广西大王岭和大明山自然保护区苏云金芽孢杆菌收集与鉴定

Bacillus thuringiensis Collection and Isolates Identification from Damingshan and Dawangling Natural Reserves in Guangxi Province

从广西大王岭和大明山两个自然保护区共采集到土样264份,共分离出597株芽孢杆菌,通过光学和电子显微镜检观察,16株分离株观察到伴胞晶体蛋白,初步确定为苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt),出菌率为6.06%。在16株Bt分离株中,有4株在芽孢形成过程中能产菱形晶体蛋白,其余12株能产圆形和其他形状的晶体蛋白。利用PCR-RFLP方法和SDS-PAGE方法对16株Bt分离菌进行了蛋白和基因型的鉴定,结果表明,16株分离株中含有4株cry1Ac基因,表达约130kD的晶体蛋白,其中含有cry30基因和cry40基因的菌株分别1株和3株,表达大约75kD的晶体蛋白;另外8株Bt菌株表达蛋白大小不一,其基因型尚不能确定,有待进一步分析。生物测定表明,产菱形晶体含有cry1Ac基因的4株Bt分离株对鳞翅目小菜夜蛾幼虫有很强的毒杀活性,而其它分离株对小菜夜蛾没有毒杀活性。

A total of 264 soil samples were collected from Damingshan and Dawangling Natural Reserves in Guangxi Province of southern China. From those 264 soil samples, 597 Bacillus isolates were harvested and among them, 16 isolates were further identified to be Bacillus thuringiesis using oil lens optical microscope and scanning electronic microscope. The isolation rate of these 264 soil samples is 6.06%. Among the 16 B. thuringiensis isolates, four isolates were observed to produce diamond parasporal crystal proteins and others produce different shapes of crystal proteins including circular and irregular shapes. The selected 16 isolates were analysed using PCR-RFLP and SDS-PAGE to identify their protein and genotype. The results showed that four B. thuringiensis isolates were identi- fied as crylAc-genotype and encoding about 130 kD parasporal crystal protein; three isolates as cry40-genotype and one B. thuringiensis isolate as cry30-genotype, in which both encode about 75 kD parasporal crystal protein. The remaining eight isolates produce different sizes of proteins and genotype cannot be determines, thus further analysis is. Toxicity bioassay was carried out and results indicated that the four B. thuringiensis isolates with cryl genotype exhibit high specific toxicity to Plutella xylostella larvae, but not the other 12 isolates.