淡色库蚊表皮蛋白CpCPR117基因克隆及表达分析

Molecular cloning and expression analysis of cuticular protein gene CpCPR117 in Culex pipiens pallens

目的克隆淡色库蚊表皮蛋白CpCPR117基因,分析其不同发育阶段的表达模式,为探索其抗药性功能奠定基础。方法利用同源克隆从淡色库蚊Ⅲ龄末Ⅳ龄初幼虫中克隆CpCPR117基因编码区序列,并用邻接法与其他昆虫同源序列构建系统发育树。利用实时荧光定量PCR(RT-qPCR)检测该基因在淡色库蚊敏感和氯氰菊酯抗性品系不同发育阶段(Ⅰ和Ⅱ龄幼虫、Ⅲ和Ⅳ龄幼虫、蛹、羽化3 d成蚊)的表达情况。表达差异分析采用独立样本t检验。结果克隆获得表皮蛋白CpCPR117基因,该基因全长459 bp,编码152个氨基酸,含有表皮蛋白CPR家族的RR-2基序。序列同源性比对和系统发育树分析显示,淡色库蚊与致倦库蚊同源蛋白亲缘关系最近,其氨基酸序列一致性为98.00%。RT-qPCR结果显示,CpCPR117在淡色库蚊敏感和抗性品系各龄期均有表达,并且在Ⅰ和Ⅱ龄幼虫、蛹期表达量的差异均有统计学意义(Ⅰ和Ⅱ龄幼虫:t=-17.177,P<0.001;蛹期:t=-3.964,P=0.001)。结论CpCPR117基因在淡色库蚊敏感和抗性品系不同发育阶段的表达水平存在差异,为进一步探索其在淡色库蚊氯氰菊酯抗药性的功能机制奠定了基础。

Objective To clone the cuticular protein gene CpCPR117 of Culex pipiens pallens and analyze its expression patterns in different developmental stages,and to lay a foundation for exploring its function in the insecticide resistance of Cx.pipiens pallens.Methods Homology cloning was performed to clone the coding sequence of CpCPR117 from the late 3rd instar and early 4th instar larvae of Cx.pipiens pallens.The neighbor-joining method was used to construct a phylogenetic tree with the homologous sequences of other insects.Quantitative real-time PCR was conducted to measure the expression of CpCPR117 in different developmental stages(1st-2nd instar larvae,3rd-4th instar larvae,pupae,and 3-day-old adults)of the cypermethrin-sensitive and cypermethrin-resistant strains of Cx.pipiens pallens.Differential expression was analyzed by the independent samples t-test.Results A clone of the cuticular protein gene CpCPR117 was acquired,whose full length was 459 bp.It encoded 152 amino acids and contained the RR-2 motif of CPR family of cuticular proteins.According to sequence homology alignment and phylogenetic tree analysis,CpCPR117 had the closest genetic relationship with the homologous protein of Cx.pipiens quinquefasciatus,with 98.00%amino acid sequence identity.Quantitative real-time PCR showed that CpCPR117 was expressed in all developmental stages of both cypermethrin-sensitive and cypermethrin-resistant strains of Cx.pipiens pallens,with statistical differences in the expression level between the two strains in the 1st-2nd instar and pupal stages(1st-2nd instar:t=-17.177,P<0.001;pupae:t=-3.964,P=0.001).Conclusion CpCPR117 shows different expression levels in different developmental stages between the cypermethrin-sensitive and cypermethrin-resistant strains of Cx.pipiens pallens.This finding lays a foundation for further exploring the function of CpCPR117 in resistance to cypermethrin of Cx.pipiens pallens.