cDNAs for a chymotrypsinogen-like protein from two strains of Plodia interpunctella.
Gut proteinases are involved in the solubilization and activation of insecticidal toxins produced by Bacillus thuringiensis and may also be involved in resistance development. Approximately threefold lower chymotrypsin-like enzyme activity was observed in a Bt(entomocidus)-resistant strain of the Indianmeal moth, Plodia interpunctella, than that in the Bt-susceptible strain. Because chymotrypsin-like proteinases are involved in Bt protoxin activation in P. interpunctella, we compared cDNA sequences, mRNA expression levels, and genomic DNA for chymotrypsin-like enzymes in Bt-susceptible and Bt-resistant strains of P. interpunctella. To isolate cDNA coding for chymotrypsinogen-like proteinases, a probe was developed using polymerase chain reaction (PCR) amplification of a cDNA library from the Bt-susceptible strain using a vector primer and a degenerate primer corresponding to a conserved sequence in the active site of serine proteinases. This probe was used to screen cDNA libraries from resistant and susceptible strains. Predicted amino acid sequences from cDNA clones of each strain share similarity with sequences of chymotrypsin-like proteinases and are most similar to a chymotrypsin-like proteinase from the tobacco hornworm, Manduca sexta. cDNAs for putative chymotrypsinogen-like proteins, from both Bt-susceptible and Bt-resistant strains of P. interpunctella share an identical open reading frame of 846 nucleotides. The encoded proteins contain amino acid sequence motifs of serine proteinase active sites, disulfide-bridge cysteine residues, and both zymogen activation and signal peptides. A difference between these cDNAs was observed only in the untranslated region where a substitution of guanine for adenine occurred in the Bt-resistant strain. Southern and Northern blotting analyses indicated that there are no major differences in chymotrypsinogen-like genomic organization and mRNA expression in the two strains. These data suggest that chymotrypsinogen-like proteinase genes and their transcription are similar in the Bt-susceptible and Bt-resistant strains of P. interpunctella.
Zhu
Y C
YC
Grain Marketing and Production Research Center, U.S. Department of Agriculture, Manhattan, KS 66502-2736, USA.
Oppert
B
B
Kramer
K J
KJ
McGaughey
W H
WH
Dowdy
A K
AK
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002919:Chymotrypsinogen / Q000235:genetics*; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D018402:Manduca / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D002919:Chymotrypsinogen; D012697:Serine Endopeptidases