【作者】 吴洪福；

【导师】 高继国；

【作者基本信息】 东北农业大学 ， 植物学， 2007， 硕士

【摘要】 金龟子属于鞘翅目金龟总科(Scarabaeoidae)，其幼虫(俗称蛴螬)是一类重要的世界性分布的地下害虫，可危害多种农林植物。20年来全国各地的大量调查表明，蛴螬在地下害虫中危害居首位。本文围绕对鞘翅目暗黑鳃金龟(Holotrichia parallela)铜绿丽金龟(Anomala carpulenta)的幼虫有活性的苏云金芽孢杆菌Cry8Ea1和Cry8Ca2蛋白的表达和纯化进行了研究。HD8E和HBF-1菌株是苏云金芽孢杆菌的菌株，分别含有cry8Ea1和cry8Ca2的基因，编码Cry8Ea1和Cry8Ca2的原毒素(伴孢晶体)。研究表明，两个菌株表达的伴孢晶体均为球形晶体，相对分子质量为130kU。用胰蛋白酶和胰凝乳蛋白酶消化得到60kU的活性毒素，利用分子筛层析(Superdex200)收集60kU的毒素，然后使用阴离子交换层析进一步纯化得到纯的60kU的毒素。对纯蛋白进行生物活性测定，结果表明Cry8Ea1的原毒素与毒素对暗黑鳃金龟幼虫均具有较高活性，Cry8Ea1原毒素的LC50值为9.47μg／克土，胰凝乳蛋白酶消化后纯化的毒素的LC50值为35.39μg／克土；Cry8Ca2的原毒素和毒素对铜绿丽金龟幼虫也具有很高的活性，Cry8Ca2原毒素的LC50值为0.47μg／克土，胰凝乳蛋白酶消化后纯化的毒素的LC50值为0.08μg／克土，胰蛋白酶消化后纯化的毒素的LC50值为0.003μg／克土。本研究建立了Cry8Ea1与Cry8Ca2活性毒素蛋白的纯化方法，通过该方法可大量获得该毒素的纯品，为进一步研究该蛋白的结构与功能，揭示毒素的杀虫机理奠定了良好的基础。研究中还发现Cry8Ea1和Cry8Ca2蛋白是与DNA相结合的。在本实验中对DNA的作用进行了研究，用透析的方法分别对两种毒素的DNA复合体和去除了DNA的毒素在不同pH条件及不同缓冲液条件下的稳定性进行了比较，结果表明，Cry8Ea1和Cry8Ca2毒素与DNA的复合体的稳定性要比不含DNA的毒素稳定，不易发生沉淀，DNA在维持毒素稳定性中起到重要作用。更多还原

【Abstract】 The Cockchafer larvae are important underground pest that make damage in agriculture.Cry8Ea1and Cry8Ca2 protein expressed in Bacillus thuringiensis are toxic to Cockchafer larvae.In the studs; we investigated the expression and purification of these two proteins and the lethalconcentration 50 percent of both protein was also evaluated.HD8E and HBF-1 are Bt strains, with genes that encoding Cry8Ea1and Cry8Ca2 protoxinseperatly. The protoxin expressed by these two Bt strains were analysed by SDS-PAGE. One majorband with an estimated molecular mass of 130 kU was detected. Cry8Ea1 and Cry8Ca2 protoxinwere activated by trpsin or chymotrypsin and the toxin with molecular mass of 60kU were obtained.Both toxin was purified by size exclusion chromatography first and further by anion exchangechromatography. The purification method of Cry8Ea1 and Cry8Ca2 toxin were established in thisstudy. Great amount of pure toxin of Cry8Ea1 and Cry8Ca2 can be obtained by using this method.It was a great foundation for futher investigation on structure and function of these two protein.The insecidical activity of both pure toxin was tested by bioassays against scarab. Cry8Ea1protoxin and toxin are both toxic to Holotrichia parallela (scarab). The lethal concentration 50percent of of Cry8Ea1 protoxin was 9.46μg per gram soil, while that of the Cry8Ea1 toxin activatedby chymotrypsin was 35.39μg per gram soil. Cry8Ca2 protoxin and toxin are both toxic toAnomala carpulenta (scarab). The lethal concentration 50 percent of Cry8Ca2 protoxin was 0.47μgper gram soil, while that of Cry8Ca2 toxin activated by trypsin was 0.003μg per gram soil,Cry8Ca2 toxin activated by chymotrypsin was 0.08μg per gram soil.更多还原