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

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 9.47 microg/mL, nearly equal to the original strain YBT-1518 (10.74 microg/mL).