细胞色素P450基因AsCYP6Z2在中华按蚊溴氰菊酯抗性维持中的作用

Role of the cytochrome P450 gene AsCYP6Z2 in the maintenance of deltamethrin resistance in Anopheles sinensis(Diptera:Culicidae)

【目的】探究细胞色素P450基因AsCYP6Z2是否与中华按蚊Anopheles sinensis抗拟除虫菊酯有关。【方法】克隆中华按蚊AsCYP6Z2基因的编码区。采用实时定量PCR(real-time quantitative PCR,qPCR)检测该基因在中华按蚊溴氰菊酯敏感品系(WX-LS)和抗性品系(YN-LR)雌蚊不同发育阶段的表达和在敏感品系雌蛹不同组织中的表达。雌蛹腹部后端分别在25 mg/mL溴氰菊酯溶液和纯丙酮溶液(对照)中体外培养后,采用qPCR检测AsCYP6Z2在溴氰菊酯处理组和丙酮对照组中的表达差异。于化蛹后10 h分别注射ds AsCYP6Z2(RNAi组)和ds EGFP(对照组),采用qPCR检测AsCYP6Z2在RNAi组和对照组中的表达差异;采用生物测定方法测定RNAi组和对照组中雌成蚊接触0.05%溴氰菊酯药膜1 h内的击倒率及雌成蚊接触0.05%溴氰菊酯药膜1 h并恢复24 h时的死亡率。通过分子对接预测该基因与溴氰菊酯相互作用的氨基酸残基。【结果】克隆获得了AsCYP6Z2基因(GenBank登录号:MT840336)的编码区,其开放阅读框(ORF)长1251 bp,编码416个氨基酸,无信号肽和跨膜结构域。发育表达谱表明,AsCYP6Z2基因主要在化蛹30 h至成蚊3 h期间高表达,且抗性品系的基因表达量明显高于敏感品系的;组织表达谱显示该基因在腹部后端的表达量较高,其次是在腹部前端和胸部,在头部的表达量最低。雌蛹腹部后端在25 mg/mL溴氰菊酯溶液中培养12 h和24 h后,处理组中AsCYP6Z2的表达量相比于对照组(纯丙酮培养组)分别上调4倍和13倍。RNAi干扰AsCYP6Z2后,RNAi组中AsCYP6Z2基因的表达量相较于对照组(ds EGFP注射组)下调了约80%。雌成蚊接触0.05%溴氰菊酯药膜1 h后,RNAi组中的个体比对照组中的约提前20 min出现明显的击倒现象,且击倒率明显高于对照组;雌成蚊接触0.05%溴氰菊酯药膜1 h并恢复24 h后,RNAi组中的个体死亡率相比对照组增加了17%,表明沉默AsCYP6Z2基因导致蚊虫对溴氰菊酯的敏感度显著增加。溴氰菊酯与AsCYP6Z2预测蛋白的分子对接研究结果显示,溴氰菊酯可以进入AsCYP6Z2蛋白的结合口袋,并且与Cys-155形成Pi-硫相互作用以及产生较稳定的氢键,侧链也可与AsCYP6Z2的Ala-165,Val-72,Leu-76,Leu-82和Ala-24等氨基酸残基形成稳定的疏水相互作用网络。【结论】研究结果表明AsCYP6Z2基因参与中华按蚊溴氰菊酯抗性表型的维持,这为进一步探究该基因在拟除虫菊酯类杀虫剂代谢过程中的分子机理奠定了前期基础。

【Aim】To investigate whether the cytochrome P450 gene AsCYP6Z2 is related to the pyrethroid resistance in Anopheles sinensis.【Methods】The coding region of the AsCYP6Z2 gene of An.sinensis was cloned.The expression profiles of this gene in different developmental stages of female mosquitoes of the deltamethrin-susceptible strain(WX-LS)and resistant strain(YN-LR)of An.sinensis and its expression profiles in different segments of female pupae of WX-LS were detected by real-time quantitative PCR(qPCR).The posterior segment of female pupal abdomen was incubated with 25 mg/mL deltamethrin solution and acetone(control),respectively,and the consequent expression response of CYP6Z2 between the deltamethrin treatment group and the acetone control group was compared by qPCR.After injection with ds AsCYP6Z2(the RNAi group)and ds EGFP(the control group)into the 10 h old pupa,the difference in the expression level of AsCYP6Z2 between the RNAi group and the control group was compared by qPCR,the knockout rate of female adults after exposure to 0.05%deltamethrin film in 1 h and the mortality of female adults after exposure to 0.05%deltamethrin film for 1 h and then recovered for 24 h in the RNAi group and the control group were determined by bioassay method.Furthermore,the amino acid residues interacting with deltamethrin were predicted by using molecular docking.【Results】The coding region of AsCYP6Z2 gene(GenBank accession no.:MT840336)was cloned,and its ORF is 1251 bp in length,encoding 416 amino acids without signal peptide and transmembrane domain.Developmental expression profiles revealed that AsCYP6Z2 gene was highly expressed in the period from 30 h old pupa to 3 h old adult,and its expression level in the resistant strain was significantly higher than that in the susceptible strain.Tissue expression profiles revealed that AsCYP6Z2 was highly expressed in the posterior segments of abdomen,followed by in the anterior segments of abdomen,throax and head.At 12 h and 24 h after incubation of the posterior segments of female pupal abdomen with 25 mg/mL deltamethrin solution,the expression level of AsCYP6Z2 increased by 4-fold and 13-fold as compared with the control(incubated with acetone),respectively.After RNAi of AsCYP6Z2,the expression level of AsCYP6Z2 gene in the RNAi group decreased by about 80%compared with the control group(ds EGFP injection group).When the female adults were exposed to 0.05%deltamethrin film in 1 h,the individuals in the RNAi group showed significant knockdown phenomenon about 20 min in advance,and the knockdown rate was significantly higher than that in the control group.In addition,the mortality of individuals in the RNAi group after exposure to 0.05%deltamethrin film for 1 h and then recovered for 24 h increased by 17%compared to the control group,indicating that silencing the AsCYP6Z2 gene results in a significant increase in mosquito sensitivity to deltamethrin.The molecular docking between deltamethrin and the predicted AsCYP6Z2 protein showed that deltamethrin can enter the binding pocket of AsCYP6Z2 protein,forming a Pi-sulfide interaction with Cys-155 and generating more stable hydrogen bonds.The side chain can also form a stable hydrophobic interaction network with Ala-165,Val-72,Leu-76,Leu-82,Ala-24 and other amino acid residues of AsCYP6Z2.【Conclusion】The results indicate that the AsCYP6Z2 gene is involved in maintaining the deltamethrin resistance phenotype of An.sinensis,laying a foundation for further exploring the molecular mechanism of AsCYP6Z2 gene in the metabolism of pyrethroid insecticides.