Yc. Zhu et al., cDNA sequence, mRNA expression and genomic DNA of trypsinogen from the Indianmeal moth, Plodia interpunctella, INSEC MOL B, 9(1), 2000, pp. 19-26
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).