Molecular characterization of anionic defensin-like peptide in immune response of silkworm, Bombyx mori L. (Lepidoptera)

We have previously identified a defensin-like peptide (BmDp, Genbank accession number DQ118523), in the larvae of a silkworm, Bombyx mori ( B. mori ) using wdS20658 cDNA in a Bombyx EST database (Silk Base: http://www.ab.a.u-tokyo.ac.jp/silkbase ). A full-length of BmDp cDNA was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and expressed in Escherichia coli cells in order to obtain a functional peptide. Amino acid sequence comparison of cysteine-rich defensin-like peptides with those of the BmDp showed to be identical protein as BmDefensinA designated as BmdefA gene deposited in the DDBJ database under the accession number AB367525. Sequence alignments revealed that the BmDp belongs to the insect defensin family and is close to galiomicin and spodoptericin, a defensin and a defensin-like protein having antimicrobial activities in phylogenetic analysis. Thus, we evaluated the effect of immune challenge and antimicrobial activity to understand the nature of this anionic BmDp. Semiquantitative RT-PCR analysis showed that BmDp gene expression was inducible by bacterial injection and peaked at a highest level at 8h after bacterial injection. This result suggests that BmDp is related with immune response against bacteria. A purified recombinant GSTBmDp fusion protein did not show direct antimicrobial activity against bacteria and fungi, which is differed from cationic antibacterial insect defensins and neutral antifungal defensin-like peptides.