Study of two midgut aminopeptidases from Ostrinia nubilalis Hubner

Abstract

Aminopeptidases N (APNs) have been identified as Bacillus thuringiensis endotoxins receptor candidates in several Lepidopteran species. Employing the RACE PCR technique we obtained two complete cDNAs corresponding to two APNs expressed in the midgut of Ostrinia nubilalis larvae. One of the sequences was 3624 bp long, and the predicted protein was composed by 940 aminoacids, whereas the other cDNA was 3226 nucleotides long, leading a putative protein composed by 994 aminoacids. The in silico study of the sequences, showed in both proteins a signal peptide, a GPI-anchor domain, a zinc-binding region HEXXHX18E and a GAMEN motif, characteristic of the gluzincin aminopeptidases. Moreover, several glicosilation sites were identified. The phylogram tree derived from ClustalW alignment grouped Lepidopteran APNs in five classes. The first sequence would belong to class 1 while the second one would belong to class 2. The expression of the two O. nubilalis APNs was studied during the larval development. Total RNA was purified from neonate larvae and from larvae 5, 10, 15 and 25 days old. RT-PCR reactions showed that both APNs where expressed during the whole larval growth.