Identification and initial characterization of the 3′ end of gene transcripts encoding putative members of the pheromone receptor subfamily in Lepidoptera

Abstract

Abstract Semiochemicals, including pheromones and kairomones, used in pest management programs reduce the need for chemical insecticides, and understanding their interactions with their membrane receptors may help make them more effective in the field. Identification of odorant receptors in the Lepidoptera has mainly been achieved using bioinformatics to search DNA sequences generated by genome or expressed sequence tag (EST) sequencing projects. This study reports a rapid method to identify members of the pheromone receptor subfamily in Lepidoptera. Degenerate oligonucleotide primers were designed against a conserved amino acid sequence in the carboxyl terminus of known lepidopteran pheromone receptors, and the primers were used in a 3′ rapid amplification of complementary DNA (cDNA) ends procedure. Polymerase chain reaction products generated from seven different lepidopteran species were TA cloned and sequenced. The cDNA sequences of 25 transcripts were determined to encode potential members of the pheromone receptor subfamily. These cDNAs ranged from 238 to 642 bp and encoded 49–54 amino acids of the carboxyl terminus. Analysis of the 3′ untranslated region reveals that most of the transcripts contain multiple polyadenylation signal sequences, and in the case of Manduca sexta, an alternate polyadenylation signal appears to be used in transcript processing. The 3′ untranslated region was also useful in determining unique receptors encoded by transcripts having highly similar nucleotide and amino acid sequences. Overall, this technique provides a complementary method of pheromone receptor identification in EST sequencing projects, or can be used as a stand‐alone method in conjunction with 5′ rapid amplification of cDNA ends procedures.