BIOCHEMICAL CHARACTERIZATION OF MIDGUT RECEPTOR OF ASIAN CORN BORER Ostrinia furnacalis Guenee (Lepidoptera: Pyralidae) AND MODIFICATION OF DOMAIN III ß-sheet 13 of Bacillus thuringiensis Cry1Ab δ-endotoxin

Edwin P. Alcantara, and Aileen N. Bayot

Abstract


Brush border membrane vesicles (BBMVs) were prepared from 4th instar larvae of Asian corn borer Ostrinia furnacalis Guenee. SDS-PAGE analysis showed major protein bands with molecular weight in the range of 80 kDa to 250 kDa. Ligand blot analysis showed that biotinylated Cry1Ab - endotoxin strongly binds to a ~250 kDa receptor protein from O. furnacalis BBMV. Another receptor protein of ~110 kDa weakly binds to Cry1Ab δ- endotoxin. At least two isoforms of the ~250 kDa receptor protein have a pH range of 5.5 to 6.5 as determined by 2D-electrophoresis. Post-translational modification of O. furnacalis BBMV proteins was also detected as revealed by the presence of two glycosylated proteins with molecular weights of >250 kDa and 70 kDa, respectively. Alanine scanning mutations were introduced in domain III β-sheet 13 of Cry1Ab δ-endotoxin. Results of bioassays showed a 5 to 7 fold reduction in insecticidal activity in Serine Alanine, Serine Alanine and Serine Alanine- 556 557 556 Serine Alanine mutants of Cry1Ab protein. The decrease in insecticidal 557 activity suggests that Serine and Serine are both important residues in the 556 557 toxicity of Cry1Ab δ-endotoxin to O. furnacalis.

Key Words: Ostrinia furnacalis, midgut receptor, Bacillus thuringiensis, Domain III, brush border membrane vesicle (BBMV)

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