Identification and Phylogenetic Analyses of Two Isoforms of the Antibacterial Gene Diptericin from the Larval Tissue of Musca domestica (Diptera: Muscidae)
Department of Biology, Aljouf University College of Science, Sakaka, KSA; Department of Entomology, Cairo University Faculty of Science, Cairo, Egypt
Department of Zoology, Menoufia University Faculty of Science, Al-Minufya, Egypt
Erciyes Med J 2017; 39: 24-31
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Key Words: AMPs, diptericin, musca domestica, diptera, immune response
Objective: Insect immune system has a potent arsenal of antimicrobial peptides (AMPs) that cooperate to clear microbial invasions. Here we aimed to explore the immune response of Musca domestica larvae when bacterially challenged and pick up induced antibacterial genes. These genes can be used in the production of novel antibiotics to compensate for the increasing demand of antibiotics in the era of resistant bacterial strains.
Materials and Methods: Hemolymph and whole body of third instar larvae were collected at 2-h intervals for 24 h postinfection. Integer and pure total RNA were transcribed into cDNA. Differential display technique was used to identify differentially expressed genes. Ten reproducible bacterial-induced bands were sequenced. Sequenced DNA fragments were deposited in GenBank under KM205630 and Hl205631 accession numbers.
Results: Sequence analyses indicated that two DNA fragments designated as MdDipWB and MdDipHL were identified as diptericin-related sequences, for which single open reading frame (orf) encoding 99 and 80 amino acids were detected, respectively. Signal peptide was predicted only for MdDipWB. Meanwhile, prosequence was predicted only for MdDipHL. Calculated molecular masses of mature MdDipWB and MdDipHL were 8.8 and 6.97 Kilo Daltons (KDa), respectively. Propeptides of MdDipWB and MdDipHL were more stable than mature peptides. Comparing MdDipWB and MdDipHL nucleotide sequences, 26 substitutions and 4 deletions were observed in MdDipWB. Despite the 90% identity between MdDipWB and MdDipHL nucleotide sequences, no significant similarity was observed between their deduced amino acids. Nucleotide and deduced amino acids of MdDipWB and MdDipHL created significant similarity with other diptericins isolated from M. domestica. On comparing amino acid sequences of our putative polypeptides to their corresponding sequences, overexpression of many specific amino acid residues was observed.
Conclusion: Our findings suggested that MdDipWB and MdDipHL are two isoforms of the same gene.