赤拟谷盗乙酰胆碱酯酶生化及毒理学特性研究

摘要/Abstract

摘要： 为了阐明赤拟谷盗[Tribolium castaneum (Herbst)]体内乙酰胆碱酯酶(AChE)在其各虫态间的变化关系，以期为该虫的综合治理提供一定的理论依据，本试验采用微量滴度酶标板法，分别对其10d龄和20d龄幼虫、蛹及成虫体内AChE的生化及毒理学特性进行了较为系统的研究。结果表明，赤拟谷盗各虫态AChE酶源蛋白含量、酶活力和比活力差异显著，酶源蛋白含量从低到高依次为：10d龄幼虫<20d龄幼虫<成虫<蛹；AChE活力和比活力依次为：成虫<蛹<10d龄幼虫<20d龄幼虫。酶动力学分析发现，AChE米氏常数Km值大小依次为：蛹<成虫<10d龄幼虫<20d龄幼虫，但成虫和蛹之间的差异不显著。此外，幼虫AChE的最大反应速度Vmax亦显著高于成虫和蛹。通过对幼虫AChE离体抑制作用测定表明，毒扁豆碱、马拉氧磷和西维因对20d龄幼虫的抑制中浓度I50均高于10d龄幼虫，说明20d龄幼虫体内的AChE对3种抑制剂的抑制作用更不敏感。

关键词: 核酸疫苗, 核酸疫苗, 作用机制, 影响因素, 应用前景

Abstract: To clarify the acetylcholinesterase (AChE) changes among different developmental stages of Tribolium castaneum (Herbst), the toxicological and biochemical characterizations of AChE in 10-d larvae, 20-d larvae, pupae and adults were investigated, respectively with the microplate reader method. The results showed that there were significant differences of protein content and the AChE activity and specific activity among the four developmental stages of T. castaneum. The protein contents of AChE were 10-d larvae < 20-d larvae < pupae < adults, while the AChE activity and specific activity were adults < pupae < 10-d larvae < 20-d larvae. The kinetic analysis of AChE revealed that the apparent Michaelis-Menten constants (Km) by magnitude were as follows: pupae < adults <10-d larvae < 20-d larvae. And no significant difference appeared between the Km values of pupae and adults. In addition, the maximum velocity (Vmax) in larvae was significant higher than those in pupae and adults. In vitro inhibition studies of larvae AChE indicated that the I50 values of 20-d larvae were higher than those of 10-d larvae when treated with eserine, malaoxon and carbaryl, respectively, suggesting that AChE in 20-d larvae was less sensitive to the inhibitors.

中图分类号:

Q965.9
Q966

刘国瑛,柴玉鑫,魏丹丹,王进军. 赤拟谷盗乙酰胆碱酯酶生化及毒理学特性研究[J]. 中国农学通报, 2006, 22(12): 303-303.

GuoYingliu, Yu Xinchai, Dan Danwei, Jin Junwang. Toxicological and Biochemical Characterizations of Acetylcholinesterase in Tribolium castaneum (Herbst)[J]. Chinese Agricultural Science Bulletin, 2006, 22(12): 303-303.

参考文献

[1] Spratt E C. The effects of atmospheres containing 5%~20% oxygen with and without 10% carbon dioxide on the productive of Tribolium castaneum (Herbst) and T. confusum Du Val. Journal Stored Product Research, 1984,4:199-203.
[2] 白旭光.储藏物害虫与防治.北京:科学出版社,2002. 287-290.
[3] 陈启宗,黄建国.仓库昆虫图册.北京:科学出版社,1983.57.
[4] 曹阳,王殿轩.米象和赤拟谷盗不同品系成虫的磷化氢击倒时间与其抗性之间的关系.郑州粮食学院学报,2000,21(2):1-5.
[5] 曹阳.我国谷蠹、赤拟谷盗、锈赤扁谷盗和土耳其扁谷盗磷化氢抗药性调查.河南工业大学学报(自然科学版),2006,27(1):1-6.
[6] Rajendran S. Insect resistance to phosphine-challenges and strategies. International Pest Control, 2001.43:1l8-123.
[7] Fournier D, Mutero A. Modification of acetylcho1inesterase as a mechanism of resistance to insecticides. Comparative Biochemistry and Physiology, 1994, 108c:19-31.
[8] 李滕武,沈福英,高希武.不同种群小菜蛾乙酰胆碱酯酶性质研究.河北北方学院学报,2005,21(1):32-35.
[9] 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. Pesticide Biochemistry and Physiology,1997,57:28-35.
[10] Wang J J, Cheng W X, Ding W, et al. The effect of insecticide dichlorvos on esterase activity extracted from the psocids, Liposcelis bostrychophila and L. entomophila. Journal of Insect Science,2004,4:23-27.
[11] Nayak M K, Collins P J, Kopittke R A. Residual toxicities and persistence of organophosphorus insecticides mixed with carbaryl as structural treatments against three liposcelidid psocid species (Psocoptera: Liposcelididae) infesting stored grain. Journal Stored Product Research, 2003, 39: 343-353.
[12] Cheng W X, Wang J J, Ding W, et al. Inhibition kinetics on carboxylesterase and acetylcholinesterase of Liposcelis bostrychophila and L. entomophila (Psocop., Liposcelididae) of two insecticides. Journal of Applied Entomology, 2004,128 (4): 292-297.
[13] 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):35-41,89.
[14] Ellman G L, Courtney K D, Andres Jr V. A new and rapid colorimetric determination of a acetylcholinesterase acticity. Biochemical Pharmacology, 1961,7:88-94.
[15] Moores G D, Gao X W, Denholm I, et al. Characterization of insensitive acetylcholinesterase in the insecticide-resistant cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae). Pesticide Biochemistry and Physiology, 1996,56:102-110.
[16] Wilkinson G N. Statistical estimations in enzyme kinetics. Biochemical Journal, 1961,80:324-332.
[17] Bradford M M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 1976,72:248-254.
[18] 陶卉英,王进军,丁伟,等.嗜卷书虱抗性与敏感品系乙酰胆碱酯酶的比较.西南农业大学学报(自然科学版),2005,27(2):151-154,192.
[19] Casida J E. Compatative enzymology of certain insect acetylesterases in relation to poisoning by organophosphorous insecticides. Biochemical Journal, 1955,6:487-496.
[20] Al-Hakkak Z S, Al-Azzawa M J, Al-Adhamy B W, et al. Inhibitory action of phosphine on acetylcholinesterase of Ephestia. Cautella (Lepidoptera: Pyralidae). Journal Stored Product Research, 1989,25:171-174.
[21] Gao X W, Zheng B Z, Zhang F, et al. Substrate specificity and developmental changes of acetylcholinesterase in cotton bollworm. Entomologia Sinca, 1996,3(1):80-89.