Cloning and expression analysis of three genes encoding nicotinic acetylcholine receptor α subunit in Leptinotarsa decemlineata

Field: Agricultural and Biological Sciences (Life Sciences)

Nicotinic acetylcholine receptors(nAChRs) play an important role in excitatory synaptic transmission in insects and are also one kind of the most important insecticide targets. Colorado potato beetle(CPB,Leptinotarsa decemlineata) is a serious defoliator of potato and often causes great yield loss. Cloning and investigation of the expression patterns of the nAChR α genes from L. decemlineata may provide important basis for understanding of the function of nAChR subunits and neonicotinoid resistance in L. decemlineata. The gene fragments encoding nAChR subunit proteins were isolated by bioinformatic analysis of L. decemlineata transcriptome dataset and polymerase chain reaction(PCR) amplification. Full-length cDNAs were further obtained by rapid amplification of cDNA ends(RACE). Sequence analysis was conducted using BLAST programs on the National Center for Biotechnology Information(NCBI). Phylogenetic analysis was carried out using ClustalW program and neighbor joining method in MEGA 5. 0 program. Quantitative real-time PCR was employed to investigate the expression levels of the three α subunit genes at various developmental stages(1to 4 instar larvae,pupa and adult) and different body parts in adults(head,thorax and abdomen). Results: 1) Three α subunit gene fragments encoding nAChR α4, α7 and α9 subunit proteins were cloned from L. decemlineata and full-length cDNAs of these genes were further obtained, correspondingly designated as Ldα4,Ldα7 and Ldα9 respectively. The complete cDNA sequence of Ldα4,Ldα7 and Ldα9 contained 1 607 bp,2 342 bp,1 227 bp,and encoded a protein of 348,529 and 341 amino acids respectively. 2) Sequence analysis showed that these genes possessed the typical structure characteristics of nAChR gene family and shared high similarities with other insect nAChR α subunit proteins,especially with those from the coleopteran insect,Tribolium castaneum(87%,89% and 47% for Ldα4,Ldα7,Ldα9, respectively). The analysis of phylogenetic tree indicated that Ldα4,Ldα7 and Ldα9 were clustered into the same class with those from T. castaneum respectively,confirming the close evolutionary relationship between L. decemlineata and T. castaneum. 3) All the threegenes,Ldα4,Ldα7 and Ldα9,exhibited the higher expression levels in the head and thorax compared with those in abdomen for adults(P0. 05). These genes also showed different expression levels among different developmental stages. Ldα4 had the highest expression levels in adult and pupa stages,while lowest levels in larvae. However,no significant difference was found between the pupa and larval stages. For Ldα7,the highest level of transcription was observed in the 3rd instar larvae,followed with the 1st instars larvae,and the lowest level was in the 2nd and 4th instar larvae,pupae and adults. For Ldα9,expression level in pupae was higher than those in the adults and larvae(P0. 05). Three full-length cDNAs encoding nAChR α subunit proteins were obtained from L. decemlineata,named Ldα4,Ldα7 and Ldα9,respectively. The expression levels of each gene in larva,pupa and adult stages were different,suggesting that the nAChR subunit genes may play specific roles in different developmental stages of L. decemlineata.