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Cloning of bacillus thuringiensis var kurstaki insecticidal protein gene


, : Cloning of bacillus thuringiensis var kurstaki insecticidal protein gene. Han Guk Journal of Genetic Engineering 1(1): 29-36

Eight varieties of Bacillus thuringiensis contained extrachromosomal (covalently closed circular) DNAs with various molecular weights. The number and masses of the various plasmids harboring in the varieties were determined by 0.7% agrose gel electrophoresis. B. thuringiensis var. finitimus contained one DNA element by the gel analysis var. galleriae two elements, var. tolworthi four elements, var. kyushuensis eleven elements, var. darmstadiensis one large DNA element, var. parkistani two small DNA elements, var. israelensis eight elements and var. kurstaki twelve elements. Their relative molecular weights ranged from 0.81 to 275.421 Md. B. thuringiensis var. kurstaki produced insecticidal crystal protein in their cells during sporulation. The insecticidal crystals were observed under phase contrast microscope, and separated and purified successfully with 50 to 80% renografin gradient centrifugation. The plasmid DNA elements harbouring in B. thuringiensis var. kurstaki were isolated and digested with restriction endonucleases, Pst I, Bam HI, Eco RI, and Sal I. The 25.41 Md Eco RI DNA fragment from the digested plasmid fragments was ligated into vector pUC 8 Eco RI site. The recombinant plasmid was named pKL-3, 5 and 7. The sonificated three clones successfully killed Bombyx mori larvae as much as the wild type kurstaki strain. The crystal protein gene in the pKL-3, 5 and 7 clones was expressed and produced the insecticidal proteins in the E. coli cells, which were aggregated, not crystalline, and were visible under a light microscope.

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