意大利蜜蜂幼虫细胞色素P450单氧酶CYP9q1在吡虫啉胁迫下的表达分析

Expression Analysis of Cytochrome P450 Monooxidase AmCYP9q1 in Honeybee under Imidacloprid Stress

【目的】通过生物信息学方法鉴定一种意大利蜜蜂(Apis mellifera)细胞色素P450单氧酶(CYP450)即AmCYP9q1,分析吡虫啉暴露后不同时间点蜜蜂幼虫体内该蛋白编码基因的表达模式,探讨在吡虫啉胁迫下该蛋白的解毒作用。【方法】利用生物信息学程序,分析AmCYP9q1的氨基酸序列特征、结构域、高级结构以及蛋白互作网络,预测该蛋白的生物学功能;构建系统进化树,分析AmCYP9q1在昆虫CYP450家族中的分类和进化关系;通过RT-qPCR测定幼虫经吡虫啉口服暴露后24,72 h时AmCYP9q1基因的表达情况。【结果】AmCYP9q1由510个氨基酸残基组成,相对分子质量为58.8 kDa,等电点为8.91。AmCYP9q1为亲水性蛋白,不具有信号肽和跨膜结构域。多重序列比对和系统进化分析结果表明,AmCYP9q1为CYP450家族中CYP3 Clan成员,具有典型的CYP450家族共有的保守结构域特征。RT-qPCR分析表明,与对照组相比,吡虫啉口服暴露组的AmCYP9q1基因均呈统计学意义上的上调表达(p<0.05),并且暴露后72 h时的相对表达量显著高于暴露后24 h。【结论】AmCYP9q1属于CYP450家族中的CYP3 Clan,可能在意大利蜜蜂幼虫对吡虫啉的代谢解毒过程扮演了重要的角色。

[Purposes]Cytochrome P450 monooxidase(CYP450) has been widely confirmed to play important role in insect detoxification pesticides. The purpose was to characterize a CYP450, AmCYP9 q1, from honey bee Apis mellifera. To analyze its encoding gene expression pattern in A. mellifera larvae under imidacloprid stress and explore its detoxification effect under imidacloprid stress. [Methods]Using bioinformatic methods analyzed the sequence characteristics, domain, advanced structure, and interaction network, predicted biological function of AmCYP9 q1 protein. Construct a phylogenetic tree to analyze the classification and evolution relationship of AmCYP9 q1 protein in the insect CYP450 family. Use the real-time quantitative PCR technology to analyze the AmCYP9 q1 gene expression pattern after imidacloprid treated 24 h and 72 h in A. mellifera larvae. [Findings]AmCYP9 q1 protein consists of 510 amino acids residues, is predicted to have a molecular mass of 58.8 kDa and a theoretical pI of 8.91. The hydrophilic-hydrophobic analysis showed that this protein was hydrophilic but without the signal peptide and the transmembrane domains. Multiple sequence alignment and phylogenetic analysis showed that AmCYP9 q1 could be classified into the CYP450 family of the CYP3 Clan with the typical domain characteristics of the CYP450 family. The real-time quantitative PCR analysis showed that compared with the control group, the expression of AmCYP9 q1 genes of A. mellifera larvae after imidacloprid treated 24 h and 72 h are statistically significantly increased(p<0.05). And the expression of AmCYP9 q1 genes was significantly higher at 72 h than 24 h(p<0.05). [Conclusions]AmCYP9 q1 protein belongs to the CYP3 Clan, a member of the CYP450 family, and may play an essential role in imidacloprid metabolic detoxification of A. mellifera larvae.