Cloning, prokaryotic expression and insecticidal assay of cry1Ac-N gene from Bacillus thuringiensis

• 1、College of Plant Protection，Nanjing Agricultural University, Nanjing 210095

Abstract：In this study, we ascertained the existence of cry1Ac-N gene in one Bacillus thuringiensis strain HD-73 by PCR-RFLP. The ORF and full-length of this gene were amplified by PCR, and the Accession No. in Genbank is GU294785. With the pET expression system, the protein Cry1Ac-N was expressed successfully. Results indicated that the cry1Ac-N-encoding protein has amino acid sequence homology of 99.7% with several other anounced Cry1Ac-N-like sequences, and the molecular weight was 133 KDa. Protein structural analysis showed that Cry1Ac-N contains three domains: endotoxin_N、endotoxin_M and endotoxin_C. We also extracted Cry1Ac-N protoxin from Bacillus thuringiensis HD-73 by means of isoelectric precipitation, finding that it had identical molecular weight to the recombinant protein. Subsequent bioassays for insecticidal activity of expression products were carried out using leaf-soaking method. They both showed high toxicities against Spodoptera litura and Plutella xylostella with LC50 of 2.334 μg/ml and 4.444 μg/ml, respectively.

Keywords： plant protection Bacillus thuringiensis cry1Ac-N gene cloning and prokaryotic expression insecticidal bioassay