Papapostolou Kyriaki Maria, Riga Maria, Samantsidis George-Rafael, Skoufa Evangelia, Balabanidou Vasileia, Van Leeuwen Thomas, Vontas John
Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, GR-700 13, Heraklion, Crete, Greece; Department of Biology, University of Crete, Vassilika Vouton, 70013, Heraklion, Greece.
Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, GR-700 13, Heraklion, Crete, Greece; Department of Biology, University of Crete, Vassilika Vouton, 70013, Heraklion, Greece.
Insect Biochem Mol Biol. 2022 Mar;142:103709. doi: 10.1016/j.ibmb.2021.103709. Epub 2022 Jan 4.
Cytochrome P450 mediated metabolism is a well-known mechanism of insecticide resistance. However, to what extent qualitative or quantitative changes are responsible for increased metabolism, is not well understood. Increased expression of P450 genes is most often reported, but the underlying regulatory mechanisms remain widely unclear. In this study, we investigate CYP392A16, a P450 from the polyphagous and major agricultural pest Tetranychus urticae. High expression levels of CYP392A16 and in vitro metabolism assays have previously associated this P450 with abamectin resistance. Here, we show that CYP392A16 is primarily localized in the midgut epithelial cells, as indicated by immunofluorescence analysis, a finding also supported by a comparison between feeding and contact toxicity bioassays. Silencing via RNAi of CYP392A16 in a highly resistant T. urticae population reduced insecticide resistance levels from 3400- to 1900- fold, compared to the susceptible reference strain. Marker-assisted backcrossing, using a single nucleotide polymorphism (SNP) found in the CYP392A16 allele from the resistant population, was subsequently performed to create congenic lines bearing this gene in a susceptible genetic background. Toxicity assays indicated that the allele derived from the resistant strain confers 3.6-fold abamectin resistance compared to the lines with susceptible genetic background. CYP392A16 is over-expressed at the same levels in these lines, pointing to cis-regulation of gene expression. In support of that, functional analysis of the putative promoter region from the resistant and susceptible parental strains revealed a higher reporter gene expression, confirming the presence of cis-acting regulatory mechanisms.
细胞色素P450介导的代谢是一种众所周知的杀虫剂抗性机制。然而，尚不清楚定性或定量变化在多大程度上导致了代谢增加。最常报道的是P450基因表达增加，但其潜在的调控机制仍不清楚。在本研究中，我们研究了CYP392A16，一种来自多食性主要农业害虫二斑叶螨的细胞色素P450。此前，CYP392A16的高表达水平和体外代谢试验表明该细胞色素P450与阿维菌素抗性有关。在这里，我们通过免疫荧光分析表明，CYP392A16主要定位于中肠上皮细胞，摄食和接触毒性生物测定之间的比较也支持了这一发现。与敏感参照品系相比，在高抗性二斑叶螨种群中通过RNA干扰沉默CYP392A16可使杀虫剂抗性水平从3400倍降至1900倍。随后进行了标记辅助回交，利用在抗性种群的CYP392A16等位基因中发现的单核苷酸多态性（SNP），在敏感遗传背景下创建携带该基因的近交系。毒性试验表明，与具有敏感遗传背景的品系相比，来自抗性品系的等位基因赋予了3.6倍的阿维菌素抗性。CYP392A16在这些品系中的表达水平相同，表明基因表达存在顺式调控。支持这一点的是，对来自抗性和敏感亲本菌株的推定启动子区域进行功能分析，发现报告基因表达更高，证实了顺式作用调控机制的存在。
