Differentially expressed microRNAs associated with changes of transcript levels in detoxification pathways and DDT-resistance in the Drosophila melanogaster strain 91-R

Dichloro-diphenyl-trichloroethane (DDT) resistance among arthropod species is a model for understanding the molecular adaptations in response to insecticide exposures. Previous studies reported that DDT resistance may involve one or multiple detoxification genes, such as cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), esterases, and ATP binding cassette (ABC) transporters, or changes in the voltage-sensitive sodium channel. However, the possible involvement of microRNAs (miRNAs) in the post-transcriptional regulation of genes associated with DDT resistance in the Drosophila melanogaster strain 91-R remains poorly understood. In this study, the majority of the resulting miRNAs discovered in small RNA libraries from 91-R and the susceptible control strain, 91-C, ranged from 16–25 nt, and contained 163 precursors and 256 mature forms of previously-known miRNAs along with 17 putative novel miRNAs. Quantitative analyses predicted the differential expression of ten miRNAs between 91-R and 91-C, and, based on Gene Ontology and pathway analysis, these ten miRNAs putatively target transcripts encoding proteins involved in detoxification mechanisms. RT-qPCR validated an inverse correlation between levels of differentially-expressed miRNAs and their putatively targeted transcripts, which implies a role of these miRNAs in the differential regulation of detoxification pathways in 91-R compared to 91-C. This study provides evidence associating the differential expression of miRNAs in response to multigenerational DDT selection in Drosophila melanogaster and provides important clues for understanding the possible roles of miRNAs in mediating insecticide resistance traits.节肢动物对滴滴涕（Dichloro-diphenyl-trichloroethane, DDT）的抗性，是探究生物应对杀虫剂暴露的分子适应性机制的经典模型。既往研究显示，DDT抗性的形成可能涉及一个或多个解毒基因的表达变化，例如细胞色素P450单加氧酶（cytochrome P450 monooxygenases, P450s）、谷胱甘肽S-转移酶（glutathione S-transferases, GSTs）、酯酶以及ATP结合盒（ATP binding cassette, ABC）转运蛋白，或是电压门控钠通道的序列改变。然而，微小RNA（microRNAs, miRNAs）在黑腹果蝇（Drosophila melanogaster）抗性品系91-R的DDT抗性相关基因转录后调控中是否发挥作用，目前仍有待深入解析。本研究从黑腹果蝇抗性品系91-R及其敏感对照品系91-C的小RNA文库中鉴定得到的miRNA，绝大多数长度介于16~25 nt，共包含163个前体与256个成熟已知miRNA，以及17个潜在新型miRNA。定量分析预测，91-R与91-C品系间存在10个差异表达miRNA；结合基因本体（Gene Ontology）与通路分析，这10个miRNA的潜在靶转录本所编码的蛋白多参与解毒通路。实时定量聚合酶链反应（RT-qPCR）验证结果显示，差异表达miRNA的表达水平与其潜在靶转录本的表达量呈负相关，提示这些miRNA在91-R与91-C品系的解毒通路差异调控中扮演重要角色。本研究为黑腹果蝇在多代DDT选择压力下的miRNA差异表达模式提供了实验证据，并为解析miRNA介导杀虫剂抗性性状的潜在机制提供了关键线索。