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在鉴定苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)Btc001菌株cry基因型的基础上,构建了Btc001菌株质粒DNA Hind Ⅲ片段的文库,并利用聚合酶链式反应-限制性酶切片段多态性(PCR-RFLP)方法筛选出含有cry1Cb全长基因的13.5kb大片段,酶切分析得到该片段的物理图谱,BamHI和EcoRI酶切完成了6.5kb含全长基因的亚克隆,并对这条6.5kb片段亚克隆、测序,序列在国际核酸序列数据库(GenBank)登记(AY007686),并由Bt杀虫晶体蛋白基因国际命名委员会命名为cry1Cb2基因。根据序列设计了一对用于扩增全长基因的引物S5B1CB和S3B1CB,扩增产物插入表达载体pET-21b中,诱导后在大肠杆菌BL21(DE3)中获得高效表达。表达产物对小菜蛾(Plutella xylostella)表现出较高活性,LC_(50)达到7.97μg/ml。
Based on the identification of the cry genotype of Bacillus thuringiensis (Bt) Btc001 strain, a library of plasmid DNA Hind Ⅲ fragment of Btc001 was constructed and its homology was analyzed by polymerase chain reaction - restriction fragment length polymorphism PCR-RFLP) screening of 13.5kb large fragment containing cry1Cb full-length gene, digested with BamHI and EcoRI to complete the 6.5kb subclone containing the full-length gene, and this 6.5 kb fragment was subcloned, sequenced and registered in GenBank (AY007686). The cry1Cb2 gene was named by the International Nomenclature Committee of Bt Insecticidal Crystal Protein Gene. A pair of primers, S5B1CB and S3B1CB, designed for amplifying the full-length gene was designed according to the sequence. The amplified product was inserted into the expression vector pET-21b and expressed efficiently in E. coli BL21 (DE3). The expression product showed high activity against Plutella xylostella, with LC 50 reaching 7.97 μg / ml.