关键词: 中华按蚊, 溴氰菊酯, 抗性, 细胞色素P450, RNAi, 分子对接

Abstract:  【Aim】 To investigate whether the cytochrome P450 gene of Anopheles sinensis (AsCYP9J5) is related to the deltamethrin resistance of An. sinensis.【Methods】 The open reading frame of AsCYP9J5 was cloned by PCR and analyzed by bioinformatics. The expression levels of AsCYP9J5 in the female pupa and female adult of deltamethrin-sensitive strain (WX-LS) and deltamethrin-resistant strains (YN-LR and CQ-LR), in the head, thorax, first 3 abdominal segments (anterior part of abdomen) and remaining parts of abdomen (posterior part of abdomen) of female pupa of WX-LS, and in the head of the WX-LS female pupa in vitro cultured with 25 mg/mL deltamethrin solution were detected by RT-qPCR. Based on the above expression levels of AsCYP9J5, dsAsCYP9J5 and dsEGFP were injected into the head of female pupa for RNAi at 20 h after pupation of YN-LR and CQ-LR, the expression level of AsCYP9J5 in adults was detected by RT-qPCR, the half knockdown time (KT₅₀), knockdown rate and mortality rate of adults after deltamethrin treatment were counted. The amino acid residues interacting between AsCYP9J5 and deltamethrin were predicted by molecular docking simulation. 【Results】 The open reading frame of AsCYP9J5 was cloned and is 1 626 bp in full-length, encoding 541 amino acids without signal peptide and transmembrane domain. The expression levels of AsCYP9J5 in YN-LR and CQ-LR at different developmental stages were different. The expression level of AsCYP9J5 in YN-LR was significantly higher than that in WX-LS from late instar pupa to the 9 h-old adult. In WX-LS and CQ-LR, AsCYP9J5 was highly expressed from the 30 h-old pupa to the 72 h-old adult. The expression levels of AsCYP9J5 in CQ-LR at different developmental stages (except 20 h-old pupa) were significantly higher than those in WX-LS. The expression level of AsCYP9J5 in the head of the WX-LS female pupa was the highest, followed by that in the throax, while those in the anterior part and the posterior part of the abdomen were similar and the lowest. The expression level of AsCYP9J5 in the head of the WX-LS female pupa at 12 h after the deltamethrin induction was up-regulated by 11-fold as compared with that in the control group, and that at 24 h was slightly lower than that in the control group. In YN-LR and CQ-LR injected with dsAsCYP9J5, the expression levels of AsCYP9J5 of the adult decreased by about 68% and 38%, the knockdown phenotype of the adult appeared about 10 and 20 min earlier, the knockdown rates of the adult were significantly increased and the mortality rates of the adult were increased by 28.6% and 24.0%, respectively, as compared to those in the control injected with dsEGFP, indicating that silencing AsCYP9J5 resulted in a significant increase in susceptibility of An. sinensis to deltamethrin. Molecular docking simulation result showed that deltamethrin can enter the binding pocket of AsCYP9J5 and form a binary Pi-cation interaction with Arg-488, a stable T-type Pi-Pi stack with Phe-109 of AsCYP9J5, and Pi-sulfur and Pi-lone pair electron interactions with Cys-348 and Thr-344. The side chain of deltamethrin can also form a stable hydrogen bonding interaction with Gln-224 of AsCYP9J5, as well as a stable hydrophobic interaction network with Ile-227, Leu-413 and Lys-53 of AsCYP9J5.【Conclusion】 The results of this study suggest that AsCYP9J5 is involved in the maintenance of deltamethrin resistance phenotype in An. sinensis, which might lay a foundation for further investigation of the molecular mechanism of AsCYP9J5 in the metabolism of deltamethrin.

Key words: Anopheles sinensis, deltamethrin, resistance, cytochrome P450, RNAi, molecular docking