Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebrionis

摘要: Abstract The insecticidal crystal protein gene of the coleopteran-toxic Bacillus thuringiensis var. tenebrionis has been isolated, and nucleotide sequence determined. A total DNA library from was made in plasmid vector pUC12. By using a synthetic 27-base oligonucleotide corresponding to stretch nine N-terminal amino acids tryptic fragment purified as probe, recombinant colonies were screened by situ hybridization for presence gene. Positive clones obtained this screening further tested toxicity. One recombinant, NSBP544 (which contained 5.9-kilobase BamHI insert), toxic larvae Colorado potato beetle. Immunoblot analysis revealed that clone produces two crystal-specific antigens 65 73 kDa do sporulating cells. However, inclusions contain primary peptide component kDa. 1932-base-pair open reading frame with coding capacity 73,119 Da identified sequencing cloned protein. In addition, mung bean nuclease mapping indicates transcription initiates 130 base pairs upstream translational start site. Southern blot an internal 0.7-kilobase EcoRI pNSBP544 probe is located on 90-MDa plasmid.