Mahmood Khalid, Højland Dorte H, Asp Torben, Kristensen Michael
Department of Agroecology, Aarhus University, Slagelse, Denmark.
Department of Molecular Biology and Genetics, Aarhus University, Slagelse, Denmark.
PLoS One. 2016 Mar 28;11(3):e0151434. doi: 10.1371/journal.pone.0151434. eCollection 2016.
Insecticide resistance in the housefly, Musca domestica, has been investigated for more than 60 years. It will enter a new era after the recent publication of the housefly genome and the development of multiple next generation sequencing technologies. The genetic background of the xenobiotic response can now be investigated in greater detail. Here, we investigate the 454-pyrosequencing transcriptome of the spinosad-resistant 791spin strain in relation to the housefly genome with focus on P450 genes.
The de novo assembly of clean reads gave 35,834 contigs consisting of 21,780 sequences of the spinosad resistant strain. The 3,648 sequences were annotated with an enzyme code EC number and were mapped to 124 KEGG pathways with metabolic processes as most highly represented pathway. One hundred and twenty contigs were annotated as P450s covering 44 different P450 genes of housefly. Eight differentially expressed P450s genes were identified and investigated for SNPs, CpG islands and common regulatory motifs in promoter and coding regions. Functional annotation clustering of metabolic related genes and motif analysis of P450s revealed their association with epigenetic, transcription and gene expression related functions. The sequence variation analysis resulted in 12 SNPs and eight of them found in cyp6d1. There is variation in location, size and frequency of CpG islands and specific motifs were also identified in these P450s. Moreover, identified motifs were associated to GO terms and transcription factors using bioinformatic tools.
Transcriptome data of a spinosad resistant strain provide together with genome data fundamental support for future research to understand evolution of resistance in houseflies. Here, we report for the first time the SNPs, CpG islands and common regulatory motifs in differentially expressed P450s. Taken together our findings will serve as a stepping stone to advance understanding of the mechanism and role of P450s in xenobiotic detoxification.
家蝇（Musca domestica）的杀虫剂抗性研究已开展60多年。随着家蝇基因组的公布以及多种新一代测序技术的发展，该研究将进入一个新时代。现在可以更详细地研究异生物质反应的遗传背景。在此，我们利用454焦磷酸测序技术研究了与家蝇基因组相关的多杀菌素抗性791spin品系的转录组，重点关注P450基因。
对clean reads进行从头组装得到35,834个重叠群，其中包含21,780个多杀菌素抗性品系的序列。3,648个序列用酶代码EC编号进行了注释，并映射到124条KEGG通路，其中代谢过程是最主要的通路。120个重叠群被注释为P450，涵盖家蝇44个不同的P450基因。鉴定出8个差异表达的P450基因，并对其启动子和编码区的单核苷酸多态性（SNP）、CpG岛和常见调控基序进行了研究。代谢相关基因的功能注释聚类和P450的基序分析揭示了它们与表观遗传、转录和基因表达相关功能的关联。序列变异分析产生了12个SNP，其中8个在cyp6d1中发现。CpG岛的位置、大小和频率存在差异，并且在这些P450中还鉴定出了特定基序。此外，使用生物信息学工具将鉴定出的基序与基因本体（GO）术语和转录因子相关联。
多杀菌素抗性品系的转录组数据与基因组数据一起为未来了解家蝇抗性进化的研究提供了基础支持。在此，我们首次报道了差异表达P450中的SNP、CpG岛和常见调控基序。综合我们的研究结果将为进一步了解P450在异生物质解毒中的机制和作用奠定基础。
