Molecular Cloning And Characterization Of Peritrophic Matrix Protein(MdPM-17)in Musca Domestica Larvae

Objective: Study on molecular characteristics and biological characteristics of peritrophic matrix protein MdPM-17 in housefly larvae.Methods:The full-length cDNA of MdPM-17 gene was acquired from housefly larvae by RT-PCR.The gene and its encoded proteins were predicted and analyzed with the bioinformatics analysis technology.The gene recombinant plasmid was constructed with a prokaryotic expression vector pET-32a(+),and transformed into Escherichia coli(E.coli)Transetta(DE3)competent cells for inducing expression and protein purification.The New Zealand rabbit was immunized with recombinant MdPM-17 protein and the polyclonal antibody was obtained.The chitin binding activity of MdPM-17 was examined by chitin binding assay.We used RNA interference to inhibit MdPM-17 and measured the interference effect with quantitative real-time PCR(qPCR).Then we explored the preferential time and effect,detected the expression of defensin,cecropins and diptericin,and studied the anti-microbial infection response after RNAi.Results: MdPM-17,from the PM of housefly larvae was cloned successfully with a full-length cDNA of 635 bp and an ORF of 477 bp,containing a signal peptide in the N-terminal,encoding 158 amino acids.The molecular weight was 17 kD and the pI 4.55.The deduced amino acid sequence of the cDNA contained the Chitin binding type-2 domains(ChtBD2).By RT-PCR MdPM-17 could be primarily detected in the fat body,foregut,midgut,malpighian tubule and trachea,with a highest expression in the midgut.The double enzyme digestion and SDS-PAGE analysis showed that the recombinant plasmid pET-32a(+)-MdPM-17 was successfully constructed and it was expressed successfully.The chitin binding assay demonstrated that MdPM-17,the class 3protein of PM,could strongly bind to chitin and the chitin-bound protein could only be released from the chitin by 6M urea.MdPM-17 was knocked down in the first 24 h after the MdPM-17 dsRNA microinjection,the survival rate and pupation rate ofMdPM-17-depleted housefly larvae was severely suppressed compared with GFP dsRNA and blank control group.The expression levels of all the three AMP genes were up-regulated in the gut of MdPM-17-depleted housefly larvae comparing to controls.And then,there were significantly a high expression of diptericin and cecropins after Escherichia coli infection,a high expression of diptericin,defensin and cecropins after Staphylococcus aureus infection,a high expression of diptericin after Candida albicans infection.Conclusion: The recombinant plasmid pET-32a(+)-MdPM-17 was successfully constructed.The recombinant MdPM-17 protein showed a high chitin-binding activity and belonged to the class 3 PM protein.MdPM-17 was highest synthesized in the midgut.The MdPM-17 gene of housefly larvae was effectively silenced in the 24 th hour with RNAi,which caused a high expression of antimicrobial peptide genes.The expression of antimicrobial peptides gene in MdPM-17 RNAi group had a compensatory and up-regulating at different levels,indicating that MdPM-17 played an important role in housefly larval intestinal immune against intestinal infection.