A thioredoxin-like protein (txl) gene was cloned from the bumblebee, Bombus ignitus. The B. ignitus txl (Bitxl) gene spans 1777 bp and consists of three introns and four exons coding for 285 amino acid residues with a conserved active site (CGPC). The deduced amino acid sequence of the Bitxl cDNA was 65% similar to the Drosophila melanogaster txl. Northern blot analysis revealed the presence of Bitxl transcripts in all tissues examined. When H2O2 was injected into the body cavity of B. ignitus workers, Bitxl mRNA expression was up-regulated in the fat body tissue. In addition, the expression levels of Bitxl mRNA in the fat body greatly increased when B. ignitus workers were exposed to low (4°C) or high (37°C) temperatures, or injected with lipopolysaccharide (LPS), which suggests that the Bitxl possibly protects against oxidative stress caused by extreme temperatures and bacterial infection.
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.
A serine protease gene was cloned from the bumblebee, Bombus ignitus. The B. ignitus serine protease (BiSP) gene spans 1702 bp and consists of four introns and five exons coding for 250 amino acid residues. Southern blot analysis of genomic DNA suggested that BiSP gene is a single copy gene. The cDNA encoding BiSP was expressed as a 28-kDa polypeptide in baculovirus-infected insect cells and the recombinant BiSP showed activity in a protease enzyme assay. BiSP was specifically expressed in the midgut of B. ignitus queens, males, and workers, suggesting that the BiSP is a gut enzyme involved in the digestion of dietary proteins.
The diamondback moth, Plutella xylostella (L.), is a notorious insect pest of cruciferous plants. To examine the pattern and magnitude of genetic variation in this species in China a portion of the mitochondrial (mt) COI gene of P. xylostella, collected at six Chinese and two Korean localities, which cover ~2,151,600 km2, was sequenced. Sequence analysis of the 681-bp mt COI gene from 80 individuals resulted in 16 haplotypes, ranging in sequence divergence from 0.1% (one nucleotide) to 0.9% (six nucleotides). One nucleotide position among 16 variable sites was a transversional substitution and the remaining positions were transitional substitutions. No position resulted in amino acid substitution. Phylogenetic analysis showed that all haplotypes were highly interrelated and no discernable haplotype group was found. From a geographical perspective, most haplotypes were found singly at one or two localities, with three haplotypes widely distributed. Little genetic differentiation (FST = -0.038-0.309) and a high rate of female migration (Nm = 1.117 - infinite) between Chinese populations suggests that dispersal over long distances is a major factor in the demography of this species.
The complete nucleotide sequence and the exon-intron structure of the luciferase gene of the firefly, Luciola lateralis (Coleoptera: Lampyridae) is described. The luciferase gene of the L. lateralis firefly spans 1,971 bp and consists of six introns and seven exons coding for 548 amino acid residues. From samples collected at Boun and Muju, Korea, three isoforms, with identical exon-intron structure, named BU, MJ1 and MJ2, respectively, were obtained. Although the amino acid sequences of MJ1 and MJ2 were identical to those of known luciferase genes of Korean origin, the BU type was novel, differing from each of the MJ1 and MJ2 types by one amino acid. The luciferase sequences of the Korean samples, including those previously revealed, differed only by one - two amino acid residues, but differed by five - six amino acid residues from that of the luciferase gene recorded from specimens from Japan, which suggest genetic divergence has occurred in this species. Phylogenetic analyses using both amino acid and nucleotide sequences further showed that the luciferase gene of the Japanese L. lateralis firefly is genetically distinguishable from that of Korean L. lateralis, suggesting the presence of a genetic subdivision between the L. lateralis dwelling in the Korean Peninsula and Japanese Islands.
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.