马铃薯甲虫3个烟碱型乙酰胆碱受体α亚基基因的克隆及表达分析

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

通过克隆马铃薯甲虫(Leptinotarsa decemlineata)烟碱型乙酰胆碱受体(nAChR)基因的全长序列并分析其时空表达量差异,为马铃薯甲虫nAChR亚基功能以及对新烟碱类药剂靶标抗性机制的研究提供重要基础。根据马铃薯甲虫转录组数据,采用RACE克隆技术获得3个马铃薯甲虫nAChRα基因的cDNA全长序列;通过相似性和进化树分析验证这3个基因的亲缘关系;利用实时荧光定量PCR技术分析这些基因在马铃薯甲虫不同发育阶段和成虫不同部位的表达情况。本试验验证并获得3条nAChRα亚基片段序列,经BLAST比对得出其分别属于nAChRα4、nAChRα7和nAChRα9亚基,进一步克隆得到这些基因的全长序列,分别命名为Ldα4、Ldα7和Ldα9。3个亚基基因编码的氨基酸序列与已知的昆虫同源序列有较高的相似性。其中Ldα4、Ldα7和Ldα9与赤拟谷盗的相似性最高,分别为87%、89%和47%;Ldα4、Ldα7和Ldα9亚基基因在马铃薯甲虫成虫中主要集中于头部和胸部,在蛹期、幼虫和成虫中均有表达,但表达量有明显差异,Ldα4、Ldα7和Ldα9分别在成虫、3龄幼虫和蛹期阶段的表达量最高。克隆获得的3个基因Ldα4、Ldα7和Ldα9具备nAChRα基因的典型结构特征,且与赤拟谷盗的相应基因位于同一分支,进一步验证了同为鞘翅目昆虫的亲缘关系。Ldα4、Ldα7和Ldα9亚基基因在马铃薯甲虫不同生长阶段的表达量有差异,推测这些亚基基因在马铃薯甲虫的不同生长发育中发挥特定作用。

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.