Bacillus thuringiensis (Bt) crystal proteins are effective in controlling agriculturally and biomedically harmful insects. However, the mechanism of Bt protein pesticidal action is not well understood. It is assumed that the pesticidal protein has affinity for specific receptors in the midgut of the susceptible larvae and binds irreversibly to create holes in the gut leading to eventual death of the target larvae.
The main aim of the research is to identify, purify and characterize Bt delta endotoxin receptor in agronomically important pest, American bollworm (Helicoverpa) armigera). The study presented in this thesis is endeavored to: discovery of a receptor protein(2) purification of the receptor. (3) cloning of the newly discovered protein gene in E.coli.(4)expression studies on recombinant protein, (5) Complete sequencing of the cloned gene.
Biological activities of different Bt crystal protein, namely Cry Aa, Cry 1 Ab and Cry1 Ac and Cry 2A were determined by in vitro mortality assays on neonatal and 2nd instar larvae of Helicoverpa armigera in the order of Cry 1Ac> Cry 1Ab> Cry1 Aa> Cry 2A. presence of a 65 kDa protein is reported here, in the extract of the larval midgut membrane of Helicoverpa armigera as putative receptor fro Bt Cry 1 A delta-endotoxins, on the basis of binding affinity to Cry 1Aa, Cry1Ab and Cry 1 Ac but not to Cry 2A.
It was observed that the sugar N-acety1 galactosamine (Gal NAc) showed no effect on binding of Cry Ac toxin to receptor protein or in other words, toxin binding to receptor was not inhibited by GalNAc. This finding suggests that Gal NAc might not be a component of Cry 1 Ac toxin receptor.
The protein has been highly purified by a combination of gel-filtration, protoxin affinity and anion exchange chromatography. When the purified proteins were assayed for amino-peptidase activity, it showed 2.5 fold enrichment when compared to the activity in crude extract f Helicoverpa armigera brush border membrane vesicles. The stability of receptor protein was analyzed by proteolysis with trypsin and gut juice of Helicoverpa armigera. The results showed that 65 kDa protein was resistant to trypsin and gut juice and has binding affinity to CrylAc in ligand blots, whereas pronase and proteinase-K showed digestion of the receptor protein.
Binding assays were performed with 125I-labeled toxin (CrylAc) and brush border membrane vesicles (BBMVs) prepared from H. armigera. CrylAc toxins produced saturable, high-affinity binding to H. armigera. BBMVs. Homologous and Heterologous competition binding assays were performed to investigate the binding site cross reactivity. The results suggest that CrylAa, CrylAb, and CrylAc recognize the same binding site, which is different from Cry2A.
N-terminal sequence of the purified protein does not show any homology toe 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 complimentarily. The structural gene of this purified protein was cloned in pGEMT-easy vector and pGEX-4T-3 expression vector.
The cloned gene was characterized through sequencing and studying expression in E.Coli. The expressed GST fusion protein was purified through Glu5tathione sepharose 4-Fast Flow column. The cloned, expressed and purified receptor protein was found to have 3-fold increase in APN activity than crude lysate, exhibited binding properties, in Western & ligand bolts and other characteristics of native protein of Helicoverpa armigera.
Larval mortality of Helicoverpa armigera to Cry 1A toxin was considerably presumably due to blocking of the receptor sites in BBMVs.
A new method for quick and easy detection of Bt-receptor in crude BBMVs extract, was established, which was based on sandwich ELISA technique. By applying that method, the types of Cry protein in Bt isolates and Bt Cry 1A receptor varieties in crude BBMVs extracts of target pests can be detected within one hour, before going into the tedious work of receptor purification.
The predicted amino acid sequence of cloned gene possess APN conserved motif (zinc biding site) and N-glycosylation site. The sequence has novelty because in has no significant homology to already existing sequences, The N-terminal sequence of the purified protein (65 kDa) has been entered in the Gen Bank (NCBI) protein database( Accession # A 59445). On Sep 13; 2005, sequence version replaced ads Gen Bank Accession( Q 7M4K6).