论文标题:杀虫剂对松墨天牛几种酶和蛋白质磷酸化的影响
Effects of Insecticides on Some Enzymes and Protein Phosphorylation in Monochamus Alternatus Hope
论文作者 刘杰
论文导师 高希武；伍一军,论文学位 硕士,论文专业 农业昆虫与害虫防治
论文单位 中国农业大学,点击次数 145,论文页数 75页File Size10713k
2005-06-01论文网 http://www.lw23.com/lunwen_1565337/ 松墨天牛;杀虫剂;乙酰胆碱酯酶;羧酸酯酶酶;谷胱甘肽S-转移酶;蛋白磷酸化;原代细胞培养
Monochamus alternatus;insecticide;acetylcholinesterase(AChE);glutathione-s-transferase (GST);carboxylesterases (CarE) ;protein phosphorylation; primary cell culture
松墨天牛是一种钻蛀性的林业害虫,主要危害松属植物,而且其传播松材线虫造成的危害远甚于其自身。近年来人们在其生物学、生态学以及防治方面对其进行了大量的研究。本文从毒理学角度研究了甲胺磷、溴氰菊酯、吡虫啉等几种代表性的杀虫剂对松墨天牛的乙酰胆碱酯酶、羧酸酯酶、谷胱甘肽S-转移酶的体外影响,并使用同位素标记的方法研究了溴氰菊酯和甲胺磷两种代表性杀虫剂对松墨天牛的脂肪体和头部蛋白磷酸化的影响情况。还进行了松墨天牛体内的神经毒性酯酶活性的测试,并进行了松墨天牛细胞原代培养的探索性试验。结果如下: 甲胺磷对松墨天牛乙酰胆碱酯酶、羧酸酯酶酶、谷胱甘肽S-转移酶均有抑制作用其I_(50)值分别为8.19×10~(-4)mol/L,1.95×10~(-7) mol/L,0.908 mol/L。 可以确定松墨天牛体内存在NTE活力,以头部活力较高,但甲胺磷在体外处理时不能抑制其活力,表现出与高等动物NTE不同的性质。 溴氰菊酯和甲胺磷对松墨天牛幼虫的蛋白磷酸化的影响非常明显,放射自显影图谱上可观察到许多发生改变的条带,而相对来说两种杀虫剂对成虫蛋白磷酸化的影响较小。 溴氰菊酯使来自幼虫脂肪体的分子量137.8 kD蛋白质和来自幼虫头部的166.5 kD蛋白质磷酸化水平增强,使来自幼虫脂肪体的分子量为111.4 kD、95.3 kD和43.8 kD蛋白质和来自幼虫头部的113.2 kD、97.5 kD和50.6 kD蛋白质以及来自成虫脂肪体的51.8 kD、44.3 kD蛋白质和来自成虫头部的43.3 kD蛋白质磷酸化水平减弱。 甲胺磷使来自成虫脂肪体的分子量为127.7 kD、44.3 kD和238.9 kD蛋白质磷酸化水平增强,使来自幼虫脂肪体的95.3 kD和43.8 kD蛋白质以及来自幼虫头部的166.5 kD和124.5 kD蛋白质磷酸化水平减弱。 通过使用杀虫剂与cAMP和钙调蛋白(CaM)共同处理对蛋白磷酸化的影响情况进行研究,对自显影图谱上的一些蛋白质磷酸化反应的蛋白激酶的性质进行分析,说明杀虫剂对天牛蛋白质磷酸化影响的复杂性和多样性。由于所测试的杀虫剂对天牛体内的某些蛋白的作用情况非常复杂,还需要进一步研究其性质才能得以确定。 本研究的结果对于松墨天牛的防治提供了毒理学参考,杀虫剂对天牛蛋白质磷酸化影响部分的研究结果说明了杀虫剂具有广泛的生物活性,除了一般意义上使昆虫快速致死的作用靶标之外,杀虫剂还可以作用于蛋白激酶、蛋白磷酸酶等非致死性作用靶标。本项研究结果对全面了解杀虫剂的毒性机理以及关于杀虫剂对蛋白磷酸化影响的进一步研究具有重要的意义。
Monochamns alternatus Hope (Coleoptera:Cerambycidae) is not only a serious pest to Pinus but also the main vector of pine wood nemadote Bursaphelenchus xylophilus, which causes pine wilt disease. There"s many researches on this insect about its biology, ecology, and the methodology of its control. In this thesis, methamidophos, deltamethrine, and imidacloprid were chosen to be the representatives of three kinds of insecticides to test its effect on acetylcholinesterase (AChE), glutathione-s-transferase (GST), and carboxylesterases (CarE). The author studied the effect of methamidophos and deltamethrine on protein phosphorylation of this insect using radioactive isotope labeling method. In addition, we assayed activity of neurotoxic esterase (NTE) in this pest insect, and tried to do primary culture of the hemocytes and fat body cells from this insect. The details of the results are as follows:Methamidophos could inhibit not only AChE,and CarE but also GST, in vitro; the I_(50) values of the inhibition concentrations are 8.19×10~(-)M, 1.95×10~(-7) M, and 0.908 M for the three enzymes respectively.It is certain that there is NTE activity in Monochatnus alternatus. The activity of the head of the insect is higher than that of other parts. It has not been observed that methamidophos can inhibit NTE activity in vitro, which is quite different from the situation found in vertebrates.Methamidophos and deltamethrin have significant effect on the phosphorylation of proteins from Monochamus alternatus larva; however, the effect of the two insecticides on the protein phosphorylation of the adult is less significant than that of the larva.Deltamethrin enhance the phosphorylation of the proteins with molecular weight of 137.8 kD and 166.5 kD from fat body and head of the insect larva, respectively. It inhibits the phosphorylation of proteinsof 111.4kD, 95.3kD and 43.8kD from larva fat body, 113.2kD, 97.5kD, 50.6kD from larva head, 51.8kD, 44.3kD from adult fat body and 43.3kD from adult head.Methamidophos enhance the phosphorylation level of the proteins with molecular weight of 127.7 kD, 44.3 kD, and 238.9 kD from adult fat body but it inhibit the phosphorylation of proteins of 95.3 kD and 43.8 kD from larva fat body and 166.5kD and 124.5kD protein from larva head.We acquired many information of the protein kinase related with protein detected by autoradiography through using cAMP and calmodulin in the assay system. Because of the complicacy of the protein phosphorylation system, further research work is needed to gain more information about the protein kinase and protein phosphatase.The result of our research will be benificial to the control of Mnonchamus alternatus. The effects of methamidophos and deltamethrin on protein phosphorylation indicated that the two insecticides may have more other targets than the fatal targets which could kill insect in short time.
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