cDNA sequence, mRNA expression and genomic DNA of trypsinogen from the Indianmeal moth, Plodia interpunctella

Trypsin-like enzymes are major insect gut enzymes that digest dietary prote ins and proteolytically activate insecticidal proteins produced by the bact erium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indi anmeal moth, Plodia interpunctella, was linked to the absence of a major tr ypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like pr oteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt- susceptible and -resistant strains of the Indianmeal moth were compared. Pr oteinase activity blots of gut extracts indicated that the susceptible stra in had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides fro m the susceptible strain and 848 nucleotides from the resistant strain cont ained an open reading frame of 783 nucleotides which encoded a 261-amino ac id trypsinogen-like protein. There was a single silent nucleotide differenc e between the two cDNAs in the open reading frame and the predicted amino a cid sequence from the cDNA clones was most similar to sequences of trypsin- like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid s equence motifs of serine proteinase active sites, conserved cysteine residu es, and both zymogen activation and signal peptides. Northern blotting anal ysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the s trains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were diffe rent. Based on an enzyme size comparison, the cDNA isolated in this study c orresponded to the gene for the smaller of two trypsin-like proteinases, wh ich is found in both the Bt-susceptible and -resistant strains of the India nmeal moth. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF064525 for the RC688 strain and AF06 4526 for HD198).