两株杀鞘翅目害虫Bt菌株的生物活性及杀虫蛋白基因鉴定

Identification of Insecticidal Protein Genes from Two Bacillus thuringiensis Strains with Insecticidal Activity Against Coleopteran Larvae

发掘对鞘翅目害虫具有活性的苏云金芽孢杆菌(Bt)菌株和杀虫蛋白基因在生物防治中日益受到人们的重视。利用杀鞘翅目Bt基因通用引物进行PCR检测与生物测定相结合的方法,从山东省各地土壤中分离的66株Bt中筛选获得了两株对鞘翅目昆虫具有杀虫活性的菌株B-DLL和B-JJX,并采用SDS-PAGE和PCR-RFLP方法对其杀虫晶体蛋白和杀虫基因类型进行了鉴定。结果表明,菌株B-DLL和B-JJX对华北大黑鳃金龟1龄幼虫14 d的校正死亡率分别为86.7%和60.0%,对暗黑鳃金龟1龄幼虫14 d的校正死亡率分别为83.3%和76.7%,对柳蓝叶甲3龄幼虫5 d的校正死亡率分别为33.3%和46.7%。菌株B-DLL的伴孢晶体呈圆形,B-JJX的晶体为近方形,两株Bt的伴孢晶体主要为130 kDa的蛋白,两株Bt均含有新的cry8类基因,而不含cry1、cry2、cry3、cry4/10和cry7等基因。

The development of Bacillus thuringiensis（Bt） strains and their insecticidal protein genes have played a significant role in biological control of insect pests.Based on PCR detection with special cry-gene primers and bioassay,two Bt strains,B-DLL and B-JJX,with insecticidal activity against coleopteran larvae were screened from 66 strains isolated from soil in Shandong Province of China.The expression of insecticidal crystal proteins and the types of cry gene of the two strains were identified with SDS-PAGE and PCR-RFLP method.Results showed that the B-DLL and B-JJX were toxic to the first instar larvae of Holotrichia oblita with the corrected mortality of 86.7% and 60.0%,to the first instar larvae of Holotrichia parallela with the corrected mortality of 83.3% and 76.7%,and to the third instar larvae of Plagiodera versicolora with the corrected mortality of 33.3% and 46.7%,respectively.The B-DLL has parasporal crystal inclusions with spherical shape whereas B-JJX has crystals with cuboidal morphology.Both strains expressed 130 kDa protein and contained novel cry8-type gene,did not contain cry1,cry2,cry3,cry4/10 and cry7 genes.