Function Of Chitin-Binding Proteins From Bacillus Thuringiensis

Bacillus thuringiensis（Bt） is Gram-positive bacteria widely distributed, whose mother cell can produce insecticidal crystal protein of growth cycle. Chitin binding protein（CBP） is a group of proteins that interact highly specifically with chitin. Chitin is the important part of insect peritrophic matrices（PM）. PM can protect insect from damage caused by viruses, bacteria and other organisms. Some study show that the chitin binding protein can promote insecticidal activity and efficiency of a variety of insecticidal proteins.Bt HD73 encodes two chitin binding protein（CBP-3189 and CBP-3152）. Preliminary study by our laboratory showed that the CBP-3189 can bind with the PM and the cell wall of Bt HD73 and it is not with chitinase activity. The strain HD73（pHT-3189-gfp）, which has CBP-3189 protein and GFP protein fusion expression vector was obtained. The purpose of this paper is to study the function of chitin binding protein. First, using bioinformatic analysis tools to provide a variety of structural and physicochemical properties of proteins, which can provide a basis for experimental research. Secondly, analysis the localization of the protein through the laser confocal microscopy. At the same time, laser confocal microscopy and qPCR were used to study the protein expression induced by alkaline in the simulated insect midgut environment. Finally, the genic function can be studied by constructing mutant strains and analysising the characteristics of mutant strains. These results will provide certain theoretical basis and reference for the further research on the insect infection mechanism of Bacillus thuringiensis.The bioinformatic analyses showed CBP-3189 contains a signal peptide, an N-terminal chitin_binding₃ domain（CBD₃）, two copies of a fibronectin-like domain 3（FN3） and a C-terminal carbohydrate binding domain classified as CBM₅₁2. It moreover predicted the protein’s associated localization site is cell wall. The ligand site prediction has shown that amino acid residues GLU-312, TRP-334, ILE-341 and VAL-382 of CBP-3189 exposed on the surface of the target protein have polar interactions with the substrate. CBP-3152 contains only one CBD₃ domain and has not signal peptide. The protein’s associated localization site of CBP-3152 can’t be predicted. The ligand site prediction has shown that amino acid residues TRP-183, GLU-184, ILE-185 and PHE-192 of CBP-3152 exposed on the surface of the target protein have polar interactions with the substrate.Observation the localization of CBP-3189 shows that the protein began to express in the T8 period and concentrated at the edge of the cell. It is at the one side closed to Cry1 AC. The expression of the CBP-3189 was increasingly to a maximum before the bacteria cell lysis. The expression of the CBP-3189 was gradually decreased when the bacteria cell begin to lyse and then stop expressing. The analysis of the protein expression induced by alkaline shows that the CBP-3189 was expressed after induced by alkaline and the expression was gradually increased with the increase of the induction time. The gene 3189 knock-out vector pRN5101Ω3189 and the gene 3152 knock-out vector pRN5101Ω3152 have been successfully constructed. The 3189 gene knock-out mutant HD73（?3189） was obtained. The phenotype analysis of the mutant strain HD73（?3189） shows that there was no visible different between their growth curve. This result suggests that the gene have no close relationship with the growth of the bacteria. The binding capacity with PM of the mutant strain HD73（?3189） have non-significant difference with the wild-type.