Abstract: Nicotinic acetylcholine receptors (nAChR) mediate the fast action of excitatory neurotransmitter at cholinergic synapses in the central nervous system of insects and are also the target site for neonicotinoids and spinosad. By using RT-PCR and RACE technique, the full-length cDNA of a novel nAChR α subunit (Pxα8) was cloned from Plutella xylostella (GenBank accession no. EU914853). Sequence analysis showed that the full-length cDNA is 1 744 bp in length and contained an open reading frame (ORF) of 1 602 bp which encodes 534 amino acid residues. The deduced amino acid sequence of Pxα8 shared typical features of nAChR α subunits and showed 77%-96% amino acid similarity with insect nAChR α8 subunit orthologues. Interestingly, it also showed 76% amino acid similarity with the nAChR β2 subunit of Drosophila melanogaster. There are many single nucleotide polymorphism (SNP) sites in the ORF of Pxα8, some of which resulted in amino acid substitution. There are more SNPs in female 4th-instar larvae than in male 4th-instar larvae, and the SNP sites are different between the female and male 4th-instar larvae. Semi-quantitative RT-PCR analysis indicated that the mRNA expression level of Pxα8 was higher at adult stage than at either pupal or 4th-instar larval stage. These findings provide important basis for further study on diversity of nAChR subunits and mechanism of target resistance to spinosad in P. xylostella.

Key words: Plutella xylostella, nicotinic acetylcholine receptor, α-subunit, cDNA cloning, semi-quantitative RT-PCR