Cloning and expression of nematicidal crystal protein gene cry6Aa of Bacillus thuringiensis

Abstract:

On the basis of the sequencing of the N-terminal amino acid of the crystal protein, a nucleotide acid fragment harboring a novel nematicidal gene cry6Aa2 was obtained from Bacillus thuringiensis strain YBT-1518. This fragment contains two ORFs: orf1 and orf2, while a “stem-loop" exists between orf1 and orf2. BLAST showed the similarity of orf1 nucleotide acid sequence with cry6Aa1 is 98%, and has been deposited in the Genbank database (Acc. No. AF499736). The cloning fragment was transferred to crystal negative mutation strain BMB171 by E. coli-Bt shuttle vector pHT304. A 54kDa protein with similarity to strain YBT-1518 was detected in recombinant strain, and rice shaped crystal was detected with transmission electron microscope. Bioassay indicated the LC₅₀ of recombinant strain against second lavae juvenile of Meloidgyne hapla is 9.47μg/mL, nearly equal to the original strain YBT-1518 (10.74μg/mL).

Key words:Bacillus thuringiensis; Nematode; cry6Aa2 gene; gene cloning; expression