Bacillus thuringiensis (Bt) is a gram-positive bacterium that produces parasporal crystal proteins known as endotoxins or Cry proteins. The Cry protoxins are then cleaved by insect midgut proteinases to form active Bt toxins. The activated Cry protein then binds to specific receptors at the midgut epithelium. Cadherin-like and aminopeptidase N (APN) proteins are involved in Bt toxin binding by interacting sequentially with different toxin structures. Aminopeptidase N (APNs) from several insect species have been shown to be putative receptors for these toxins. We have characterized four different midgut APNs(APN1, APN2, APN3, APN4) cDNAs from S. exigua. Forward primers and reverse primers for confirmation of four different midgut APNs were designed based on their sequences cloned from the cDNA libraries. Quantitative RT-PCR procedures includes 42℃ for 20min (cDNA synthesis), 99℃ for 5min, and 35 cycles (94℃ for 1min, and 60℃ for 50 s) for collection. Four aminopeptidase N isoforms were confirmed with qRT-PCR. Sequence analysis was performed by BlastX search the National Center for Biotechnology Information(NCBI) nucleotide. Furthermore, double-stranded RNAs(dsRNAs) were synthesized. DsRNAs were determined for bioassay.

The diamondback moth, Plutella xylostella, is one of the most important pests of cole crops in the world and is the first insect to evolve resistance to Bt toxins in open-field populations. To search for useful molecular markers for Bt reistance monitoring, the PCR-based restriction fragment length polymorphism (RFLP) profiles of three aminopeptidase N (PxAPN1, PxAPN2 and PxAPN4) were determined for 15 representative regional field populations of P. xylostella. Most regional samples had similar RFLP patterns, whereas PxAPN1 from four regions and PxAPN4 from two regions showed different banding patterns after restriction enzyme treatment, but no differences were found in PxAPN2 among populations. The DNA sequence analysis revealed that a point mutation at the restriction site was responsible for the polymorphism of PxAPN1 but no mutations were observed in PxAPN4. Comparing amino acid sequences of PxAPNs from regional populations with reference PxAPNs (GenBank accession no. AAB70755) revealed that four regional populations possessed a point mutation in the Cry1A binding site of PxAPN1 and five regional populations possessed a deletion of eight amino acids in PxAPN4. These RFLP patterns were consistently observed in Southern regions of Korea, including Kyungsangnam-Do and Jeju-Do. The functional association of these RFLP with Bt resistance is currently under investigation