Highly toxic and broad-spectrum insecticidal localBacillusstrains engineered using protoplast fusion

Abstract

Protoplast fusion was performed between a local Bacillus thuringiensis UV-resistant mutant 66/1a (Bt) and Bacillus sphaericus GHAI (Bs) to produce new Bacillus strains with a wider spectrum of action against different insects. Bt is characterized as sensitive to polymyxin and streptomycin and resistant to rifampicin and has shown 87% mortality against Spodoptera littoralis larvae at concentration of 1.5 × 107cells/mL after 7 days of feeding; Bs is characterized as resistant to polymyxin and streptomycin and sensitive to rifampicin and has been shown to have 100% mortality against Culex pipiens after 1 day of feeding at the same concentration as that of Bt. Among a total of 64 Bt::Bs fusants produced on the selective medium containing polymyxin, streptomycin, and rifampicin, 17 fusants were selected because of their high mortality percentages against S. littoralis (Lepidoptera) and C. pipiens (Diptera). While Bt harboured 3 plasmids (600, 350, and 173 bp) and Bs had 2 plasmids (544 and 291 bp), all the selected fusants acquired plasmids from both parental strains. SDS–PAGE protein analysis of the 17 selected fusants and their parental strains confirmed that all fusant strains acquired and expressed many specific protein bands from the 2 parental strains, especially the larvicidal proteins to both lepidopteran and dipteran species with molecular masses of 65, 70, 80, 88, 100, and 135 kDa. Four protein bands with high molecular masses of 281, 263, 220, and 190 kDa, which existed in the Bt parental strain and did not exist in the Bs parental strain, and 2 other protein bands with high molecular masses of 185 and 180 kDa, which existed in the Bs parental strain and did not exist in the Bt parental strain, were expressed in most fusants. The results indicated the expression of some cry genes encoded for insecticidal crystal proteins from Bt and the binary toxin genes from Bs in all fusant strains. The recombinant fusants have more efficient and potential values for agricultural application compared with both the insecticidal Bt and the mosquitocidal Bs strains alone against S. littoralis and C. pipiens larvae, respectively.