Inducible gene expression of moricin, a unique antibacterial peptide from the silkworm (Bombyx mori)

Molecular cloning of cDNAs encoding moricin, a novel antibacterial peptide from the silkworm (Bombyx mori), was performed using a fat-body cDNA librar y. A reverse-transcription PCR product encoding a partial nucleotide sequen ce of moricin was used as a probe. Nucleotide sequencing of four positive c lones revealed two types of moricin cDNAs designated moricin 1 and 2. cDNAs for moricin 1 and 2 shared 97.2% identity in their nucleotide sequences. A lthough one amino acid residue (Phe(6)) of moricin I in the putative signal peptide was replaced with Lys(6) in moricin 2, amino acid sequences of the ir mature portions were identical. Moricin gene expression in B. mori larva e injected with Escherichia coli was observed in fat-bodies, haemocytes and the Malpighian tubule, but not in other tissues like the midgut and silk g lands. Accumulation of moricin gene transcripts induced by E. coli reached a maximum level 8 h after injection and persisted up to 48 h. It was confir med that lipopolysaccharide (LPS) and lipid A, which are cell-wall componen ts of E. coli, triggered moricin gene expression. Comparison of gene expres sion between moricin 1 and 2 by PCR using specific primers indicated that m oricin 2 gene was more strongly expressed than moricin 1 gene. A genomic cl one encoding moricin 2 was screened from a B. mori genomic library using a moricin cDNA as a probe. Regulatory motifs for gene expression such as nucl ear-factor-kappa B-binding-site-like sequence (KB site) and nuclear-factor- interleukin-6-binding-site-like sequence (NF-IL-6 site) were found in the 5 '-upstream regulatory region. An electrophoretic-mobility-shift assay revea led that there are bacterial LPS-inducible nuclear proteins that can bind t o the kappa B Site and other sites in the regulatory region.