拟黑多刺蚁5-HT2A受体基因cDNA的克隆及表达

摘要

[目的]克隆拟黑多刺蚁(Pol yrhachis vicina Roger) 5-HT2A受体基因的cDNA序列,并对其核苷酸序列和系统进化进行分析,检测其在拟黑多刺蚁不同发育阶段幼虫和不同品级成虫mRNA的表达情况,为探究5-HT2A受体基因对拟黑多刺蚁生长发育的调控规律提供证据.[方法]从拟黑多刺蚁中提取总RNA,RT-PCR扩增并克隆Pv5-HT2A受体基因;用ProtParam预测蛋白质序列,Blast比较蛋白质序列同源性,用Clustal X进行核酸和氨基酸序列比对,MEGA 6.0比邻法生成系统进化树.利用荧光实时定量PCR检测Pv5-HT2A受体基因在拟黑多刺蚁不同发育阶段幼虫及不同品级成虫mRNA的表达水平.[结果]克隆得到Pv5-HT2A受体基因的cDNA序列,全长2 965 bp,开放阅读框长1 926 bp,编码641个氨基酸.疏水性分析显示,Pv5-HT2A受体氨基酸具有7个跨膜疏水区,属于典型的G蛋白偶联受体类型.系统发育分析表明,拟黑多刺蚁与红收获蚁的亲缘关系最近,相似度达69％.荧光实时定量PCR结果显示,拟黑多刺蚁5-HT2A mRNA在不同发育阶段和不同品级成虫中均有表达,其中卵、3龄幼虫及蛹期的表达量较高;成虫中,雄蚁表达量最高,雌蚁的表达量最低.[结论]拟黑多刺蚁与其他昆虫的5-HT2A受体基因存在相关性,推测5-HT2A受体基因对拟黑多刺蚁的生长发育具有一定调节作用.%[Objective] Polyrhachis vicina Roger Pv5-HT2A gene was cloned,its nucleotide sequence and system evolution were analyzed,and its expressions in different developmental stages were investigated in this paper to provide evidence for the participation of Pv5-HT2A receptor in modulation of P.vicina development and further exploration of Pv5-HT2A functions.[Method] The total RNA was extracted from P.vicina,the Pv5-HT2A receptor gene was amplified and cloned,and the protein sequence was predicted by ProtParam.The homology was compared by Blast,nucleic acids and amino acid sequence alignment were conducted by Clustal X and phylogenetic tree was generated by MEGA 6.0 nearest neighbor method.The Pv5-HT2A receptor mRNA expressions were detected in different developmental stages and castes using real-time PCR.[Result] The obtained full length cDNA of Pv5-HT2A receptor gene was 2 965 bp.The open reading frame (ORF) of 1 926 bp encoded 641 amino acids.Hydrophobic analysis showed that the Pv5-HT2A receptor had seven transmembrane hydrophobic regions.The phylogenetic tree analysis revealed that P.vicina had close relationship with Pogonomyrmex barbatus.The real-time PCR results showed that Pv5-HT2A mRNA was detected at all tested development stages.Pv5-HT2A mRNA was expressed highly in embryo,3rd larvae and pupae.The Pv5-HT2A expression level in male was higher than in female.[Conclusion] The 5-HT2A receptor of P.vicina was cloned successfully and it had correlation with the genes of other insects.It is speculated that 5-HT2A receptor gene plays certain role in growth and development of P.vicina.