Analysis of the 5'-upstream regulatory region of a gene encoding coleoptericin, an antibacterial protein from the beetle, Allomyrina dichotoma(Coleoptera: Scarabaeidae).

Abstract

A gene encoding coleoptericin, an antibacterial protein from the beetle, Allomyrina dichotoma was cloned and the nucleotide sequence determined. The A. dichotoma (A. d.) coleoptericin genomic clone was full size and contained an intron between a putative transcription initiation site and a signal sequence region. The 5′-upstream regulatory region of the A. d. coleoptericin had a TATA box and other regulatory motifs such as a NF-κB site, a region 1 (R1), a NF-IL6 site, 2 GATA motifs and 3 CATT(A/T) motifs. Expression vectors consisting of different lengths of the regulatory region and a luciferase gene as a reporter were constructed to analyze the induction of gene expression by bacterial lipopolysaccharide (LPS), a strong trigger for insect antibacterial protein genes. A silkworm Bombyx mori cell line was transfected with the expression vectors treated with or without LPS and the luciferase activity measured. Results showed that luciferase activity was dramatically reduced when NF-κB site was deleted. Deletion of R1 resulted in 64% reduction of luciferase activity but no marked effect was observed with GATA motif mutation. These results suggest that the NF-κB site is indispensable for A. d. coleoptericin gene expression and R1 is also necessary for full gene expression.