绿盲蝽P450基因的克隆及增效醚对P450酶活性的抑制作用  

Cloning of the P450 genes of Apolygus lucorum and the inhibition of piperonyl butoxide by cytochrome P450

作　　者：许新新[1] 谭瑶[1] 高希武[1] 

机构地区：[1]中国农业大学昆虫系,北京100193

出　　处：《应用昆虫学报》2012年第2期324-334,共11页Chinese Journal of Applied Entomology

基　　金：国家重点基础研究发展规划项目(973计划)(2009CB119200);公益性行业(农业)科研专项(201103012)

摘　　要：为探讨P450介导的绿盲蝽Apolygus lucorum(Meyer-Dür)抗药性机制,合理使用杀虫药剂,本研究通过活体和离体抑制实验发现,增效醚(PBO)对绿盲蝽P450酶活性有显著的抑制作用:在处理时长为24h时,P450酶活性由未处理时的12.02pmol/min/mgPro.下降至1.63pmol/min/mgPro.,PBO对P450酶的抑制中浓度为0.256mmol/L。生物测定结果表明,PBO对三氟氯氰菊酯具有显著增效作用,增效7.2倍,而对吡虫啉、灭多威、马拉硫磷无显著增效作用。利用RT-PCR及RACE技术对绿盲蝽P450基因进行克隆,获得了2条CYP4家族基因,全长均为1631bp,含有完整的开放阅读框,编码501个氨基酸;序列比对表明这是一对等位基因,含有CYP4家族所有保守特征序列;同源性比较及系统发育分析显示这2个基因编码的氨基酸序列与褐飞虱Nilaparvata lugens CYP4CE1亲缘关系最近,同源性分别为41.5%和41.1%。The inhibition of cytochrome P450 activity by piperonyl butoxide （PBO）,and its synergistic effects against insecticides,were investigated in vivo and in vitro in Apolygus lucorum （Meyer-Dür）.Results show that PBO synergized cyhalothrin toxicity;the synergistic ratio was 7.2.PBO possessed the potential to inhibit the activity of cytochrome P450 in vitro and in vivo.The IC50 of PBO was 0.256 mmol/L.24 h after the topical application of PBO,the activity of cytochrome P450 decreased from 12.02 pmol/min/mg Pro.to 1.63 pmol/min/mg Pro.Two novel full-length CYP4 cDNAs of cytochrome P450 were isolated from A.lucorum using the RACE method.Each CYP4 cDNA sequence is 1 631 bp and contains an open reading frame encoding 501 amino acids.Multiple sequence alignment demonstrated that the consistent homology rate of the prognosis within the two amino acid sequences was 98.7%,indicating that they are a pair of alleles.Phylogenetic analysis showed that the two genes were most homologous with CYP4CE1 of Nilaparvata lugens with homologies of 41.5% and 41.1%,respectively.

关 键 词：绿盲蝽 增效醚 P450 基因克隆 

分 类 号：S433.3[农业科学—农业昆虫与害虫防治]