摘要
乙酰胆碱酯酶(AChE，EC 3．1．1．7)是生物神经传导中的一种关键性的酶，是有机磷和氨基甲酸酯类杀虫剂的靶标酶，AChE敏感性的降低是害虫对有机磷和氨基甲酸酯类杀虫剂产生抗性的重要机理之一。害虫AChE的纯化及其cDNA克隆对于AChE性质的深入研究、杀虫剂合理设计和合理使用、害虫抗药性治理等都具有重要的科学意义。作为目前蔬菜的主要害虫之一，黄曲条跳甲的抗药性问题日益得到关注，但其AChE的纯化、基因克隆和序列分析在国内外未见报道。本文以黄曲条跳甲为材料，在比较分析3种纯化方法的基础上，采用CEA亲和层析法对黄曲条跳甲AChE进行纯化，并对AchE的生化性质进行研究；利用RT-PCR的方法对黄曲条跳甲AchE的cDNA片断进行克隆并序列分析，推导出氨基酸，并与其它昆虫的氨基酸序列的进行同源性比较。
研究结果表明，在(NH_4)_2SO_4分级沉淀、Sephadex G-200凝胶层析和CEA亲和层析3种纯化方法中，以CEA亲和层析的纯化效果最好，其纯化倍数为651.20，产率为41.80％，Sephadex-200层析分离法效果次之，纯化倍数为12.60，产率为48.50％，(NH_4)_2SO_4分级沉淀法效果最差，其纯化倍数仅为1.50，产率62.70％；CEA亲和层析纯化产物经SDS-PAGE电泳后得到两条明显的条带，分子量分别为45kD，37kD。对纯化前后的AChE的生化特性初步研究的结果表明，纯化后的AChE反应最适温度为37℃，反应最适pH为7.0，底物ATChI浓度为2500μM，酶促反应速度达到最大值；纯化前的AChE受温度影响的变化趋势与纯化后的AChE相似，AChE活性也是随着pH值的升高而变大，但直到pH9.5时活性才稍有降低，底物ATChI浓度1500μM，反应速度达到最大值；在设定的实验条件下，纯化后酶的活性水平比纯化前明显提高，这表明在粗酶中存在未知因子影响AChE的反应活性。
根据已发表的AChE基因序列及氨基酸序列的保守区域，分别设计上游引物和下游引物，利用RT-PCR的方法对黄曲条跳甲的AChE基因cDNA片断进行克隆和序列分析，通过上、下游引物扩增了AChE基因750bp的cDNA片断。同源性分析表明，获得的750bp核苷酸序列与其它昆虫AChE基因序列具有较高的同源性，应为编码黄曲条跳甲AChE部分氨基酸序列。
Acetylcholinesterase (AChE, EC3. 1.1.7) played an important role in the function of nerve impulse transmission and was the target sit of organophosphorus and carbamate insecticides. Reducing AChE sensitivity was one of the key mechanism of insect pests resistance to organophosphorus and carbamate insecticides. Purification and cDNA cloning were helpful to further studies on AchE, and were good to insecticides rational design and application. As one of destructive insect pests, the resistance to Phyllotreta striolata (F.) insecticides was attracted more and more concern. However, there were no information about purification and cDNA cloning of P. striolata AchE. Reported the comparison of three purification methods in this paper, CEA was selected for AChE purification. The biochemical characteristics were also studied. In addition, RT-PCR was used to clone the AchE partial cDNA. cDNA sequence and its homologous analysis were dicussed subsequently in the paper.
The results showed that the CEA affinity chromatography was the best method which accounted to the purification fold of 651.20 and yield of 41.80%. The purification fold and yield by the method of sephadex G-200 chromatography procedure were 12.60 and 48.50% respectively, and by ammonium sulfate method were 1.50 and 62.74% respectively. The separated fraction was detected by SDS-PAGE, and corresponded to molecular weights of 45kD and 37kD. Properties of crude extract and purified AChE were studied. The results showed the optimal temperature, pH and the substrate of ATChI of purified AChE were 37 C, 7.0 and 2500 M respectively. The crude extract conducted differently to varied temperature , pH and ATChI. Investigation revealed that crude extract and purified AChE had different characteristics. We infered that one or more factors in the crude extract could influence AChE activity.
With RT-PCR, the partial cDNA sequence of AChE gene in the P. striolata was cloned and analyzed. Using a pair of primers based on the conserved gene sequence of AChE gene, the fragment of 750bp was amplified. Homologous analysis revealed that there was high homology degree of amino acid sequence between the P. striolata and other insects.
引文
高希武，周序国，王荣京，郑炳宗．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(增刊)：19—25．
高希武．乙酰胆碱酯酶(AChE)的毒理学、生物化学和分子生物学．见：冷欣夫，唐振华，王荫长，杀虫蓟剂分子毒理学及昆虫抗药性．北京：中国农业出版社，1996，30-33．
彭宇，王荫长，韩召军等．二化螟体内乙酰胆碱酯酶的分布及纯化方法．昆虫学报，2002，45：(2)209—214．
任晓霞，韩召军等．棉铃虫乙酰胆碱酯酶cDNA片断的克隆和序列分析．动物学报，2002，48(1)：121—124．
王建军，韩召军等．小菜蛾乙酰胆碱酯酶cDNA片断的克隆和序列分析．昆虫学报，2002，45(4)：544—547．
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．
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．
Barak D., Kronman C., Ordentlich A., et al.. Suitability of textural parameters for characterization of Trappistcheeseripening. Biol. Chem., 1994, 269: 6296-6305.
Bonning B.C., Malcol C.A., Hamingway J..Purification and characterization of acetylcholinesterase from Culex popoens(Say). Pestic.Sci., 1988, 24: 278-280.
Brestkin A.P., Maizel E.B, Moralev S.N., Novozhilov K.V., Sazonova I.N.. Cholinesterase of aphids-1: Isolation partial purification and some properties of cholinesterase of spring grain aphid Schizaphis gramina(Rond.).Insect Biochem., 1985, 15(2): 309-314.
Brown T.M., Bryson P.K.. Selective inhibitors of methylrathion-resistant acethlcholinesterase from Heliothis Virescens. Pestic. Biochem. Physiol., 1992, 44: 155-164.
Bull D. L., Lundquist D.A..Cholinesterase in bollweevils Anthonomus grandis: Distribution
and some properties of the crude enzyme. Comp.Biochem.Physiol., 1968, 25: 639-645.
Combes D., Fedon Y., Grauso M., et al.. Four genes encode acetylcholinesterase in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, cDNA sequences, genomic structures, mutations and in vivo expression. Mol. Biol., 2000, 300(4): 727-742.
Devonshire A.L.,Moores G.D.. A earboxylesterase with broad substrate specificity causes organophosphorous, carbamate and pyrethroid resistance in peach potato aphids(Myxus persicae), Pistic.Biochem.Physiol., 1982, 18: 235-246.
Devonshire A.L., Moores G.D., Different forms of insensitive acetylcholinesterase in insecticide-resistant house flies(Musca domestica). Pestic. Biochem. Physiol., 1984, 21: 336-340.
Fournier D., Bride J., Hoffmann F., et al.. Solitonlike pulse shortening in a femtosecond parametric amplifier. Biol.Chem., 1992, 267: 14270-14274.
Fournier D., Mutero A.. Mini review: modification of acetylcholinesterase as a mechanism of resistance to insecticides. Comp. Biochem. Phylsiol., 1994, 108 c(1): 19-31.
Gnagey A.L., Forte M., Rosenberry T.L., Isolation and characterization of acetylcholinesterase from Drosophila. Biol. Chem., 1987, 262: 13290-13298.
Hama H., Twata T., Miyata T., Saito T.. Some properties of acetylcholinesterase partially purified from susceptible ans resistant green rice leafhoopers, Nephotettix cincticeps Ulder(Hemiptera:Deltocephalidae). Appl. Entomol. Zool., 1980, 15: 249-264.
Hoffmann F., Fournier D., Spierer P.. Proline reduces Acetylcholinesterase activity in cerebral cortex of rats. Mol.Biol., 1992, 223: 17-22.
Laster D.S., Gilbert L.T.. Characterization of acetylcholinesterase activity in the larval brain of Minduca Sexta.Insect Biochem., 1987, 17: 99-109.
Lenoir-Rousseaux J.J., Arpagaus M., Toutant J.P.. Separation and characterization of hydrophilie and detergent interacting components of brain acetylcholinesterase from
Tenebrio molitor L. (Insecta, Coleoptera). Comp.Biochem.Physiol., 1988, 90B: 29-35. Li Y., Camp S., Rachinsky T.L., et al.. Biol.Chem., 1991, 226: 23083-23090.
Massoulie J., Anselmet A., Bon S., et al..The polymorphism of acetylcholinesterase: post-translational processing, quaternary associations and localization. Chem Biol Interace, 1999, 119-120(1): 29-42.
Massoulie J., Pezsementi L.. Bon S., et al.. Tenth International Symposium on Cholinergic Mechanisms～Final rept. Prog.Neurobiol., 1993, 41: 31-91.
Mutero A.M., Pralavorio J.M., Bride and Fournier D.. Resistance-associated point mutions insecticide-insensitive acetylcholinesterase. Proc.Natn. Acad. Sci. USA, 1994, 91: 5922—5926。
Ordentlich A., Barak D., Kronman C., et al.. A fluorometric method to detect insensitive Acetylcholinesterase in resistantpests. Biol. Chem., 1993, 268:
Perrier A.L., Massoulie J., Krejci E.. PRIMA: the membrane anchor of acetylcholinesterase in the brain. Neuron, 2002, 33(2): 275-285.
Sussman J.L., Harel M., Frolow F., et al.. Cloning, Mutagenesis, and Expression of the Acetylcholinesterase Gene from a strain of Musca domestica; the Change from a Drug-resistant to a Sensitive Enzyme. Science, 1991, 253: 872-879.
Taylor P., Radio Z.. A new method for ranking Tacrine derivatives binding affinities with Acetylcholinesterase via finite difference thermodynamic integration. Annu Rev Pharmacol toxicol., 1994, 34: 281-321.
Vellow D.C., Radic Z., Li Y., et al.. Acetylcholinesterase activity in copepods(Tigriopus brevicornis)from theVilaine River estuary, France, as a biomarker of neurotoxic contaminants. Biochemistry, 1993, 32:12-17.
Xu G., Bull D.L.. Acetylcholinesterase from the horn fly(Diptera: Muscidae): distribution and purification. Econ. Entomol., 1994, 87(1): 22-26.
Zhu K.Y., Brindely W.A.. Molecular properties of acetylcholinesterase purified from Lygus
Zhu K.Y., Brindely W.A.. Molecular properties of acetylcholinesterase purified from Lygus Hesperus Knight(Hemiptera: Miridae). Insect Biochem. Mol.Biol., 1992, 22: 253-260.
Zhu K.Y., Clark J.M.. Addition of a competitive primer can dramatically improve the specificity of PCR amplification of specific alleles(PASA). Biotechniques, 1996, 21: 586-594.
Zhu K.Y., Clark J.M.. Purification and characterization of acetylcholinesterase from the Colorado potato beetle, Leptinota decemlineata(Say). Insect Biochem. Mol.Bio., 1994, 24(5): 453-461.
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.