›› 2009, Vol. 52 ›› Issue (1): 17-26.

• 研究论文 • 上一篇    下一篇

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

赵宇,杨亦桦,武淑文,吴益东   

  • 出版日期:2009-01-20 发布日期:2009-01-20
  • 通讯作者: 吴益东

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

  • Online:2009-01-20 Published:2009-01-20

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-PCRRACE技术,克隆了小菜蛾Plutella xylostella nAChR α亚基的一个新基因(Pxα8)的全长cDNA(GenBank登录号为EU914853)Pxα8cDNA序列全长1 744 bp,开放阅读框为1 602 bp,编码534个氨基酸,具有nAChR α亚基的典型特征,与其他昆虫nAChR α8亚基具有77%96%的相似性,与果蝇nAChR β2亚基具有76%的相似性。Pxα8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,Pxα8 mRNA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。

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

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