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

ABSTRACT: Molecular cloning of cDNAs encoding moricin, a novel antibacterial peptide from the silkworm (Bombyx mori), was performed using a fat-body cDNA library. A reverse-transcription PCR product encoding a partial nucleotide sequence of moricin was used as a probe. Nucleotide sequencing of four positive clones 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. Although one amino acid residue (Phe6) of moricin 1 in the putative signal peptide was replaced with Lys6 in moricin 2, amino acid sequences of their mature portions were identical. Moricin gene expression in B. mori larvae injected with Escherichia coli was observed in fat-bodies, haemocytes and the Malpighian tubule, but not in other tissues like the midgut and silk glands. 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 confirmed that lipopolysaccharide (LPS) and lipid A, which are cell-wall components of E. coli, triggered moricin gene expression. Comparison of gene expression between moricin 1 and 2 by PCR using specific primers indicated that moricin 2 gene was more strongly expressed than moricin 1 gene. A genomic clone 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 nuclear-factor-kappaB-binding-site-like sequence (kappaB 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 revealed that there are bacterial LPS-inducible nuclear proteins that can bind to the kappaB site and other sites in the regulatory region.

SUBMITTER: Furukawa S

PROVIDER: S-EPMC1220245 | biostudies-other | 1999 May

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Carboxylesterase is a multifunctional superfamily and ubiquitous in all living organisms, including animals, plants, insects, and microbes. It plays important roles in xenobiotic detoxification, and pheromone degradation, neurogenesis and regulating development. Previous studies mainly used Dipteran Drosophila and mosquitoes as model organisms to investigate the roles of the insect COEs in insecticide resistance. However, genome-wide characterization of COEs in phytophagous insects and comparative analysis remain to be performed. RESULTS: Based on the newly assembled genome sequence, 76 putative COEs were identified in Bombyx mori. Relative to other Dipteran and Hymenopteran insects, alpha-esterases were significantly expanded in the silkworm. Genomics analysis suggested that BmCOEs showed chromosome preferable distribution and 55% of which were tandem arranged. Sixty-one BmCOEs were transcribed based on cDNA/ESTs and microarray data. Generally, most of the COEs showed tissue specific expressions and expression level between male and female did not display obvious differences. Three main patterns could be classified, i.e. midgut-, head and integument-, and silk gland-specific expressions. Midgut is the first barrier of xenobiotics peroral toxicity, in which COEs may be involved in eliminating secondary metabolites of mulberry leaves and contaminants of insecticides in diet. For head and integument-class, most of the members were homologous to odorant-degrading enzyme (ODE) and antennal esterase. RT-PCR verified that the ODE-like esterases were also highly expressed in larvae antenna and maxilla, and thus they may play important roles in degradation of plant volatiles or other xenobiotics. CONCLUSION: B. mori has the largest number of insect COE genes characterized to date. Comparative genomic analysis suggested that the gene expansion mainly occurred in silkworm alpha-esterases. Expression evidence indicated that the expanded genes were specifically expressed in midgut, integument and head, implying that these genes may have important roles in detoxifying secondary metabolites of mulberry leaves, contaminants in diet, and odorants. Our results provide some new insights into functions and evolutionary characteristics of COEs in phytophagous insects.

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

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