PJB-2006-84
IDENTIFICATION, PURIFICATION, CLONING AND EXPRESSION OF A NOVEL RECEPTOR FOR BACILLUS THURINGIENSIS CRY1A DELTA-ENDOTOXINS IN THE BRUSH BORDER MEMBRANES OF THE HELICOVERPA ARMIGERA (LEPIDOPTERA: NOCTUIDAE)
KAUSAR MALIK, SHEIKH AMER RIAZUDDIN AND SHEIKH RIAZUDDIN
Abstract
Insecticidal crystal proteins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects. However, little is known about the mechanism of insecticidal activity of these proteins. We report here a 65 kDa Protein present in the extract of the larval midgut membrane of Helicoverpa armigera as putative receptor for Bt Cry1A delta-endotoxin, on the basis of binding affinity to Cry1Aa, Cry1Ab and Cry1Ac but not to Cry2A. The protein has been highly purified by a combination of chromatography, electrophoresis and isoelectrifocussing techniques. The isolated protein exists as an oligodimer in its native form. The purified protein exhibits amino-peptidase activity. N-terminal sequence of the purified protein shows no homology to protein sequences in the Gen bank (NCBI) protein database. Degenerate primers were designed, based on N-terminal sequence of the purified protein and hybridization of Probe with mRNAs of Helicoverpa armigera indicated sequence complementarity. The structural gene of this purified protein was cloned in pGEX-4T-3 expression vector. The cloned Protein exhibited binding properties, aminopeptidase activity and other characteristics of native protein of Helicoverpa armigera. Larval mortality of Helicoverpa armigera to Cry1A toxin was considerably reduced when the larvae were pre-fed a diet containing antibodies to the 65 kDa protein, presumably due to blocking of the receptor sites in BBMVs.
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