苏云金芽胞杆菌4.0718菌株中杀虫晶体蛋白基因的定位及鉴定

Localization and identification of crystal protein genes in Bacillus thuringiensis strain 4.0718

摘要:

【目的】对苏云金芽胞杆菌（Bacillus thuringiensis, 简称Bt）4.0718菌株中的杀虫晶体蛋白基因（insecticidal crystal protein gene，简称cry基因）进行定位和鉴定, 系统分析高毒力Bt 4.0718菌株的杀虫基因背景。【方法】采用脉冲电泳（PFGE）分离Bt 4.0718菌株的基因组DNA，确定该菌株的PFGE图谱和质粒图谱；采用Southern 杂交分析该菌株中cry基因的定位，并使用PCR及PCR产物限制性片段长度多态性分析（PCR-RFLP）方法鉴定该菌株染色体和质粒上含有的cry基因类型。【结果】确定了Bt 4.0718菌株的PFGE图谱和质粒图谱，鉴定到Bt 4.0718菌株的染色体和质粒上均定位有cry基因，且分别包含cry1Aa、cry1Ac、cry2Aa和cry2Ab 4种基因成分。但染色体上含有的cry基因可能不如质粒上含有的cry基因具有完整的开放阅读框。【结论】首次在Bt 4.0718菌株的染色体上发现有丰富的cry基因，且与质粒上含有的cry基因类型一致。

关键词:苏云金芽胞杆菌;cry基因;定位;脉冲电泳（PFGE）;限制性片段长度多态性分析（RFLP）

Abstract:

[Objective] To localize and identify the insecticidal crystal protein genes (cry genes) in high-toxic Bacillus thuringiensis strain 4.0718. [Methods] The genomic DNA of B. thuringiensis strain 4.0718 was isolated by pulse field gel electrophoresis (PFGE), and the PFGE pattern and plasmid pattern were characterized. The cry genes in the strain were analyzed by Southern blot and the cry gene types on the chromosome and plasmid were identified by PCR and restriction fragment length polymorphism patterns of the PCR-amplified fragments (PCR-RFLP). [Results] Southern blots showed that the cry genes in B. thuringiensis strain 4.0718 are present on both chromosome and plasmid. PCR and RFLP patterns showed that the chromosome and plasmid contain four cry genes, cry1Aa, cry1Ac, cry2Aa, and cry2Ab. However, the cry genes on the chromosome may be incomplete, while the cry1Aa and cry1Ac genes localized on the plasmid were found to contain the full coding sequence. [Conclusion] The abundant cry genes were first found on chromosome of B. thur-ingiensis strain 4.0718, and the cry-type genes are the same as the plasmid.苏云金芽胞杆菌；cry基因；定位；脉冲电泳（PFGE）；限制性片段长度多态性分析（RFLP）

Key words:Bacillus thuringiensis; cry gene; localization;pulse field gel electrophoresis (PFGE);restriction fragment length polymorphism (RFLP)