四川盆地部分地区土壤中苏云金芽胞杆菌分离与cry基因鉴定

摘要

[Objective] The resources of Bacillus thuringiensis in Sichuan Basin were studied to understand their distribution characteristics and prepared for their use in the future. [Method] Soil samples were collected from five regions of Sichuan Basin to screen the Bt strains. The genotypes and insecticidal crystal protein (ICPs) of the strains isolated were identified by PCR-RFLP and SDS-PAGE, respectively. The toxicity of the Bt strains to Plutella xylostella and Colaphellus bowringii were evaluated in the lab. [Result] Twenty-six wild strains of Bacillus thuringiensis were isolated from 207 soil samples, which accounted for 12.56％ of the soil samples tested. Various shapes of parasporal crystals, including spherical, square, bipyramidal and irregular, were observed in the strains.The genes cry1, crylI, cry2 and cry9 were found in 21 strains, and most of them contained more than one gene.However, no known genes were detected in the other five strains. Nine strains expressed the protein of 130 ku,and two strains expressed the protein of 124 ku, and two strains expressed the protein of 45 ku. But six strains expressed both 130 ku and 60 ku proteins, four strains expressed both 124 ku and 60 ku proteins, two strains expressed both 135 ku and 60 ku proteins, and one strain simultaneously expressed 130 ku, 90 ku and 75 ku proteins.The bioassay results of the 26 strains indicated that nine of them were highly toxic to Plutella xylostella and two of the nine strains showed some toxicity to Colaphellus bowringii larvae. [Conclusion] There are rich Bt resources in Sichuan Basin, and 2 Bt stains isolated from there showed high prospective value.%[目的]对四川盆地土壤中Bt资源进行研究,了解其分布特点,为我国苏云金芽胞杆菌的储备奠定基础.[方法]从四川盆地5个地区采集土样,分离苏云金芽胞杆菌野生菌株.利用PCR-RFLP及SDS-PAGE方法对Bt 分离株进行墓因型鉴定和蛋白分析,并对其室内杀虫活性进行初步测定.[结果]从207份土样中获得26株苏云金芽胞杆菌吁生菌株,出菌率为12.5%.油镜观察可看到球形、方形、菱形及不规则形的伴胞晶体.在21株Bt分离株中检测到.cryl、crylI、cry2、cry9四类基因,且多数菌株都是同时含有多个基因类型,有5株Bt分离株未发现已知墓因;SIBS-PAGE显示有9株菌株只表达了130 ku的蛋白,2株只表达了124 ku的蛋白,2株只表达了45 ku 的蛋白,6株同时表达了130 ku和60 ku的蛋白,4株同时表达了124 ku和60 ku的蛋白,2株同时表达了135 ku 和60 ku的蛋白,1株同时表达了130、90 ku及75 ku的蛋白.毒力测定结果表明:9株菌株对小莱蛾具有高毒力,2株对大猿叶虫有一定的杀虫活性.[结论]四川盆地土壤中Bt资源多样性丰富,其中有2株有一定的研究前景.