Highly toxic and broad-spectrum insecticidal Bacillus thuringiensis engineered by using the transposon Tn917 and protoplast fusion

The chromosome of the Bacillus thuringiensis strain S184 that was toxic aga inst the third instar larvae of Spodoptera litura with the LC50 of 9.74 mug /ml was successfully integrated into two genes of cyt1Aa and cry11Aa using the transposon Tn917, yielding the primary engineered strain TnX. The strai n TnX was highly toxic to the third instar larvae of Culex pipiens fatigans with the LC50 of 5.12 ng/ml which was 1.82-fold higher than that of B. thu ringiensis subsp. israelensis, but lowly toxic to lepidopterous larvae. By the protoplast fusion of the strain TnX and the strain S184-Tet(r) (resista nce to tetracycline), the target engineered strain TnY was obtained. Agains t the third instar larvae of S. litura, the strain TnY LC50 was of 4.68 mug /ml and increased by 2.08-fold in comparison with the parent strain S184. A gainst the third instar larvae of C. pipiens fatigans, the strain TnY LC,, was of 103.20 ng/ml. The two target genes of cyt1Aa and cry11Aa integrated into the chromosome were extremely stable and had little possibility of a s econd transposition. It was unclear whether some factors existing in the pa rent strain, S184, contributed to the high toxicity of the strains TnX and TnY.