The Expression of a Recombinant cry1Ac Gene with Subtilisin-Like Protease CDEP2 Gene in Acrystalliferous Bacillus thuringiensis by Red/ET Homologous Recombination

Abstract

A novel cDNA encoding the subtilisin-like serine protease gene CDEP2 was isolated from Beauveria bassiana by reverse transcription polymerase chain reaction (RT-PCR). It contained an 1137 bp ORF that predicted a protein of 379 amino acids with M = 38863 Da and pI = 8.21. In an attempt to improve insecticidal activity, the CDEP2 gene and the cry1Ac gene from Bacillus thuringiensis were co-fused into the vector pHT315 as pHAc–CDEP2 plasmid by Red/ET homologous recombination. The co-fusion gene was attempted under the control of the native cry1Ac promoter. Plasmid pHAc–CDEP2 was electro-transformed into the B. thuringiensis subsp. kurstaki Cry⁻B. Analyzed by SDS-PAGE and Western blotting, the transformant Cry⁻B–pHAc–CDEP2 strain produced a 130 kDa Cry1Ac protein and 39 kDa CDEP2 protein. The 50% lethal concentration values (LC₅₀) of Cry⁻B–pHAc–CDEP2 strain (8.5 μl/ml) to Helicoverpa armigera third instars larvae was clearly higher than the Cry⁻B–pHAc strain (16.7 μl/ml) at 72 h.