| Pak. J. Bot., 38(3): 767-778, 2006. | Back to Contents | ||||
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Updated: 09-07-09 | ||||
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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. National Center of Excellence in Molecular Biology, University of the Punjab 87-West Canal Bank Road, Thokar Niaz Baig Lahore-53700, Pakistan |
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