A gut-specific chitinase gene was cloned from the mulberry longicorn beetle, Apriona germari. The A. germari chitinase (AgChi) gene spans 2894 bp and consists of five introns and six exons coding for 390 amino acid residues. AgChi possesses the chitinase family 18 active site signature and three N-glycosylation sites. Southern blot analysis of genomic DNA suggests that AgChi is a single copy gene. The AgChi cDNA was expressed as a 46-kDa polypeptide in baculovirus-infected insect Sf9 cells and the recombinant AgChi showed activity in a chitinase enzyme assay. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-linked glycosylation, revealed that AgChi is N-glycosylated, but the carbohydrate moieties are not essential for chitinolytic activity. Northern and Western blot analyses showed that AgChi was specifically expressed in the gut; AgChi was expressed in three gut regions, indicating that the gut is the prime site for AgChi synthesis in A. germari larvae.
Two glutathione S-transferase (GST) cDNAs, GSTD2 and GSTS2, were cloned from the silkworm Bombyx mori. The B. mori GSTD2 (BmGSTD2) gene spans 4371 bp and consists of four introns and five exons that encode 222 amino acid residues. The deduced amino acid sequence of BmGSTD2 showed 58% protein sequence identity to the Delta-class GST of Maduca sexta. The B. mori GSTS2 (BmGSTS2) gene spans 3470 bp and consists of three introns and four exons that encode 206 amino acid residues. The deduced amino acid sequence of BmGSTS2 revealed 67%, 63%, and 61% protein sequence identities to the Sigma-class GSTs from B. mori, Platynota idaeusalis, and M. sexta, respectively. The BmGSTD2 and BmGSTS2 cDNAs were expressed as 25 kDa and 23 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot and Western blot analyses showed that BmGSTD2 and BmGSTS2 were specifically expressed in three gut regions, indicating that the gut is the prime site for BmGSTD2 and BmGSTS2 synthesis in B. mori larvae.