ISOLATION, CDNA CLONING AND GENE-EXPRESSION OF AN ANTIBACTERIAL PROTEIN FROM LARVAE OF THE COCONUT RHINOCEROS BEETLE, ORYCTES RHINOCEROS

An antibacterial protein, designated rhinocerosin, was purified to hom ogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoce ros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reve rse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a r esult, a 279-bp fragment was obtained. The complete nucleotide sequenc e was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids w ithout cystein residues and was shown to be rich in glycine (11.1%) an d proline (11.1%) residues. Comparison of the deduced amino acid seque nce of rhinocerosin with those of other antibacterial proteins indicat ed that it has 77.8% and 44.6% identity with holotricin 2 and coleoptr ecin, respectively. Rhinocerosin had strong antibacterial activity aga inst E, coli, Streptococcus pyogenes, Staphylococcus aureus but not ag ainst Pseudomonas aeruginosa. Results of reverse-transcriptase PCR ana lysis of gene expression in different tissues indicated that the rhino cerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gen e expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the m idgut.