Analysis Of Bmduox Gene And Identification Of Midgut Peritrophic Membrane Proteins In Silkworm

Dual oxidase (Duox) belongs to the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family members, and its production of such oxidase activation is reactive oxygen species (ROS) which play important roles in maintaining the normal physiological function.In this study, the cDNA of dual oxidase (BmDuox) gene was cloned by RACE technique from silkworm, Bombyx mori. It was of5,545bp in length, including a28-amino acid residues signal peptide at N-terminal, a5’-terminal untranslated region (UTR) of240bp, a3’-terminal UTR of802bp, which contains9ATTTA motifs, and an ORF of4,503bp encoding a polypeptide of1,500amino acids. Structural analysis indicated that BmDuox contains a typical peroxidase domain at the N-terminal, followed by a calcium binding motif, a ferric-reductant domain, six transmembrane regions, binding domains for a flavin adenine dinucleotide (FAD) and a nicotinamide adenine dinucleotide (NAD), respectively. Transcriptional analysis revealed that BmDuox mRNA was expressed at higher level in the head, testis and trachea. Subcellular localization showed that BmDuox was mainly detected in the periphery of the cells, with less signals in the nucleus. Expression of BmDuox was significantly induced in the larval midgut upon challenge by E.coli and virus. BmDuox-deleted larvae showed a marked increase in microbial proliferation in the midgut compared with that of the control after ingestion of fluorescence-labeled bacteria. Hence, it suggested that BmDuox play an important role in inhibiting microbial proliferation and maintain homeostasis in the midgut of silkworm.The insect peritrophic membrane (PM) is secreted by intestinal epithelial cells with a colorless transparent semi-permeable membrane, similar to the vertebrate intestinal mucosa. It is mainly composed of protein, proteoglycan and chitin. The PM located between the intestinal contents and the intestinal epithelium to prevent intestinal epithelial cells from mechanical injury of exotic food, invasion of pathogenic microbial and toxins intrusion.In this study, shotgun approach was applied to investigate the composition of PM proteins. Total47proteins were identified, of which most of them were found to be closely related to catalytic activity, binding activity, transport function, as well as larval nutrients metabolism and innate immunity. Two unknown functional protein-encoding gene(BmMtch and BmPM-24) were further cloned and analyzed. The open reading frame (ORF) of BmMtch is888bp in length, encoding295amino acid residues consisting of two domains (Mito_carr domains) and three transmembrane regions. qRT-PCR revealed that BmMtch was mainly expressed in larval fat bodies, Malphigians tubules, testis and ovaries. Western blotting analysis showed that BmMtch were detected in total proteins of PM and larval midgut. The characteristics of BmMtch indicated that BmMtch is a novel insect PM proteins, without chitin-binding domains. Another new peritrophic membrane protein, designated as BmPM-24, was also characterized. Its ORF of BmPM-24was684bp in length, encoding227amino acid residues with a15-amino acid signal peptide at N-terminal. Western blotting analysis showed that positive signals were detected in total proteins of PM and larval midgut. qRT-PCR revealed that BmPM-24was mainly expressed in larval midgut, fat bodies and Malpighian tubules. BmPM-24has also no chitin-binding domain.The deeper study on the composition and functions of the insect PM proteins will help us getting more understanding their potential physiological functions, as well as developing more efficient biological insecticide.