关键词: 家蝇, 8-羟基鸟嘌呤糖苷酶, 基因克隆, RNA干扰, 氧化应激

Abstract: 【Aim】 The objective of this study is to clone and identify the 8-hydroxyguanine glycosylase 1 gene from Musca domestica (Mdogg1), so as to verify its function in regulating redox balance. 【Methods】 Based on the previous transcriptome data of M. domestica, the cDNA sequence of Mdogg1 was cloned by RT-PCR and then subjected to bioinformatics analysis. The expression levels of Mdogg1 at different developmental stages (egg, 1st-3rd instar larva, pupa and adult), in different tissues (hemocyte, muscle, gut, and fat body) of the 3rd instar larvae and in the 2nd instar larvae of M. domestica after different stresses ［exposed to 2.5-100 mmol/L CdCl₂ for 24 h, soaked in 0.1 g/L doxorubicin hydrochloride (DOX) solution for 30 min and then recovered for 6, 12 and 24 h, and exposed to 280-315 nm ultraviolet (UV) at the dose of 5 J/cm² for 5-30 min, respectively］ were detected by qRT-PCR. The expression of Mdogg1 in the 2nd instar larvae of M. domestica was knocked down by RNAi, and the changes in the malondialdehyde (MDA) content, reactive oxygen species (ROS) level, superoxide dismutase (SOD) activity, and 8-oxoguanine (8-oxoG) content were determined. 【Results】 The full-length cDNA sequence of Mdogg1 (GenBank accession number: AYK27449.1) was obtained. It is 1 062 bp in length, encoding 353 amino acid residues, with the theoretical molecular weight of 41.08 kD and the isoelectric point of 9.02. The amino acid sequence of MdOGG1 includes a HhH (Helix-hairpin-Helix) motif and the conserved endonuclease III domain ENDO3c domain. The qRT-PCR results showed that Mdogg1 was highly expressed in the egg stage and the fat body of the 3rd instar larvae of M. domestica. When the 2nd instar larvae of M. domestica were exposed to 2.5-100 mmol/L CdCl₂, the expression level of Mdogg1 increased first and then decreased, and the 8-oxoG content increased. The expression levels of Mdogg1 in the 2nd instar larvae soaked in 0.1 g/L of DOX solution for 30 min and then recovered for 12 and 24 h, and those exposed to UV for 30 min, were significantly up-regulated as compared to that in the untreated control. After knockdown of Mdogg1 in the 2nd instar larvae of M. domestica by RNAi, the ROS level, MDA content and 8oxoG content in M. domestica larvae increased significantly as compared to the control (dsGFP injection group), while the SOD activity decreased significantly. 【Conclusion】 Mdogg1 is involved in maintaining redox balance in M. domestica, playing an important role in protecting the insect from oxidative damage.

Key words: Musca domestica, 8-hydroxyguanine glycosylase, gene cloning, RNA interference; oxidative stress