苏云金芽孢杆菌与蜡状芽孢杆菌亲缘关系研究

Study on genetic relationship between bacillus thuringiensis and bacillus cereus

目的探讨苏云金芽孢杆菌(Bt)与蜡状芽孢杆菌(Bc)的亲缘关系,为Bt鉴定、安全性评价及降低其致病风险等提供科学依据。方法采用肠杆菌基因间重复一致序列-PCR(ERIC-PCR)技术,对6株苏云金芽孢杆菌和3株蜡状芽孢杆菌及对照菌株的基因组DNA进行扩增,对其指纹图谱进行分析;回收并克隆重复性好的苏云金芽孢杆菌基因组DNA扩增片段,以其为探针,分别与供试菌株基因组DNA进行杂交。结果与蜡状芽孢杆菌相比,苏云金芽孢杆菌菌株间基因组DNA指纹图谱较一致;所有供试苏云金芽孢杆菌菌株均扩增产生一条250 bp左右的片段;苏云金芽孢杆菌与蜡状芽孢杆菌均可扩增产生600 bp左右的共有DNA片段。此外,以苏云金芽孢杆菌500 bp片段为探针与苏云金芽孢杆菌基因组DNA杂交有很好的特异性。结论肠杆菌基因间重复一致序列-PCR指纹图谱可以正确反映苏云金芽孢杆菌与蜡状芽孢杆菌亲缘关系;500 bp片段可以作为苏云金芽孢杆菌鉴定探针。

Objective To study the genetic relationship between bacillus and bacillus cereus, and lay the foundation for identification and safety use of B. thuringiensis. Methods The genome DNA of 6 B. thuringiensis, 3 B. cereus and other contrast strains was amplified by the enterobacteia repetitive intergenic consensus sequences - based PCR. DNA fingerprints were got and analyzed. Amplified fragments from B. thuringiensis were retrieved, cloned and marked as probes to hybridizate with tested DNA. Results DNA fingerprints were varied between B. thuringiensis and B. cereus. DNA fingerprints of B. thuringiensis were consistent comparatively and about a 250bp fragment could be amplified from all tested B. thuringiensis strains. DNA fingerprints of B. cereus varied from each other, but a 600bp fragment could be got both in B. thuringiensis and B. cereus strains. The hybridization result of 500bp fragment with the genome DNA of B. thuringiensis showed a great specificity. Conelusion Genetic relationship between B. thuringiensis and B, cereus can be obtained by enterobacteia repetitive intergenic consensus sequences - based PCR fingerprint, which can be used in the study on the genetic relationship of B. thuringiensis and B. cereus. B, thuringiensis can be identified according to 500bp amplified fragment.