【作者】 叶雅杰；

【导师】 马逊风；

【作者基本信息】 东北师范大学 ， 环境科学， 2006， 硕士

【摘要】 光肩星天牛[Anoplophora glabripennis(Motsoh)]因其幼虫钻蛀木材,是著名的林业蛀干害虫。近年来,我国部分地区的城市园林绿化也深受其害。1998年美国政府宣布将禁止我国带有木质包装和铺垫材料的产品进入美国境内,以防止一种甲虫—光肩星天牛危害美国的森林。其报告指出,光肩星天牛一旦在美国传播开来,将对美国槭糖业、旅游业和生态环境造成约1380亿美元的直接经济损失。2002年的中美蛀干害虫研讨会上强调了关于以光肩星天牛、黄斑星天牛为代表的蛀干害虫是我国林木的重要害虫,对林业生产构成严重威胁,特别是在以杨柳榆为主的“三北”地区,曾造成毁灭性的灾害。蛀干害虫的危害性越来越受到人们的关注。以昆虫多酚氧化酶为靶标的生物杀虫剂方面研究不多,尤其蛀干害虫方面尚未见报道。本试验选取蛇床子素、槲皮素、姜黄素、穿心莲内酯、绿原酸、厚朴酚、补骨脂、咖啡酸八种不同生物活性的植物化学物质作为多酚氧化酶的效应物来尝试蛀干害虫防治技术的研究。本实验选取蛀干害虫光肩星天牛为受试生物实验结果表明,以光肩星天牛幼虫为材料提取多酚氧化酶,经40%饱和硫酸铵处理的沉淀纯化后,总活力回收率为30.08%,酶活力提高4.486倍。在分别对以光肩星天牛幼虫为材料的粗酶液进行活性比较的实验中发现,蛹期幼虫的PPO的活力明显高于其它各龄期。参照甜菜夜蛾等昆虫的多酚氧化酶反应的最适条件:PH=6.5、T=37℃、最佳反应时间为2min,研究了蛇床子素、槲皮素、姜黄素、穿心莲内酯、绿原酸、厚朴酚、补骨脂、咖啡酸八种化合物对该酶活力的影响,结果表明,其中以甲醇为溶剂配制的效应物对光肩星幼虫多酚氧化酶的活性影响试验中,槲皮素抑制力最强,其次酶活性从大到小顺序是蛇床子素、绿原酸、姜黄素、穿心莲内酯、咖啡酸,且都小于溶剂对照。其中以磷酸缓冲液-甲醇为溶剂的效应物对光肩星幼虫多酚氧化酶的活性影响试验中,蛇床子素的抑制力最强,其次酶活性从大到小的顺于是蛇床子素、姜黄素、穿心莲内酯、绿原酸、咖啡酸、槲皮素,且都小于溶剂对照。根据文献资料和时间的情况,最后选取槲皮素和姜黄素对该酶活力的抑制机理分析,设定两组,其一在底物浓度固定不变的情况下,测得不同酶液量0.02、0.05、0.1、0.15、0.20随不同效应物浓度0.1、0.3、0.5、0.7、0.95μg/mL的酶活力变化,其二在酶液量固定不变的情况下,测得不同底物浓度0.001、0.005、0.01、0.05、0.1mol/L随不同效应物浓度0.1、0.3、0.5、0.7、0.95μg/mL的酶活力变化。用Lineweaver-Burk(双倒数作图法)作图,槲皮素和姜黄素的抑制动力学结果表明,该两种化合物对该酶的影响均是Vmax值的变化对Km值几乎没有任何影响,因此断定槲皮素和姜黄素都是光肩星天牛幼虫多酚氧化酶的非竞争性抑制剂,抑制常数分别为1.8860μg/mL和1.4549μg/mL。槲皮素和姜黄素作抑制剂很早就有人研究,在昆虫领域的研究报道不多,槲皮素和姜黄素二效应物对光肩星天牛幼虫多酚氧化酶的更多还原

【Abstract】 The Motsoh (Anoplophora glabripennis) is a notorious Trunk borer, which eats the woodand settles in wood. Recently, part of gardens has fallen deeply into the victim of the Motsh.In 1998, the U.S.A government declared a bill that it would forbid imports the woodenpacking and matting material from China to prevent the hazard of a kind of pest-Trunk borer.It stated that once the pest got prevalent in U.S.A, it would have brought a direct loss of 138billion dollar in the journalism as well as eco-environment. In 2002, The Wood Eaten PestPreminar between U.S.A and China stated that the motsh have been a severer threat to thewood industry, especially to the “Three North” protecting wood belt.The harm of Trunk borer has received more and more attention .The thesis Of biologicalpesticide chosen PPO as target are still scare. The research with Psoralen, Magnolol, Osthole,Chlorogenic acid, Andrographolide, Curcumin, Quercetin , coffee acid phytochemistricalmaterial with different biological activity as the effection was conducted to validate theanti-Trunk borer technique.The Trunk borer is been selected as object to study. The result revealed that when thedeposits disposed by 40 per cent saturated ammonium sulfate solution and then purified, thetotal reclaim rate of enzyme is up to 30.08%, increased more than 4 folds comparing nothingtreatment condition . The test on crude enzyme of the Trunk borer larva discovered that , thePPO in the period of pupa is transparently higher than growing phase.Selecting the PH=6.5,T=37℃ and the optimal reaction time setting as 2min to havestudied the influence of eight substrates which including Psoralen, Magnolol, Osthole,Chlorogenic acid, Andrographolide, Curcumin, Quercetin , coffee acid to the PPO of Trunkborer. The results indicated that the strongest inhibition among the eight substrates is theQuercetin solute by methanol, and then the Osthole, Chlorogenic acid, Curcumin, coffee acid.But the PPO in all the eight substrates is minor than trial group. However, Osthole appears tobe the strongest inhibition in the test of PPO activity of worm, if the effects solutes by thephosphate-methanol buffer solutes .And following are Osthole, Chlorogenic acid,Andrographolide, coffee acid, Magnolo. And the PPO in all the eight substrates is minor thantrial group.Two groups are divided to study the enzyme inhibition mechanism analysis arefocus on the Quercetin and Curcumin. One is measuring the enzyme change with differentenzyme volume of 0.02,0.05,0.1,0.15,0.2 following the different effect concentrations of0.1,0.3,0.5,0.7,0.95μg/mL but keep the identical substrate concentration. The other group isthe measuring the enzyme change with same enzyme volume however different substrateconcentrations of0.001,0.005,0.01,0.05,0.1mol/L and following the different effectconcentration of 0.1,0.3,0.5,0.7,0.95μg/mL. The inhibition dynamics of Quercetin andCurcumin expressed by Bi-reciprocal diagram indicated that the change of Vmax almosteffected nothing to Km, Therefore , such a conclusion can be drawn that the these twoeffects is non-competition inhibitor, and the inhabitation constant are 1.886μg/mL and1.4549μg/mL respectly.Two groups are divided to study the enzyme inhibition mechanismanalysis are focus on the Quercetin and Curcumin. One is measuring the enzyme changewith different enzyme volume of 0.02,0.05,0.1,0.15,0.2 following the different effectconcentrations of 0.1,0.3,0.5,0.7,0.95μg/mL but keep the identical substrate concentration.The other group is the measuring the enzyme change with same enzyme volume howeverdifferent substrate concentrations of0.001,0.005,0.01,0.05,0.1mol/L and following thedifferent effect concentration of 0.1,0.3,0.5,0.7,0.95μg/mL. The inhibition dynamics ofQuercetin and Curcumin expressed by Bi-reciprocal diagram indicated that the change ofVmax almost effected nothing to Km, Therefore , such a conclusion can be drawn that thethese two effects is non-competition inhibitor, and the inhabitation constant are 1.886μg/mLand 1.4549μg/mL respectly. The thesis about pest-inhabitation by applying Magnolol andOsthole has proposed long time for years, however, in pest field were scarce. The inhabitationmechanism of Magnolol and Osthole to PPO of the Motsoh worm appears to benon-competition . Thereinto, the Magnolol set phosphate as buffer solution can reflect thereaction which catalyzes L dopamine. However, a further analysis about the effect of Ostholeneed judge the influence from carbinol to enzemy, under the condition that Osthole wassoluted by carbinol.更多还原