cDNAs of aminopeptidase-like protein genes from Plodia interpunctella strains with different susceptibilities to Bacillus thuringiensis toxins

Aminopeptidase N has been reported to be a Bacillus thuringiensis (Bt) Cry1 A toxin-binding protein in several lepidopteran insects, cDNAs of aminopept idase-like proteins from both Bt-susceptible RC688(s) and Bt-resistant HD19 8(r) strains of the Indianmeal moth, Plodia interpunctella were cloned and sequenced. They contain 3345 and 3358 nucleotides, respectively, and each h as a 3048 bp open reading frame that encodes 1016 amino acids. Putative pro tein sequences include 10 potential glycosylation sites and a zinc metal bi nding site motif of HEXXH, which is typical of the active site of zinc-depe ndent metallopeptidases. Sequence analysis indicated that the deduced prote in sequences are most similar to an aminopeptidase from Heliothis virescens with 62% sequence identity and highly similar to three other lepidopteran aminopeptidases from Plutella xylostella, Manduca sexta. Bombyx mori with s equence identities of 51-52%. Four nucleotide differences were observed in the open reading frames that translated into two amino acid differences in the putative protein sequences. Polymerase chain reaction (PCR) confirmed a n aminopeptidase gene coding difference between RC688(s) and HD198(r) strai ns of P. interpunctella in the PCR amplification of a specific allele (PASA ) using preferential primers designed from a single base substitution. The gene mutation for Asp(185)-->Glu(185) was also confirmed in two additional Bt-resistant P. interpunctella strains. This mutation is located within a r egion homologous to the conserved Cry1Aa toxin binding regions from Bombyx mori and Plutella xylostella. The aminopeptidase-like mRNA expression level s in the Bt-resistant strain were slightly higher than those in the Bt-susc eptible strain. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF034483 for susceptible strain RC 688(s) and AF034484 for resistant strain HD198(r)). (C) 2000 Published by E lsevier Science Ltd. All rights reserved.