Proteomic Analysis On Peritrophic Membrane And Functional Characterization Of Chitin Deacetylase In Silkworm, Bombyx Mori

Chitin is a linear polymer of (3-(1-4)-linked N-acetylglucosamines (GlcNAc), that most common natural amino polysaccharide in nature.It is is second only to cellulose in abundances and mainly synthesized by fungi, nematodes and arthropods. In insects, chitin is a constituent part of the cuticle and peritrophic membrane (PM)in insects. The chitin metabolism have been reported to change at different growth stages of insects. So the chitin is crucial for insect growth and development. The domesticated silkworm, Bombyx mori, is not only a very important economic insect that contributes to the national economy of many countries, but is also an excellent model organism of Lepidoteran insects for basic research. Chitin and protein are the major constituent of PM. The structural integrity of the PM is maintained by the protein component present in the PM. The hydrophobic of protein polysaccharides have the function to help maintain the chitin micro fiber reticular formation and its strength of PM. the PM is first line of defense against invading microorganisms. Degrading chitin or disturbing the combination of protein and chitin can inhibit peritrophic membrane formation and insect growth and development. It is become the domestic and foreign scholars study hotspots, and has great to control pests.Chitin deacetylase (CDA) is the structural peritrophic matrix proteins and could modify chitin. So, it can be an attractive target to disturb the PM. Our study use proteomic methods to identify comprehensively the proteins from the PM of silkworm. Meanwhile we perform further study on the BmCDA7gene by the molecular techniques of gene cloning, reverse-transcription PCR, western blotting and prokaryotic and eukaryotic expression. Then we used the transgene technology to analyze the function of BmCDA7gene. The main results are as follows:1. Peritrophic membrane of silkworm structure and analysis of its protein componentsThe silkworm PM that almost cover the entire midgut wrap with food bolus, and is a colorless, transparent, flexible tubular structure, which is similar to the type I PM. The PM clearly locates between the food and the midgut from cross-section of the silkworm, Which indicates that the silkworm has PM as most of the Lepidoptera. The silkworm PM has the chitin network that protect midgut from invading microorganisms.There are at least two layers can be found on the PM. And the inner surface of PM is smooth and compact, but still has an obvious fold. This structure suggests the PM not only plays important roles in facilitating food digestion and providing protection to the gut epithelium.To study the protein profiles of silkworm PM, the total proteins extracted from day3of the5th instar larvae of silkworm PM were separated by shotgun techonology, and a total of305proteins were identified. The molecular weights mostly ranged between8.02kDa and788.52kDa, except for BGIBMGA006856-PA (2002.29kDa) and BGIBMGA010471-PA (1538.87kDa). A total of79.34%of the identified proteins were smaller than100kDa. The most acidic and basic proteins identified by LC-MS/MS had pIs of3.39and12.91, respectively.The protein extracts from the PM were aslo separated by2-DE. More than60protein spots were detected. Most of the resolved protein spots had pI values between pH5and9with molecular weight of10-66kDa. We detected more spots in silkworm PM than other insects using2-DE. And then30spots from PM were excised and further investigated by MALDI-TOF MS. In addition, there were12proteins were successfully identified. This investigation revealed that these proteins were components of the PM. There were two structural peritrophic matrix proteins, chitin deacetylase and peritrophic membrane chitin binding protein2.Gene Ontology tools were used to analyze the PM proteome to present an overall view on the functional categories of PM proteins. The results indicate that216of the305identified proteins show at least one matched GO annotation. The proteins were classified into cellular component, molecular function, and biological process according to the GO hierarchy using WEGO. Majority of the proteins were assigned to the cell and cell part in terms of "cellular component." The highest distribution was associated with binding and catalytic activity in terms of "molecular function." The remaining proteins were linked to different activities, such as antioxidation, electron transport, and enzyme regulation, and among others. The proteins were classified according to different categories based on "biological process."The highest number of proteins was mapped to proteins involved in metabolic process, followed by proteins associated with cellular process.The PM proteins were mapped to the KEGG ortholog level for the KEGG analysis. Two hundred and thirty-four different pathways were linked to the PM. These pathways were classified into metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases. Among the organismal systems, the digestive system was the most active. Moreover, five pathways were related to the immune system. This phenomenon is likely because the PM not only could help the midgut in coordinating the movement of enzymes and nutrient uptake, but also plays an important role in insect defense strategy.2. Full-length cDNA cloning and sequence analysis of silkworm BmCDA7geneBased on the assembled9X coverage genome sequence, we cloned and sequenced the BmCDA7gene. The putative BmCDA7cDNA contains an open reading frame (ORF) of1,140bp. RACE experiments were performed to obtain the5’and3’ends of BmCDA7,27bp upstream-untranslated region and190bp down stream-untranslated region. So, the full length of the cDNA of BmCDA7is1357bp followed by an AT rich region with two typical polyadenylation signal signal sequence, AATAAA. The putative BmCDA7cDNA encoded for a379-amino acids protein consisting of a16-amino acid signal peptide by the software SignalP and a mature polypeptide of363amino acids. After removal of the signal peptide, the deduced protein is predicted to have a molecular weight of41.26kDa and the theoretical pI of5.12by the ExPASy server. Prediction of potential glycosylation sites using the NetNglyc1.0and NetOglyc3.1server shown that the protein contains one putative N-glycosylation site at Asn168, one putative O-glycosylation site at Thr209.,215. BmCDA7has a putative polysaccharide deacetylase-like domain (residues46-182) and15cysteine residues which maight be a basic module that combined with other protein sequences to generate new function or modify existing function by SMART analysis.8putative silkworm CD As were identified. Based on the EST, there are two genes (BmCDA3and BmCDA4)have expression evidence. BmCDA2and BmCDA5have alternative splicing. Phylogenetic analysis shows that all of the CDA-like proteins from insects originated from one root. The silkworm CDAs also grouped the proteins into five major classes, groups I through V, and BmCDA7was divided into group V.The expression of BmCDA7was also studied by RT-PCR. It show this gene has expression from9d after oviposition to day7of5th instar larva and has high expression at the molting silkworm. It is just period from the formation to the process of apoptosis of silkworm midgut. The foregut, midgut, hindgut and remaining carcasses were dissected from Day-3of the fifth instar larvae to use in this study. The signal was only detected in midgut, but no signals were found in the foregut, hindgut and remaining carcasses. This suggested that the gene should be updated with PM.3. Prokaryotic expression and tissue localization of silkworm BmCDA7geneThe complete CDS of BmCDA7gene was subcloned into the p28prokaryotic expression vector, the BmCDA7-p28plasmids were introduced into Escherichia coli BL21(DE3), and the recombinant protein was abundantly expressed as insoluble inclusion body after induction by0.2mM IPTG,37℃,4h. There was one specific band corresponding to molecular weights of BmCDA7and purified using the Ni2+-NTA affinity column. Then the purified proteins were injected into rabbit to generate polyclonal antibodies. In addition to the PM, the tissue distribution of BmCDA7in silkworm larvae was detected by western blot using the antibody to the recombinant protein. BmCDA7was detected in midgut and PM tissue, but was not detectable from the larval foregut, hindgut and remaining carcasses. This result was also confirmed by the translational levels analysis of BmCDA7, and suggested the BmCDA7was specific expression protein in the midgut and PM.4. Eukaryotic expression of silkworm BmCDA7gene and activity determinationThe recombinant expression vector pPIC9K-BmCDA7plasmid and pPIC9K plasmid were transformed into P. pastoris by electroporation. After transformation and primary screening by histidine-deficient medium and G418, The positive yeast cells containing plasmid was selected for cultivation in in BMMY medium after induction by1%methanol at30℃. SDS-PAGE analysis of the crude supernatants at the induction period from96h appeared no significant additional band corresponding to the molecular masses of BmCDA7in the induced yeast containing recombinant pPIC9K. But we detected specific band from crude supernatants of pPIC9K-BmCDA7using the BmCDA7antibodies. This result indicated that BmCDA7was successfully expressed in the yeast. Using the differences method as previous described, we demonstrated that recombinant BmCDA7is active, and the CDA activity of BmCDA7is1.85U/mL.5. Function analysis of silkworm BmCDA7geneWe have successfully constructed two transgenic vectors, the transgenic overexpression vector ([P2-BmCDA7-SV40,3×P3-EGFP-SV40]) and the transgenic RNAi vector (pBac[P2-BmCDA7S-I-BmCDA7A-SV40,3xP3-EGFP]). Comicro-inject the transgenic vectors and helper vector into the silkworm eggs which have been broken embryonic diapause. Scan the G1by fluorescence microscope and positive individuals were obtained. The inverse PCR results show that insertion site of transgenic overexpression and transgenic RNAi system are localed on nscaf2859of chromosome10and nscaf2888of chromosome15, respectively.Analysis of the PM permeability of the two transgenic system, we found the blue dextran in the transgenic system more than the control. This in the PM permeability of transgenic system has improved. In the transgenic overexpression system, the over-expression of BmCDA7maight excessive modification the chitin. In the transgenic RNAi system, the suppression expression of BmCDA7maight made chitin deformity. The chitin in PM of the two transgenic system have obvious change(reduced or smaller), and disorder arrangement. Ang the chitin content of two transgenic system have reduce. So, we verify the BmCDA7’modification of chitin play an important role in formation and arrangement of chitin.