【作者】 邹传山；

【导师】 王志英；

【作者基本信息】 东北林业大学 ， 森林保护学， 2013， 博士

【摘要】 落叶松毛虫(Dendrolimus superans Butler)又称西伯利亚松毛虫,隶属鳞翅目枯叶蛾科,是我国北方针叶树重要的食叶害虫之一,经常周期性爆发成灾,给林业生产带来严重的损失。目前有关落叶松毛虫的研究主要集中在生活习性、预测预报、化学防治、生物防治、物理机械防治以及诱导抗性选育等方面,而关于落叶松毛虫酶学及生化毒理学的研究尚未见报道。本文从生物化学角度系统分析了乙酰胆碱酯酶、羧酸酯酶、谷胱甘肽S-转移酶、超氧化物歧化酶、过氧化氢酶在落叶松毛虫6龄幼虫体内的分布特征,通过正交试验建立了落叶松毛虫幼虫乙酰胆碱酯酶的最佳反应条件；比较了落叶松毛虫幼虫5种酶对几种常用农药原药及制剂的敏感性,分析了杀虫剂处理后其体内5种酶活性的变化规律。通过离心、硫酸铵沉淀、离子交换层析、亲和层析等方法对乙酰胆碱酯酶和羧酸酯酶进行了纯化,并对纯化前后羧酸酯酶的酶学性质进行了研究,其结果为高效、低残留、环境友好型杀虫剂的筛选及落叶松毛虫生化毒理学研究提供理论依据。1、通过正交试验设计,分析了酶浓度、底物浓度、反应时间、反应温度及pH值等因素对落叶松毛虫乙酰胆碱酯酶活性的影响,最终确定落叶松毛虫乙酰胆碱酯酶反应的最佳条件为酶液质量浓度0.2g/mL、底物浓度0.8mmol/L、反应体系pH7.5、水浴温度35℃和反应时间5min；2、落叶松毛虫6龄幼虫头部、中肠、脂肪和表皮4个组织中AChE、CarE、 GST、SOD及CAT活性的分布特征为：AChE分布相对比较均匀,其中脂肪体中的含量略高于其它组织,占躯体的29.01%,而表皮含量略低于其它组织,占21.16%；CarE主要集中在中肠,占躯体的61.31%,头部分布最少,仅占躯体的6.37%；GST则主要分布在脂肪体中,占躯体的36.15%,头部分布最少,仅占躯体的6.58%；头部的SOD含量最多,占躯体的39.79%,表皮最少,占15.30%；CAT头部含量最少,仅占躯体的3.66%,脂肪体中含量最多,占38.17%；3、测定了农药原药和制剂对落叶松毛虫6龄幼虫5种酶离体活性的影响,其结果显示：在杀虫剂制剂中,2种有机磷(敌敌畏和辛硫磷)对落叶松毛虫幼虫AChE离体活性抑制程度显著强于其它制剂,其抑制中浓度分别为1.050×10-9,7.144×10-8mg/mL;辛硫磷、敌敌畏对落叶松毛虫幼虫CarE离体活性抑制作用最强；敌敌畏制剂对落叶松毛虫幼虫GST抑制作用最强(IC50=8.737×10-6mg/mL),其次为高效氯氟氰菊酯制剂(IC50=7.174×10-3mg/mL);高效氯氰菊酯和高效氯氟氰菊酯杀虫剂制剂对落叶松毛虫SOD活性抑制性最强,其IC50分别为1.879×10-1和6.115×10-1mg/mL,敌敌畏和辛硫磷对SOD离体活力抑制程度最弱,在质量浓度低于0.05mg/mL时表现为诱导激活作用；灭幼脲和阿维菌素对CAT活性抑制最强,IC50分别为2.233mg/mL和7.672mg/mL。在供试农药原药中,2种有机磷原药(敌敌畏、氧化乐果)对落叶松毛虫幼虫AChE离体活性抑制程度显著强于其它原药及制剂,其抑制中浓度分别为4.762×10-3mg/mL和2.369×10-3mg/mL;敌敌畏、氧化乐果对落叶松毛虫幼虫CarE离体活性抑制作用最强；2种拟除虫菊酯类原药(百树菊酯和高效氯氟氰菊酯)对落叶松毛虫幼虫GST离体抑制作用最强；高效氯氟氰菊酯对离体SOD活性的抑制作用最强,而吡虫啉最弱；阿维菌素和高效氯氟氰菊酯对离体CAT活性的抑制作用最强,氧化乐果抑制力最弱。4、利用点滴法低剂量农药原药和制剂处理落叶松毛虫6龄幼虫敏感品系,研究了供试昆虫体内AChE、CarE、GST、SOD和CAT5种酶活性的变化与处理时间的关系。结果表明,原药与制剂处理落叶松毛虫6龄幼虫后体内5种酶活性均发生了相应变化,且存在显著的时间效应,但处理时间与酶的比活力并非呈线性关系。在同一时间点,杀虫剂的种类、作用机理、杀虫剂作用时间与5种酶的比活力密切相关。5、采用硫酸铵沉淀、凝胶层析和亲和层析等方法分离纯化了落叶松毛虫6龄幼虫体内的AChE和CarE,并比较分析了粗酶液和纯化CarE的生化特性。结果表明,60%硫酸铵沉淀、Sephadex G-25凝胶层析、Procainamide与CEA亲和层析能够将落叶松毛虫幼虫AChE从杂蛋白中较好的纯化,但60%硫酸铵沉淀和Sephadex G-25凝胶层析纯化效率较低,纯化倍数分别为1.645和11.297倍。CEA亲和层析纯化效果好于Procainamide亲和层析,其纯化后的比活力达162.866μmol/min-mg protein,纯化的倍数为115.344倍,回收率为16.510%。利用硫酸铵梯度沉淀、凝胶层析、DEAE纤维素离子交换层析和羟基磷灰石亲和层析等技术纯化了落叶松毛虫6龄幼虫CarE,得到纯化倍数为138.348倍,回收率为2.782%的目的蛋白CarE,通过SDS-PAGE估算其分子量约为84.78kD。以a-NA和p-NA为底物,经动力学分析落叶松毛虫幼虫粗酶液CarE的Km值分别为8.601×10-5和3.775×10-4mol/L,是纯化酶液的1.989和2.337倍,而CarE纯化酶液的Kcat分别为5.824×101和2.306×102min-1,是粗酶液的106.084和135.012倍；落叶松毛虫6龄幼虫粗酶液和纯化酶液离体CarE对敌敌畏、阿维菌素、高效氯氟氰菊酯均较敏感。敌敌畏对CarE活性抑制作用最强,其对纯化酶液CarE的ICso为1.105×10-4mg/mL,是粗酶液33.394倍,而阿维菌素和高效氯氰菊酯对粗酶液CarE的IC50分别为1.259×10-2和4.529×10-1mg/mL,是纯化酶液的1.228和23.214倍。更多还原

【Abstract】 Dendrolimus superans (Butler)(Lepidoptera:Lasiocampidae) named Siberian caterpillar in English, is one of important defoliator to boreal conifer, which periodically outbroke and led to serious losses to the forestry production. Presently, researches on D. superans (Butler) focused on its living habits, prediction the forecast, chemical control, biological control, physical and mechanical prevention and treatment, and induced resistance utilization, etc. The studies on the enzymology and biochemical toxicology have not been reported to date. In this paper, activity distribution characteristics of acetylcholinesterase, carboxylesterase, glutathione S-transferase, superoxide dismutase and catalase in D. superans6th instar larvae were systematically analyzed on the biochemical perspective. The optimal reaction condition of acetylcholinesterase was established by orthogonal experiment. Sensitivities of five enzymes to several insecticide preparations and raw insecticides were compared in vitro and the activity variations of five were analyzed in vivo. Acetylcholinesterase and carboxylesterase were purified by a series steps of centrifugation, ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, etc. Enzymatic properties of purified and unpurified carboxylesterases had been investigated. All the results will provide a theoretical basis for the selection of efficient, low-residue, environment-friendly insecticide, and the studies on biochemical toxicology of D. superans.1. The impact of enzyme and substrate concentration, reaction time, reaction temperature and the pH on acetylcholinsterase activity was analyzed by orthogonal designed experiment, and the optimum conditions for acetycholinsterase had been ultimately determined as0.2g/mL enzyme concentration,0.8mmol/L substrate concentration, pH7.5reaction system,35℃bath temperature and5min reaction time.2. Activity distribution of AChE, CarE, GST, SOD and CAT in four tissues (head, midgut, fat body and epidermis) of the D. superans6th instar larvae were characterised. AChE distributed relatively uniformly, activity in the fat body was slightly higher than in other tissues, accounting for29.01%of the body, while activity in the epidermal content slightly less than other tissues, accounting for21.16%. Activity of CarE mainly concentrated in the midgut, accounting for61.31%of the body, while the head had the least, only accounting for6.37%of the body. The fat body and the head had the largest and the least activity of GST, accounting for36.15%and6.58%of the body, respectively. The most activity of SOD was detected in the head, accounting to39.79%of the body, while the least was detected in the epidermis, accounting to15.30%. For the activity of CAT, the head accounted for3.66%and the fat body accounted for38.17%of the body, which were the tissues with the highest and the lowest activity of CAT, respectively.3. The influence of several insecticide preparations and raw insecticides on five enzymes from D. superans (Butler)6th instar larvae had been measured in vitro. Results showed that among insecticide preparations, two organophosphates (dichlorvos and phoxim) had significantly stronger inhibition to AChE activity than any other tested insecticide preparations, whose IC50values were1.050×10-9and7.144×10-8mg/mL respectively; phoxim and dichlorvos also showed strongest inhibition to CarE activity; dichlorvos had the strongest inhibition to GST activity (IC50=8.737×10-6mg/mL), followed by lambda-cyhalothrin with an IC50of7.174×10-3mg/mL; cypermethrin and lambda-cyhalothrin showed the strongest inhibition to SOD activity, whose IC50werel.879×10-1and6.115×10-1mg/mL respectively. Whereas dichlorvos and phoxim showed the weakest inhibition to SOD activity. Moreover, these two insecticide formulations showed inducing activation as the concentration dropped below0.05mg/mL; diflubenzuron and avermectin showed strongest inhibition CAT activity, with IC50values of2.233and7.672mg/mL, respectively. Among raw insecticides, two organophosphorus (dichlorvos, omethoate) showed stronger inhibition than any other raw insecticides to AChE, whose IC50values were4.762×10-3and2.369×10-3mg/mL respectively. Raw dichlorvos and omethoate had the strongest inhibition to CarE activity. Two raw pyrethroid insecticides (beta-cyfluthrin and lambda-cyhalothrin) showed the strongest inhibition to GST activity. The raw lambda-cyhalothrin had the strongest inhibition to in vitro SOD activity, while raw imidacloprid had the weakest inhibition. Raw abamectin and lambda-cyhalothrin showed the strongest inhibition to CAT activity, and inhibition by the raw omethoate was the weakest.4. After being treated with low-dose insecticde preparations and raw insecticdes through topical application, AChE、CarE、GST、SOD and CAT activity of D. superans (Butler) larvae susceptible strain had been investigated in vivo. Results showed that the activities of the five enzymes changed correspondingly after being treated with either insecticde preparations or raw insecticdes, and there was not a linear relationship between treatment time and the specific activity of the enzyme. At the same time point, the specific activities of five enzymes were closely related to the kind, mechanism and treated time of insecticide.5. AChE and CarE of D. superans (Butler)6th instar larvae were separated and purified by a series of setps of ammonium sulfate precipitation, gel chromatography and affinity chromatography, and the biochemical characteristics of the crude and purified CarE were analyzed. Results revealed that AChE had been purified successfully from the crude protein. However, the fold purification of60%ammonium sulfate precipitation and Sephadex G-25gel filtration chromatography were1.645and11.297, respectively, indicating a low purification efficiency. The purification of AChE by CEA affinity chromatography was much better than by Procainamide affinity chromatography. The specific activity of the purified AChE reached162.866μmol/min-mg protein, purified-fold attained to115.344, and the yield was16.510%.CarE from D. superans6th instar larvae was purified by gradient ammonium sulfate precipitation, gel chromatography, DEAE cellulose ion exchange chromatography and hydroxyapatite affinity chromatography, and the final purified CarE was obtained with138.348-fold and2.782%-yield to the crude homogenate. The molecular weight of CarE was estimated to be84.78kD approximately by SDS-PAGE. Km values of crude CarE were8.601×10-5and3.775×10-4mol/L respectively by using alpha-NA and beta-NA as the substrate of kinetic analysis of CarE from D. superans larvae, which were1.989-fold and2.337-fold of the purified. CarE, while the Kcat values of purified CarE were5.824×101and2.306×102min-1respectively, which were106.084-fold and135.012-fold of crude one. Both the crude and the purified CarE of D. superans6th instar larvae were sensitive to dichlorvos, avermectin and lambda-cyhalothrin in vitro. Dichlorvos showed the strongest inhibition to the activity of CarE, and the IC50value to the purified CarE was1.105×10-4mg/mL, which was33.394-fold of the crude one. However, the IC50values of avermectin and lambda-cyhalothrin to the crude CarE were1.259×10-2and4.529×10-1mg/mL, which were1.228-fold and23.214-fold of the purified CarE respectively.更多还原