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研究报告 Research Report 对鳞翅目昆虫小菜蛾高毒力的苏云金芽孢杆菌的鉴定 梁照东1,2钟昊1,2周燕2* 李有志1* 1 广西大学生命科学与技术学院, 南宁, 530005 2 海南省热带农业资源研究所, 三亚, 572025 *通讯作者： 摘 要 本研究利用从黑龙江凉水国家自然保护区的土壤中分离出来的31株Bt野生菌株作为研究菌株，对重要的鳞翅目农业害虫小菜蛾进行高毒力菌株的筛选。经生物活性定性测定，筛选出对小菜蛾活性较高的10株Bt菌株。镜鉴观察表明，这些野生菌株在芽孢形成期均产生对鳞翅目小菜蛾有毒性的菱形晶体伴孢蛋白。cry基因型鉴定表明，这10株菌株均含有cry l类基因。进一步SDS分析表明，这10株菌株表达约130kDa的蛋白。对菌株的生长曲线进行分析，发现2-14h时Bt菌株处于对数生长期，16-30h时Bt菌株处于稳定期，32h时Bt菌株进入衰亡期。其中，菌株S2685-1和S2737-3产生大约130kDa的晶体蛋白起始时间分别为培养22小时及20小时，前者到培养26h时蛋白表达量基本稳定，后者其表达量随时间而逐步增加，到22h时蛋白的表达量趋于稳定状态。对小菜蛾的半致死浓度(LC50)和半致死时间(LT50)测定显示，S2685-1对小菜蛾的LC50为17.113 ng·ml-1，LT50为37.343h；S2737-3对小菜蛾的LC50为25.782 ng·ml-1，LT50为36.825h。而标准菌株HD-73对小菜蛾的LC50为15.414 ng·ml-1，LT50为35.917h。菌株S2685-1和S2737-3具有较突出的半致死浓度以及半致死时间，两者对小菜蛾有很明显的杀虫活性。从本研究的结果我们认为，Bt菌株S2685-1和S2737-3是两株杀虫活性较为突出，具有一定应用潜力的野生Bt菌株，可望作为标准菌株HD-73的替换菌株，用于作为生产防治鳞翅目害虫的Bt生物农药的出发菌株。 关键词 苏云金芽孢杆菌；小菜蛾；Bt毒蛋白；生物测定 Identification of Highly Toxic Bacillus thuringiensis Isolates to Plutella xylostella Liang Zhaodong1,2 Zhong Hao1,2 Zhou Yan2* Li Youzhi1* 1 College of Life Science and Technology, Guangxi University, Nanning, 530004 2 Hainan Institute of Tropical Agricultural Resources (HITAR), Sanya, 572025 *Corresponding Authors： Abstract In this research, we employed 31 Bacillus thuringiensis isolates from the soils collected in the Liang Shui Natural Reserve of Heilongjiang province to identify the highly toxic to Plutella xylostella, an important Lepidopteran pest in agriculture. The results showed that ten of thirty one Bacillus thuringiensis isolates have high toxicity to Plutella xylostella via the bioassay. Microscopic observation for ten seleted isolates was carried out; the results confirmed that these isolates can generate bi-diamond crystal protein which might be crystalline toxin. The further genotypic analysis by PCR and SDSindicated that the selected 10 Bt isolates were consistent with the genotype of cry1 class toxin genes that can generate 130 kD crystal protein in molecular weight. Whilst, The growth curves of