Abstract
The gene encoding the 66-kDa entomocidal protein (P2 protein or mosquito factor) from Bacillus thuringiensis var. kurstaki has been isolated by the use of a 62-mer oligonucleotide probe that encoded 21 amino acids of the P2 protein NH2 terminus. The DNA sequence of the gene, designated cryBI, was unique from the published sequences of other B. thuringiensis genes. However, the amino acid sequence of the P2 protein, as deduced from the DNA sequence of the cryBI gene, was found to contain a sequence of 100 amino acids having 37% homology to a group of B. thuringiensis entomocidal proteins, the P1 proteins. Late stationary phase Bacillus megaterium cells harboring the cloned B. thuringiensis cryBI gene contained large aggregates of the P2 protein, and the cells were highly toxic to both lepidopteran and dipteran larvae. In contrast, Escherichia coli cells harboring the cloned cryBI gene contained very low levels of the P2 protein. DNA blot hybridization experiments showed that certain B. thuringiensis strains contained at least one cryBI-related DNA sequence in addition to the cryBI gene itself.
Highlights
The gene encoding the66-kDa entomocidal protein toxicity of unsolubilized, intact P2 crystals has not been (P2 protein or mosquito factor) fromBacillus thurin- described
We report that Bacillus megaterium cells harboring the B. thuringiensis cryBI gene produce crystalline inclusions that are insecticidal for both lepidopteran and were highly toxic to both lepidopteran and dipteran dipteran species
Purification of the P2 Endotoxin and NHz-terminulAmino Acid Sequence Determinution-Two major proteins with mosize-specificDNA fragments wereexcised.DNA fragments were lecularmasses of 130 kDa (P1protein) and 66 kDa (P2 separated from gel slices byelectroelution.A cryBI-enriched plasmid library was constructedby ligating size-selectedDNA fragments into plasmid pBR322 (Bolivar et al, 1977) that had been digested with
Summary
The gene encoding the66-kDa entomocidal protein toxicity of unsolubilized, intact P2 crystals has not been (P2 protein or mosquito factor) fromBacillus thurin- described. Kurstaki P1 proteins (Schnepf et al, 1985; Adang et al, 1985;Shibano et al, 1985),nor was the cryBI gene homologous to the gene that encodes the 28-kDa protein, reported to be toxic to mosquito larvae (Ward et al, 1984), from B. thuringiensis var.
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