小菜蛾烟碱型乙酰胆碱受体α亚基cDNA的克隆、序列分析与不同发育阶段表达分析  

Cloning,sequence analysis and developmental expression of a cDNA encoding nicotinic acetylcholine receptor α subunit from Plutella xylostella(L.)(Lepidoptera:Plutellidae)

作　　者：赵宇[1] 杨亦桦[1] 武淑文[1] 吴益东[1] 

机构地区：[1]南京农业大学植物保护学院,农业部作物病虫害监测与防控重点开放实验室,南京210095

出　　处：《昆虫学报》2009年第1期17-26,共10页Acta Entomologica Sinica

基　　金：国家“973”计划项目(2006CB102003);国家“863”计划项目(2007AA10Z421);国家自然科学基金项目(30530530)

摘　　要：烟碱型乙酰胆碱受体（nAChR）介导昆虫中枢神经系统中胆碱能突触兴奋性神经递质的快速传递，也是新烟碱类杀虫剂和多杀菌素的作用靶标。本研究利用RT-PCR和RACE技术，克隆了小菜蛾Plutella xylostella nAChRα亚基的一个新基因（Pxx8）的全长cDNA（GenBank登录号为EU914853）。Pxα8的cDNA序列全长1744bp，开放阅读框为1602bp，编码534个氨基酸，具有nAChRd亚基的典型特征，与其他昆虫nAChRα8亚基具有77％～96％的相似性，与果蝇nAChR132亚基具有76％的相似性。既胡的开放阅读框存在单核苷酸多态性位点，导致多个位点氨基酸的替换。雌性4龄幼虫的多态性位点多于雄性4龄幼虫，而且雌、雄4龄幼虫的多态性位点均不相同。半定量RT—PCR研究结果表明，Pxcα8mRNA在成虫期表达量高于蛹期和4龄幼虫期。本研究结果为进一步研究小菜蛾nAChR亚基的多样性和对多杀菌素的靶标抗性机制提供重要基础。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 eDNA 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 nueleotide 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.

关 键 词：小菜蛾 烟碱型乙酰胆碱受体 Α亚基 CDNA克隆 半定量RT-PCR 

分 类 号：Q966[生物学—昆虫学]