Identification And Functional Analysis Of Cuticular Chitin Binding Protein(BmCBP) In Silkworm, Bombyx Mori

The surface of silkworm is covered by cuticle, which plays important roles in multiple physiological functions to protect its body from invasion of pathogens, physical injury and dehydration ect, In addition,, it is indispensable to keep its body shape and maintain normal activity in the process of development of silkworm. Therefore, cuticle has greatly enhanced the silkworm survival and ability to adapt enviroment.Chitin binding protein(CBP) is an important the structural component of the cuticle during molting and metamorphosis. CBP widely existed in the insect cuticle or peritrophic membrane(PM). It is can binding to chitin as compounds, which participated in the formation of the silkworm cuticle or PM. In this study, we performed a genomic-level search to identify the genes encoding chitin binding protein contain ChtBD2, and examined expression pattern of the BmCBP, which provided information for chitin binding protein during silkworm development. The mainly methods contained gene cloning, prokaryotic expression, western Blot, immunofluorescence and biological information analysis.The main results were listed as follow: 1. Identification and expression analysis BmCBP of SilkwormWe conducted a genome-wide search for genes encoding proteins with peritrophin A(Cht BD2) chitin-binding domain(CBD) of the genome of silkworm. A total of 46 genes encoding proteins containing at least one ChtBD2 domain were identified, Including 26 CPAPs(cuticle proteins analogous to peritrophins), 13 PMPs(peritrophic matrix proteins), 4 chitinase and 3 chitin deacetylase. Alignment of the CBDs from the proteins in each family showed that the six cysteine residues were highly conserved motif. The lengths of the CBDs ranged from 50 to 63 amino acids residue from the first cysteins to the sixth cysteins. The spacing between cysteines of each family also had high similarily. The gene microarray data indicated that the tissue expression patterns of the family can be divided into 3 groups, the first group is mainly expressed in the head and cuticle. The second group is mainly highly expressed in the midgut. The third group is mainly expressed in the ovary and testis. 2. Cloning, expression, purification and antibody preparation of BmCBPWe identification two coding chitin binding protein gene, named BmCBP1 and BmCBP2 respectively. BmCBP1 encoded 237 amino acids residues with predicted molecular weight of 26.4 kD, and with predicted isoelectric point of 4.9. BmCBP2 encoded 227 amino acid residues protein, with predicted molecular weight of 25.7 KD, predicted isoelectric point of 4.6. Both of them contain three ChtBD2 domains. Evolutionary analysis found that BmCBP1 and BmCBP2 have been existed early evolution in the silkworm. The gene cloning, expression of BmCBP1 and BmCBP2 show that BmCBP1 mainly expressed in the form of inclusion body, however BmCBP2 mainly expressed in soluble form. After purificated the soluble BmCBP2 protein, the purified proteins were performed to produce polyclonal antibody. The titers of BmCBP2 antibody were beyond 1: 512,000, and concentrations were 1.60 mg/ml. So the antibodies of BmCBP2 can be used in subsequent experiment. 3. Expression characteristics and tissue localization of BmCBPThe Spatio-temporal expression analysis by RT-PCR and Q-PCR show that BmCBP1 and BmCBP2 mainly expressed in head and epidermis. The temporal expression indication that BmCBP1 and BmCBP2 were highly expressed during wandering stage(larva to pupa) and the later stage of the pupa(pupa to moth).In addition, using the westeren blot experiment, we found that BmCBP2 also expressed in head and epidermis, expecially during molting and metamorphosis stage highly expression, which is constantly with the level of mRNA expression profiles. In the 20 E induced experiment, BmCBP2 has an up-regulation of expression. Immunofluorescence results showed chitin was located the epidermis layer, and BmCBP2 was uniformly distributed throughout the entire epidermis. The co-localization of BmCBP1 protein and chitin were examined at the fourth instar larval molting stage. The results indicated that BmCBP2 could participated in the formation of new cuticle studies. 4. The functional exploration of BmCBP2The soluble recombinant BmCBP2 was used for combining with chitin bead, The Western blot results show that BmCBP2 protein can capable of binding with chitin in vitro. In order to identify the parterner protein of BmCBP2, pull-down technology was used to study protein interaction. we build the BmCBP2 gene expression vector with GST tag, and further analysis of the interaction of proteins with BmCBP in vitro. By co-incubation with the blood and BmCBP2 protein, CP8 protein were considered to have potential interaction with BmCBP2, When co-incubated BmCBP2 protein with moulting fluid, we found that BmCBP2 could be degraded by moulting fluid. The result indicated some protease in the moulting fluid could degrede BmCBP2 in vitro. We also study the function of BmCBP2 using RNAi tool. The result show that the expression of BmCBP2 gene was down regulated by RNAi at the fourth instar larval molting, and the expression of BmCBP2 was down regulated at the level of nucleic acid. However, the phenotype did not change significantly, Totally, these results will provide important clues for further analyzing function of the protein.