海南岛吊罗山杀棉铃虫Bt菌株的筛选

摘要

[目的]从海南岛吊罗山原始热带雨林区筛选分离杀棉铃虫Bt菌株,为研发新的防治棉铃虫的生物农药和抗虫转基因棉花奠定基础.[方法]在吊罗山原始热带雨林区不同海拔高度采集253份土壤样品,采用醋酸钠高温处理方法从中分离各类芽孢杆菌,通过显微镜观察鉴定出产伴胞晶体蛋白的苏云金芽孢杆菌(Bt),对伴胞晶体蛋白进行SDS-PAGE电泳和质谱分析,了解其基本特性.并以棉铃虫二龄幼虫为靶标,测定获得的Bt菌株对棉铃虫的杀虫活性.[结果]从253份土壤样品中分离出芽孢杆菌786株,其中产伴胞晶体蛋白的Bt菌株33株,Bt菌株分离率为4.2％.SDS-PAGE电泳和质谱分析表明,分离Bt菌株含有的伴胞晶体蛋白与已发现的毒素蛋白不同,可能为新型晶体蛋白;利用棉铃虫二龄幼虫进行Bt分离株生物活性测定,结果表明有8株Bt菌株对棉铃虫幼虫的致死率为25％～85％.[结论]从海南岛吊罗山原始热带雨林区筛选分离获得8株杀棉铃虫的Bt菌株,这些菌种可能含有新的杀虫晶体蛋白基因.%[Objective] This research aimed to select Bacillus thuringensis (Bt) with insecticidal activity against cotton bollworm (Helicoverpa armigera) from tropical rain forest reserves of Diaoluoshan to provide new insecticidal protein genes for microbial biopesticide and transgenic crops.[Method] Soil samples were collected from Diaoluoshan at different heights above sea level.The Bacillus isolates were harvested following the approach of selection culture medium with sodium acetate at high temperature.Bt strains producing parasporal crystal were recognized by microscope.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry were used to identify Bt isolates.Then,bioassay was carried out to detect the insecticidal activity of Bt crystalline inclusion.[Result] A total of 786 Bacillus were obtained from 253 soil samples,and 33 Bacillus isolates were further identified as Bt isolates with isolation rate of 4.2 ％.The bioassay against the second instar larva of Helicoverpa armigera indicated that 8 Bt isolates out of 33 strains showed insecticidal activity of 25％-85％ against lavas of cotton bollworm.[Conclusion] Eight Bt isolates were identified with insecticidal activity against lavas of the cotton bollworm.They may have new insecticidal genes and provide application possibility for cultivating transgenic cotton.