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.
We sequenced nine introns of 25 silkworm (Bombyx mori L.) strains, assuming that the introns are particularly prone to mutation. Mean sequence divergence and maximum sequence divergence in each intronic sequence among 25 silkworm strains ranged from 0.81% (3.8 nucleotides) ~ 9.15% (85.2 nucleotides) and 1.2% (seven nucleotides) ~ 39.3% (366 nucleotides), respectively. The degree of sequence divergence in some introns is very variable, suggesting the potential of using intronic sequences for strain identification. In particular, some introns were highly promising and convenient strain markers due to the presence of a large indels (e.g., 403 bp and 329 bp) in only a limited number of strains. Phylogenetic analysis using the individual or the nine concatenated intronic sequences showed no clustering on the basis of known strain characteristics. This may further indicate the potential of the intronic sequences for the identification of silkworm strains.