Characterization of a homologue of the Rel/NF-κB transcription factor from a beetle, Allomyrina dichotoma

Abstract

A cDNA encoding a Rel/NF-κB homologue was cloned from a beetle, Allomyrina dichotoma, by reverse transcriptase-polymerase chain reactions (RT-PCR) taking advantage of the conserved Rel homology domain (RHD) to synthesize primers. The Rel/NF-κB homologue was designated A. dichotoma ( A.d.) Rel A. The amino acid sequence of the A.d. Rel A RHD was compared with those of insect RHDs. The result showed that it has 70% identity with Tribolium castaneum Dorsal, 66% with Drosophila melanogaster Dorsal, 61% with Anopheles gambiae Gambif1, and 55% with D. melanogaster Dif. A putative phosphorylation site in the RHD, RRPS, and two putative nuclear localization signals were conserved in A.d. Rel A. A recombinant fusion protein containing the A.d. Rel A RHD was confirmed to bind specifically to the NF-κB site of a gene encoding A.d. coleoptericin A, an antibacterial peptide from A. dichotoma. The activity of A.d. Rel A in modulating a gene construct of the A.d. coleoptericin A promoter-luciferase reporter by expressing the A.d. coleoptericin A cDNA in a Bombyx mori cell line was analyzed. The result showed that A.d. Rel A strongly activates the A.d. coleoptericin A gene construct, whereas A.d. Rel A failed to activate the gene construct containing the mutated NF-κB site, suggesting the importance of the interaction between the NF-κB site and A.d. Rel A in the signal transduction for gene expression of antibacterial peptides in A. dichotoma.