Abstract: Bradysia odoriphaga is an important agricultural pest, whose larvae have caused serious damage to various crops such as leeks. Bacillus thuringiensiss train with high toxicity to B. odoriphaga larvae has been identified in our laboratory, but its insecticidal protein type is not yet clear. In this study, we sequenced the genome sequence of Bt and inferred that cry39A-like and cry40-like genes might have insecticidal activities. The full length sequence of the cry gene was obtained by PCR amplification with specific primers. Sequence analysis of the cry gene was performed by bioinformatics software. Finally, the Cry protein was obtained by transferred the cry gene into E. coli Rosetta (DE3) and its insecticidal activity of the larvae of B. odoriphaga was determined. Analysis of cry39A-like gene showed that cry39A-like gene has an open reading frame (ORF) of 1986 bp encoding a protein of 661 amino acids (aa). The result of phylogenetic tree analysis showed a closely evolutionary relationship between cry39A-like gene and cry39Aa1, with the protein sequence identity of 94.10%, suggesting that cry39A-like gene belongs to the third classification class. The Cry39A-like protein has a molecular weight of 76 kDa, and has insecticidal activity against B.odoriphaga larvae, with LC50 value of 89.42 μg/mL. The ORF of cry40-like gene is 2001 bp and encodes a protein of 666 aa. The phylogenomic analysis showed that cry40-like gene is clustered with the cry40 gene family, with the highest protein sequence identity of 70.88%, which indicated the second class position of cry40-like gene. The Cry40-like protein is 74.5 kDa, and exhibited LC₅₀ value of 126.83 μg/mL against B. odoriphaga larvae. These findings indicate that cry39A-like and cry40-like genes in Bt could be used as novel insecticidal gene resource for the biological control of B.odoriphaga.

Key words: Bacillus thuringiensis, cry39A-like, cry40-like, Bradysia odoriphaga, insecticidal activity