，冯国蕾，等．微量滴度酶标法测定棉蚜 Aphis gossypii(Glover) 酯酶特性与有机磷抗性的关系．农药学学报，2000，2(1)：47-52．
60．谢佳燕，何凤琴，李梅，等．杀虫剂及不同地区寄主植物对棉蚜酯酶的影响．昆虫知识，2001，38(6)：429-435．
61．谢佳燕，何凤琴，李梅，等．不同地区棉蚜酯酶与有机磷杀虫剂抗性的关系．河北农业大学学报，2001，24(3)：43-45．
62．谢力，彭统序．小菜蛾对杀虫剂抗性的研究Ⅱ．小菜蛾幼虫的酯酶分析．昆虫天敌，1996，18(4)：23-29．
63．徐恩斌，张忠兵，谢渭芬．乙酰胆碱酯酶的研究进展．国外医学·生理、病理科学与临床分册，2003，23(1)：73-75．
64．许雄山，韩召军，王荫长．羧酸酯酶与棉铃虫对有机磷杀虫剂抗性的关系．南京农业大学学报，1999，22(4)：41-44．
65．姚洪渭，蒋彩英，叶恭银，等．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)，2001，27(1)：5-10．
66．姚洪渭，叶恭银，程家安．同翅目害虫抗药性研究进展．浙江农业学报，2002，14(2)： 63-70．
67．禹邦超，何开宇．油菜酯酶同工酶热稳定性初探．华中师范大学学报，1991，25(1)：79-84．
68．张红英，赤国彤，张金林．昆虫解毒酶系与抗药性研究进展．河北农业大学学报，2002，25(增刊)：193-195．
69．张柯，叶镇清，乔传令，等．库蚊羧酸酯酶研究进展．生命的化学，2002，22(1)：65-66
70．张友军，张文吉．乙酰胆碱酯酶分子生物学研究．昆虫知识，1997，34(4)：242～246．
71．赵国文，王标，王海军，等．十二种农药防治棉蚜效果研究．农药，1996，35(3)：38-39．
72．赵颖，高希武，胡熳华，等．棉蚜不同抗性品系羧酸酯酶比较．植物保护学报，
1997，24(4)：351-55赵国文，王标，王海军，等．十二种农药防治棉蚜效果研究．农药，1996，35(3)：38-39．
73．赵颖，高希武，胡熳华，等．棉蚜不同抗性品系羧酸酯酶比较．植物保护学报，1997，24(4)：351-355．
74．郑炳宗，高希武．北京及河北省北部瓜—棉蚜对拟除虫菊酯抗药性的研究初报．植物保护学报，1998，15(1)：55-60．
75．郑炳宗，高希武．瓜—棉蚜对有机磷及氨基甲酸酯杀虫剂抗性机制研究[J]．植物保护学报，1989，16(2)：131-138．
76．郑成．酶工程的研究进展简述．韶关学院学报，2001，22(6)：39-44．
77．周亚凤，张先恩，Anthony E．G．Cass．分子酶工程学研究进展．生物工程学报，2002，18(4)：401-406．
78．周亦红，姜卫华，赵志模，等．温度对美洲斑潜蝇及南美斑潜蝇种群增长的影响．生态学报，2001，21(8)：1276-1284．
79．朱九生，屈会选，樊建斌，等．山西省棉蚜对常用五种农药抗药性普查与监测研究．山西农业科学，1996，24(2)：18-32．
80．朱美财．乙酰胆碱酯酶的结构与功能研究进展．生物化学与生物物理进展，1992，19(5)：338-342．
81．庄占兴，韩书霞．抗灭多威棉蚜种群对其它常用药剂的交互抗性研究．农药，1997，36(10)：16-17．
82. Anthony N, Rocheleau T, Mocelin G, Lee H J and Ffrench-Constant R. Cloning, sequencing and functional expression of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti. FEBS Lett, 1995, 368(3):461-465.
83. Almirall M, Esteve G E. In vitro stability of a beta-glucanase from Trichoderma longibrachiatum and its effect in a barley based diet fed to broiler chicks. Animal Feed Science and Technology, 1995,54(1-4): 149-158.
84. Arpagaus M, Combers D, Culetto E, et al. Four acetylcholinesterase genes in the nematode Caenorhabditis elegans. J Physiol Paris,1998,92:363～367.
85. Arpagaus M, Toutant J P, Polymorphism of acetylcholinesterase in adult Pieris brassicae heads. Evidence for detergent-insensitive and Triton X-100 interacting forms. Neurochem Int, 1985,7: 793-804.
86. Baxter G D and Barker S C. Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: Characterization and role in organophosphate resistance. Insect Biochem Mol Biol, 1998, 28:581-589.
87. Baxter G D and Barker S C. Analysis of the sequence and expression of a second putative acetylcholinesterase cDNA from organophosphate-susceptible and organophosphate-resistant cattle ticks. Insect Biochem Mol Biol, 2002, 32(7):815-820.
88. Baxter G D and Barker S C. Comparison of acetylcholinesterase genes from cattle ticks. International Journal for parasitology, 1999,29:1765-1774.
89. Bonning B C, Hemingway J, Romi R and Majori G. Interaction of insecticide resistance genes in field populations of Culex pipiens (Diptera: Culicidae) from Italy in response to changing insecticide selection pressure. Bull Entomol Res, 1991, 81: 5～10.
90. Booth G M, Larsen J R, Gupta A P. Histochemistry o f acetylcholinesterase in the insect brain. Arthropod brain:its evolution, development, structure and functions, 1987: 439-456.
91. Bourguet D, Capela R, Raymond M. An insensitive acetylcholinesterase in Culex pipiens (Diptera : Culicidae) from Portugal. J Econ Entomol, 1996,89(5): 1060-1066.
92. Bourguet D, Pasteur N, Bisset J, et al. Determination of ace.1 genotypes in single mosquitoes: toward an ecumenical biochemical test. Pestic Biochem Physiol, 1996,55:122-128.
93. Bourguet D, Raymond M, Foumier D, Malcolm C A, Toutant J P, Arpagaus M. Existence of two acetylcholinesterase in the mosquito, Culex pipens (Diptera: Culicidae). J Neurochem, 1996, 67(5):2115-2123.
94. Bourguet D, Roig A, Toutant J P, et al. Analysis of molecular forms and pharmacological properties of acetylcholinesterase in several mosquito species. Neurochem Int, 1997,31 (1): 65-72.
95. Breer H, Sattelle D B. Molecular properties and functions of insect acetylcholine receptors. J Insect Physiol, 1987, 33:771-790.
96. Bull D L, Lindquist D A. Cholinesterase in boll weevils Anthonanus grandis: Ⅰ. Distribution and some properties of the crude enzyme. Comp Biochem Physiol, 1968,25: 639-649.
97. Burgess S K, Oxendine S L. Thermal inactivation of butyrylcholinesterase and acetylcholinesterase. Journal of Protein Chemistry, 1993,12(6): 651-658.
98. Byrne F J and Divonshire A L. Insensitive acetylcholinesterase and esterase polymorphism in susceptible and resistant populations of the tobacco whitefly Bemisia tabaci (Germ). Pestic Biochem Physiol, 1993, 45: 34～42.
99. Chen W, Sun C N. Purification and characterization of carboxylesterase of a rice brown planthopper Nilaparvata lugens. Insect Biochem Mol Biol, 1994,24:3472～3551.
100. Chen Z Z, N ewcomb R, Forbes E, M cKenzie J. and B atterham P. The a cetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina. Insect Biochem Mol Biol, 2001, 31(8): 805-816.
101. Combes D, Fedon Y, Grauso M, Toutant J P, Arpagaus M. Four genes encode acetylcholinesterase in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. CDNA sequences, Genomic structures, mutations and in vivo expression. J Mol Bio, 2000,300:727-742.
102. Dary O, Wedding R T. Absence of substrate inhibition and freezing-inactivation of the mosquito acetylcholinesterase are caused by alterations of hydrophobic interactions. Biochimica et Biophysica Acta P, Protection Structure and Molecular Enzymology, 1990, 1039: 1,103-109.
103. Derta R L, Collins W J. Characterization of cholinesterase activity in larval Chironomus riparius Meigen (=Thummi kiefer). Insect Biochem,1986,16: 733.
104. De Vilar, M.I.P. et al. Pestic Biochem Physiol,1983,19: 60.
105. Devi N A, Rao A G A. Effect of additives on kinetic thermal stability of polygalacturonase Ⅱ from aspergillus carbonarius: mechanism of stabilization by sucrose. Journal of Agricultural and Food Chemistry, 1998,46(9): 3540-3545.
106. Devonshire A L and Moores G D. Characterisation of insecticide-insensitive acetylcholinesterase: microcomputer-based analysis of enzyme inhibition in Homogenates of individual House Fly (Musca domestica) heads. Pestic Biochem Physiol, 1984,21: 341-348.
107. Devonshire A L and Moores G D. Different forms of insensitive acetylcholinesterase in insecticide—resistant house flies (Musca domestica). Pestic Biochem Physiol, 1984a, 21: 336～340.
108. Edwards J A, Brimijoin S. Thermal inactivation of the molecular forms of acetylcholinesterase and butyrylcholinesterase. Biochimica Et Biophysica Acta, 1983,742(3): 509-516.
109. Estrada-Mondaca S, Fournier D. Stabilization of recombinant Drosophila acetylcholinesterase. Protein Expression and Purification, 1998,12: 166-172.
110. Foumier D, Cuany A, Bride J M. Molecular polymorphism of head acetylcholinesterase from adult houseflies (Musca domestica L.). J Neurochem, 1987, 49(5): 1455-1461.
111. Foumier D, Francois K, Bride J M, Hall L M C, Berge J B. and Spierer P. Drosophila melanogaster acetylcholinesterase gene structure, evolution and mutations. J Mol Biol, 1989, 210: 15-22.
112. Foumier D, Mutero A. Modification of acetylcholinesterase as a mechanism of resistance to insecticides. Comp Biochem Physiol, 1994, 108C(1): 19-31.
113. Gao J R, Rao J V, Wilde G E, Zhu K Y. Purification and kinetic analysis of acetylcholinesterase from western corn rootworm, Diabrotica virgiferavirgifera (Coleoptera: Chrysomelidae). Arch Insect Biochem Physiol, 1998, 39:118-125.
114. Gnagey A L, Forte M, Rosenberry T L. Isolation and characterization of acetylcholinesterase from
Drosophila. J Biol Chem, 1987,262(27): 13290-13298.
115. Greenblatt H M, Silman I, Sussman J L. Structural studies on vertebrate and invertebrate acetylcholinesterase and their complex with functional ligands. Drug Development Research, 2000,50: 573-583.
116. Guilbault G G, Kuan S S, Sadar M H. Purification and properties of cholinesterase from Honeybees Apis mellifera Linnaeus and Boll Weevils Anthonomus grandis Boheman. Agr Food Chem, 1970,18: 692-697.
117. Gunning R V, Moores G D, Devonshire A L. Insensitive acetylcholinesterase and resistance to organophosphates in Australian Helicoverpa armigera. Pestic Biochem Physiol, 1998,62: 147-151.
118. Gunning R V, Moores G D, Devonshire A L. Insensitive acetylcholinesterase and resistance to thiodicarb in Australian Helicoverpa armigera Hübner (Lepidoptera: Noctuidae). Pestic Biochem Physiol, 1996,55: 21-28.
119. Gupta S, Sanyal S N, Duggal C L. Study of the acetylcholinesterase activity of Ascaridia galli: kinetic properties and the effect of anthelmintics. Acta Veterinaria Hungarica, 1991, 39(3-4): 165-174.
120. Habibulla M, Newburgh R W. Studies of acetylcholinesterase of the central nervous systerm of Galleria mellonella. Insect Biochem, 1973,3:231-242.
121. Hall L M and Malcolm C A. The a cetylcholinesterase g ene o f Anopheles s tephensi. Cell Mol. Neurobiol. 1991, 11(1): 131-141.
122. Hall L M and Spierer P. The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5' leader. EMBO J, 1986, 5(11): 2949-2954.
123. Hama H, Iwata T. Insencitive cholinesterase in the Nakagawara strain of the green rice leafhoppers, Nephotettix cincticeps, Uhler (Hemiptera: Cicadellidae), as a cause of resistance to carbamate insecticides. Appl Entmol Zool, 1971, 6: 183～191.
124. Han Z J, Moores G D, Denholm I, Devonshire A L, Association between Biochemical Markers and Insecticide in the Cotton Aphid, Aphis gossypii Glover. Pest Biochem Physi, 1998, 62: 164-171.
125. Hemingway J and Georghiou G P. Studies on the acetylcholinesterase of A nopheles albimanus resistant and susceptible to organophosphate and carbamate insecticides. Pestic Biochem Physiol, 1983, 19: 167～171.
126. Hemandez R, He H Q, Andrew C, Chen G, Wayne I, George J E. and Wagner G G. Cloning and sequencing of a putative acetylcholinesterase cDNA from Boophilus microplus (Acari: Ixodidae).
J Med Entomol, 1999, 36(6): 764-770.
127. Huang, Y., Qiao, C. L., Williamson, M. S. and Devonshire, A. L. Characterization of acetylcholinesterase gene from insecticide resistant house flies (Musca domestica). Chinese J Bioth, 1997, 13 (3): 258-263.
128. Isabelle F, Serge M, Andree B L, et al. Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine. Biochemistry, 2002,3(1): 21.
129. Iwata T, Hama H. Insensitivity of cholinesterase in Nephotettix cincticeps resistant to carbamate and organophosphorus insecticides. J Ecom Ent, 1972,65: 643～644.
130. Kozaki T., Shono T., Tomita T., and Kono Y. Polymorphism in the acetylcholinesterase gene of the housefly, Musca domestica L.(Diptera: Muscidae). Appl Entomol Zool, 2001,36(3):377-380.
131. Kristjansson M M, Kinsella J E. Protein and enzyme stability: structural, thermodynamic, and experimental aspects. Advances m Food and Nutrition Research, 1991,35:237-316.
132. Lee A H, Metcalf R L, Booth G M. House cricket acetylcholinesterase: histochemical location and in situ inhibition by o,o-dimethy S-aryl phosphorothioates. Ann Ent Soc Am, 1973,66(2): 333-343.
133. Lee R M, Batham P. The activity and organophosphate inhibition of cholinesterases from susceptible and resistant ticks (Acari). Ent Exp Appl, 1966, 9: 13～24.
134. Lenoir R J, Delbecque J, Gautron J. Developmental changes and acetylcholinesterase activity in the metamorphosing brain of Tenebrio molitor: correlation to ecdysteroid titers. Archives of Insect Biochemistry and Physiology, 1994,25(3): 207-226.
135. Li F, Han Z J. Mutations in acetylcholinesterase associated with insecticide resistance in the cotton aphid, Aphis gossypii Glover. Insect Biochem Mol Biol, 2004,34: 397-405.
136. Li F, Han Z J. Purification and characterization of acetylcholinesterase from cotton aphid, Aphis gossypii Glover. Archives of Insect Biochemistry and Physiology, 2002, 51: 37-45.
137. Li F, Han Z J. Two different genes encoding acetylcholinesterase existing in cotton aphid, Aphis gossypii Glover. Genome, 2002, 45 (6): 1134-1141.
138. Malcolm C A, Bourguet D, Ascolillo A, Rooker S J, Garvey C F, Hall L M, Pasteur C N and Raymond M. A sex-linked Ace gene not linked to insensitive acetylcholinesterase-mediated insecticide resistance in Culex pipens. Insect Mol Bio, 1998, 7(2): 107-120.
139. Melanson S W, Yun C H, Pezzementim L. Characterization of acetylcholinestemse activity from Drosphila melanogaster. Comp Biochem Physiol, 1985,81C: 87-96.
140. Moores G D, Devonshire A L, Denholm I. A microtitre plate assay for characterizing insecticides acetylcholinesterase genotypes of insecticides-resistant insects. Bull Entomol Res, 1998, 78:
537-546.
141. Moores G D, Gao X W, Denholm I, Devonshire A L. Characterization of insensitive Acetylcholinesterase in the Insecticide-Resistant Cotton Aphids, Aphis gossypii Glover (Homoperta:Aphidedae). Pestic Biochem Physiol, 1996, 56: 102-110.
142. Moores G D, Gregor J D, Devonshire A L. Insecticide resistance due to insensitive acetylcholinesterase in Myzus persicae and Myzus nicotlanae. Pests and Diseases, 1994, 4C: 413-418.
143. Motogoyama L R, Kao P, Lin T, et al. Dual role of esterases in insecticide resistance in the green rice leafhopper. Pestic Biochem Physiol, 1984, 32: 1392～1471.
144. Mutero A, Pralavorio M, Bride J M, Fournier D. Resistance-associated point mutation in insecticide-insensitive acetylcholinesterase. Proc Natl Acad Sci USA, 1994, 91: 5922-5926.
145. Nabeshima T, Kozaki T, Tomita T, Kono Y. An amino acid substitution on the second acetylcholinesterase in the pirimicarb-resistant strains of the peach potato aphid, Myzus persicae. Biochemical and Biophysical Research Communications. 2003, 307(1): 15-22.
146. Nabeshima T, Mori A, Kozaki T, et al. An amino acid substitution attributable to insecticide-insensitivity of acetylcholinesterase in a Japanese encephalitis vector mosquito, Culex tritaeniorhynchus. Biochemical and Biophysical Research Communications. 2004, 313(3): 794-801.
147. Newcomb R D, Campbell P M, Ollis D L. A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly. Proc Natl Acad Sci USA, 1997, 94: 7464-7468.
148. Oi M, Dauterman W C and Motoyama N. Biochemical factors responsible for an extremely high level of diazinon resistance in housefly strain. J Pestic Sci, 1990, 15: 217-224.
149. Oppenoorth F J, Smissaert H R, Welling W, van der Pas L J T and Hitman K T. Insensitive acetylcholinesterase, high glutathione-S-transferase, and hydrolytic activity as resistance factors in a tetrachlorvinphos-resistant strain of house fly. Pestic Biochem Physiol, 1977, 7: 34～47.
150. Oppenoorth F J. Two different paraoxon-resistant acetylcholinesterase mutants in the house fly. Pestle Biochem Physiol, 1982, 18: 26～27.
151. Owusu E O. Komi K. Horike M. Hirano C. Some properties of carboxylesterase from Aphis gossypii Glover (Homoptera: Aphididae). Appl Entomol zool, 1994, 29: 47～53.
152. Polyzou A, Debras J F, Belzunces L P. Changes in acetylcholinesterase during pupal development of Apis mellifera queen. Arch Insect Biochem Physiol, 1997,36: 69-84.
153. Pralavorio M, Foumier D. Drosophila acetylcholinesterase: characterization of different mutants
resistant to insecticides. Biochem Genet, 1992, 30: 77～83.
154. Raymond M, Fournier D, Bride J M, Cuany A, Berge J B, Magnin M and Pasteur N. Identification of resistance mechanisms in Culex pipiens (Diptera: Culcidae) from Southern France: insensitive acetylcholinesterase and detoxifying oxidases. J Econ Ent, 1986, 79: 1452～1458.
155. Ren X X, Hart Z J. Mechanism of monocrophos resistance in cotton Bollworm, Helicoverpa argmigera (Hubner). Arch. Insect Biochem, 2002, 51:103-110.
156. Saito T, Harna H, Suzuki K. Insecticide resistance in clones of the cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae), and synergistic effect of esterase and mixed-function oxidase inhibitors. Japanese Journal of Applied Entomology and Zoology, 1995,39(2): 151-158.
157. Saito T, Hama H. Carboxylesterase isozymes responsible for organophosphate resistance in the cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae). Appl Entomol zool, 2000,35(1): 171-175.
158. Saito T. Insecticide resistance of the cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae) Ⅵ. Qualitative variation of aliesterase activity. Appl Entomol zool, 1993,28(2): 263-265.
159. Sharon K, Ryan M F. Purification, characterization, and inhibition by monoterpenes of acetylcholinesterase from the waxmoth, Galleria mellonella (L.). Insect Biochem Molec Biol, 1999, 29:1097-1104.
160. Sharon Keane, Ryan M F. Purification, characterization, and inhibition by monoterpenes of acetylcholinesterase from the waxmoth, Galleria mellonella (L.). Insect Biochem Mol Biol, 1999,29: 1097-1104.
161. Siegfried B D, Ono M, Swanson J J. Purification and characterization of a carboxylesterase associated with organophosphate resistance in the greenbug, Schizaphis graminum (Homoptera: Aphididae). Arch Insect Biochem Physiol, 1997, 36: 229-240.
162. Siegfried B D, Scott J G. Properties and inhibition of acetylcholinesterase in resistant and susceptible German Cockroaches (Blattella germanica L.). Pestic Biochem Physiol, 1990,38: 122-129.
163. Smissaert H R. Cholinesterase inhibition in spider mites susceptible and resistant to organophosphate. Science, 1964,143: 129～131.
164. Sung D Y, Kang H S. The N-terminal amino acid sequences of the firefly luciferase are important for the stability of the enzyme. Photochemistry and Photobiology, 1998,68(5): 749-753.
165. Sussman J L, Harel M, Frolow F, Oefner C, Goldman A, Toker L and Silman I. Atomic structure of acetylcholinesterase from: Torpedo califomica: A prototypic acetylcholine-binding protein. Science, 1991, 253: 872.
166. Suzuki K, Hama H, Konno Y. Carboxylesterase of the cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae), responsible for fenitrothion resistance as a sequestering protein. Appl Entomol zool, 1993,28(4): 439-450.
167. Suzuki K, Hama H. Carboxlesterase of the cotton aphid, Aphis gossypii Glover ( Homoptera : Aphididae ) isoelectric point variants in organophosophorus insecticide resistant clone. Appl Entomol zool, 1998, 33(1): 11～20.
168. Takashi T, Osamu H, oshiaki K Y. Absence of protein polymorphism attributable to insecticide —insensitivity of acetylcholinesterase in the green rice leafhopper,Nephotettix cincticeps. Insect Biochem Mol Biol, 2000, 30: 325～333.
169. Tang Z H, Wood R J and Cammak S L. Acetylcholinesterase activity in organophosphorus and carbamate resistant and susceptible strains of the Culex pipiens complex. Pestle Biochem Physiol, 1990, 37: 192～199.
170. Tomita T, Hidoh O, Kono Y. Absence of protein polymorphism attributable to inscticide-insensitivity of acetylcholinesterase in the green rice leafhopper, Nephotettis cincticeps. Insect Biochem Mol Biol, 2000, 30: 325-333.
171. Toutant J P, Insect acetylcholinesterase: catalytic properties, tissue distribution and molecular forms. Prog Neurobiol, 1989,32: 423-446.
172. Tripathi P K. Relation of acetylcholinesterase sensitivity to cross-resistance of a resistant house fly strain to organophosphates and carbamates. Pestic Biochem Physiol, 1976, 6: 30～34.
173. Villatte F, Ziliani P, Marcel V, Menozzi P, Fournier D. A high number of mutations in insect acetylcholinesterase may provide insecticide resistance. Pestic Biochem Physiol, 2000, 67:95-102.
174. Villatte F, Ziliani P, Estrada-Mondaca S, Menozzi P, Fournier D. Is acetyl/butyrylcholine specificity a marker for insecticide-resistance mutations in insect acetylcholinesterase ? Pest Manag Sci, 2000,56: 1023-1028.
175. Vontas J G, Hejazi M J, Hawkes N J, et al. Resistance-associated point mutations of organophosphate insensitive acetylcholinesterase, in the olive fruit fly Bactrocera oleae. Insect Mol Biol, 2002,11(4): 329-336.
176. Voss G. Cholinesterase autoanalysis: a rapid method for biochemical studies on susceptible and resistant insects. J Econ Entomol, 1980, 73: 189.
177. Walsh S B, Dolden T A, Moores G D, Kristensen M, Lewis T, Devonshire A L, Williamson M S. Identification and characterization of mutation in the housefly (Musca domestica) acetylcholinesterase involved in insecticide resistance. Biochem J, 2001, 359: 175-181.
178. Weill M, Fort P, Berthomieu A, Dubois M P, Pasteur N, Raymond M. 2002. A novel
acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila. Proc R Soc Lond B, 2002,269: 2007-2016.
179. Whyard S, Russell R J, Walker V K. Insecticide resistance and malathion carboxylesterase in the sheep blowfly, Lucilia cuprina. Biochem Genet, 1994, 32: 1-2,9-24.
180. Xu G, Bull D L. Acetylcholinesterase from the horn fly (Diptera: Muscidae): distribution and purification. Journal of Economic Entomology, 1994,87(1): 20-26.
181. Yagüe-Guirao A, Munoz-Delgado E, Vidal C J. Thermal stability of molecular forms of acetylcholinesterase from muscle microsomes. Biochemistry International, 1986,12(5): 685-692.
182. Zahavi M, Tahori A S. Differences in acetylcholinesterase sensitivity to phosphamidon in Mediterranean fruit fly strains. Israel J Entomol V, 1970, 185～191.
183. Zhang A G, Dunn J B, Clark J M. An efficient strategy for validation of a point mutation associated with acetylcholinesterase sensitivity to azinphosmethyl in Colorado potato beetle. Pestic Biochem Physiol, 1999, 65: 25～35.
184. Zhu G P, Teng M K, Wu C J, et al. Mutation of G138P enhanced the thermostability of D-glucose isomerase. Acta Biochimica et Biophysica Sinica, 1998,30(6): 607-610.
185. Zhu K Y and Brindley W A. Acetylcholinesterase and its reduced sensitivity to inhibition by paraoxon in organophosphate-resistant Lygus Hesperus Knight (Hemiptera: Miridae). Pestic Biochem Physiol, 1990, 36: 22～28.
186. Zhu K Y, Brindley W A, Hsiao T H. Isolation and partial purification of acetylcholinesterase from Lygus Hesperus (Hemiptera: Miridae). J Econ Entomol, 1991,84(3): 790-794.
187. Zhu K Y, Brindley W A. Enzymological and inhibitory properties of acetylcholinesterase purified from Lygus hesperus Knight. Insect Biochem Mol Biol, 1992, 22(3): 245-251.
188. Zhu K Y, Clark J M. Cloning and sequence of a cDNA encoding acetylcholinesterase in Colorado potato beetle, Leptinotarsa decemlineata (Say). Insect Biochem Mol Biol ,1995, 25(10): 1129-1138.
189. Zhu K Y, Clark J M. Comparisons of kinetic properties of acetylcholinesterase purified from azinphosmethyl-susceptible and resistant strains of Colorado Potato Beetle. Pestic Biochem Physiol, 1995,51: 57-67.
190. Zhu K Y, Clark J M. Purification and characterization of acetylcholinesterase from the Colorado potato beetle, Leptinotarsa decemlineata (Say). Insect Biochem Mol Biol, 1994, 24: 453-461.
191. Zhu K Y, Clark J M. Validation of a point mutation of acetylcholinesterase in Colorado potato beetle by polymerase chain reaction coupled to enzyme inhibition assay. Pestic Biochem Physiol, 1997, 57: 28～35.
192. Zhu K Y, Gao J R. Increased activity associated reduced sensitivity of acetylcholinesterase in organophosphate-resistant greenbug, Schizaphis graminum (Homoptera: Aphididae). Pestic Sci,1999,5591): 11-17.
193. Zhu K Y, Lee S H and Clark J M. A point mutation of acetylcholinesterase associated with azinphosmethyl resistance and reduced fitness in Colorado potato beetle. Pestic Biochem Physiol, 1996, 55: 100-108.