二化螟P-糖蛋白基因的克隆、序列分析及在阿维菌素处理下的表达模式

Molecular Cloning and Sequence Analysis of the P-glycoprotein Gene in the Rice Stem Borer,Chilo suppressalis and Its Expression Patterns under the Treatment of Abamectin

【目的】P-糖蛋白是ABC转运蛋白超家族的成员,在异源物质的代谢中起着关键的作用,并且参与了多药耐药性。研究旨在明确二化螟Chilo suppressalis P-糖蛋白与阿维菌素抗性的关系,为有效防治二化螟提供理论依据。【方法】结合二化螟转录组信息,克隆二化螟P-糖蛋白基因,并进行生物信息学分析。设置3种浓度的阿维菌素(LC10、LC30和LC50)处理二化螟敏感种群,结合荧光定量PCR技术测定处理后P-糖蛋白基因的表达模式,同时也对比分析二化螟田间抗性种群和敏感种群P-糖蛋白基因的表达差异。【结果】克隆获得了二化螟CsPgp1和CsPgp2 cDNA序列,CsPgp1开放阅读框全长3780 bp,编码1259个氨基酸,预测蛋白分子量为137 ku。CsPgp2开放阅读框全长3939 bp,编码1312个氨基酸,预测蛋白分子量为144 ku。CsPgp1和CsPgp2都拥有2个跨膜结构域(TMD)和2个核苷酸结合结构域(NBD),符合ABC蛋白全转运体的典型特征。系统发育分析表明,二化螟CsPgp1和CsPgp2分属不同的进化支,分别与亚洲玉米螟Ostrinia furnacalis的2种P-糖蛋白亲缘关系最近。使用阿维菌素处理二化螟敏感种群4龄幼虫后,CsPgp1和CsPgp2的表达量均显著上调;田间抗性种群的CsPgp1和CsPgp2表达量均显著高于敏感种群。【结论】CsPgp1和CsPgp2可能参与了二化螟对阿维菌素的代谢,可能与二化螟对阿维菌素抗性有关。

[Objective]P-glycoprotein(Pgp)belongs to the ABC transporter protein superfamily and it is responsible for xenobiotic compounds metabolism and multidrug resistance.This study aims to clarify the relationship between Pgp and abamectin resistance in the Chilo suppressalis and provides theoretical basis for developing efficient management tools for C.suppressalis.[Method]Based on the transcriptome data of C.suppressa⁃lis,the full-length of C.suppressalis Pgp genes were cloned and then subjected to bioinformatics analysis.The expression levels of Pgp genes of C.suppressalis larvae,which were treated with three concentrations(LC10,LC30 and LC50 value)of abamectin,were performed by qRT-PCR.And the expression levels of Pgp genes in field resistant strain compared to susceptible strain were also detected.[Result]Two cDNA sequences of CsPgp1 and CsPgp2 of C.suppressalis were obtained.The opening reading frame(ORF)of C.suppressalis CsPgp1 was 3780 bp,which encoded 1259 amino acid proteins with a calculated molecular weight of about 137 ku.And the CsPgp2 cDNA contained an ORF of 3939 bp,which encoded a protein of 1312 amino acids residues with a molecular mass of approximately 144 ku.CsPgp1 and CsPgp2 both contained two transmembrane domains(TMD)and two nucleotide-binding domains(NBD),which is consistent with the structure motif of full ABC transporter.Phylogenetic analysis showed that CsPgp1 and CsPgp2 were clustered into different branches,those shared high homology with the Pgp of Ostrinia furnacalis.The expression levels of CsPgp1 and CsPgp2 in the 4th instar larvae of susceptible C.suppressalis increased significantly when treated with abamectin.Furthermore,the expression levels of CsPgp1 and CsPgp2 in field resistant strain were significantly higher compared to that of the susceptible strain.[Conclusion]These results indicate that CsPgp1 and CsPgp2 may be involved in xenobiotic metabolism and resistance of abamectin in C.suppressalis.