Research On The Binding Mechanism Between Bacillus Thuringiensis Insecticidal Protein Cry1Ie And Peritrophic Membrane Of The Asian Corn Borer

Bacillus thuringiensis（Bt）can produce insecticidal crystal toxins duringspore formation stage, which has high specificity to target insects. Cry1Ie gene issilent in Bt, but can be over expressed in BL21/DE3resulting with Cry1Ie proteincomposed of719amino acids, which is larvicidal to Plutella xylostella, Asiancorn borer(Ostrinia furnacalis) and soybean pod borer(Leguminivora glycinivorel).IE648toxin is a truncated Cry1Ie protein, corresponding to the648amino acids fromN-terminal, with increased toxicity against Asian corn borer (ACB). The peritrophicmembrane (PM) is an important barrier that Cry toxins must cross before binding tomidgut cells. PM is a semipermeable matrix composed of chitin fibrils andprotein-glycojugates that protect the insect gut epithelial cells from injury and infection.This research focused on the binding between Cry1Ie and PM from two sides， PMproteins and chitin, based on the components of PM.As for the ensemble of PM, previously competitive binding assay indicates thatIE648and domain Ⅲ ofCry1Ie can both bind with PM of ACB, but the contribution ofdomain Ⅱremains unclear because it’s expressed as inclusions and can’t be dissolved. Inthis study domain Ⅱ was dissolved and renatured, and purified by affinitychromatography. Competitive binding assay indicates that domain Ⅱ can bind with PM.At the same time in order to locate the binding site more accurately, many peptides weresynthesized, including conserved peptides, some non-conserved peptides, some specialpeptides that play an important role in other Cry toxin’s binding with BBMV and peptideslocate in the loop region of domain Ⅲ. Competitive binding assay indicates that D3-L8can compepte with IE648for binding with PM.As for the PM protein, binding interaction in vitro between PM and domain Ⅱindicates that domain Ⅱ can bind with PM proteins between25-35kDa, which is similarto the situation of IE648and domain Ⅲ. Binding interaction in vitro between PM andBSA indicating that Bovine Serum Albumin (BSA) can bind with PM. The molecularweight of PM proteins BSA binds is very different with that IE648and domains of Cry1Ie.It is shown that PM can bind with kinds of proteins specially and unspecially, but theyhave different binding sites.As for chitin, binding interaction between chitin and IE648as well as three domains of Cry1Ie indicates that they can all bind with chitin. The interaction between Cry toxinand chitin has never been reported before. Bioinformatics analysis of three domains ofCry1Ie were carried out, including3D ligand site predition, structural modeling, structuralfitting and so on. It corroborates that domain Ⅱ and domain Ⅲ may be involved incarbonhydrate binding, the mechanism and specifity still need more research.