Molecular cloning and expression patterns of copper/zinc superoxide dismutase and manganese superoxide dismutase in Musca domestica

• Published in: Gene Vol. 505; no. 2; pp. 211 - 220
• Main Authors: Tang, Ting, Huang, Da-Wei, Zhou, Chuan-Qi, Li, Xiang, Xie, Qi-Jing, Liu, Feng-Song
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2012
• Subjects: Activity; Amino Acid Sequence; amino acids; Animals; bacteria; cadmium; Cadmium - toxicity; Cloning, Molecular; digestive system; double-stranded RNA; Epidermis - enzymology; Escherichia coli; Escherichia coli Infections - enzymology; Expression profile; fat body; Fat Body - enzymology; Gene Expression Profiling; genes; heat stress; hemocytes; Hemocytes - enzymology; Hot Temperature; Houseflies - enzymology; Houseflies - genetics; larvae; molecular cloning; Molecular Sequence Data; Musca domestica; proteins; quantitative polymerase chain reaction; reactive oxygen species; Recombinant protein; RNA, Double-Stranded - metabolism; RNAi; SOD; Staphylococcal Infections - enzymology; Staphylococcus aureus; superoxide dismutase; Superoxide Dismutase - genetics; Superoxide Dismutase - metabolism; NADPH; Cd; NBT; ORF; RNAi; RT; Expression profile; MS; Musca domestica; SOD; Activity; β-actin; FTICR; GFP; Ct; LB; ROS; WSSV; IPTG; Recombinant protein; NJ; HPLC
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2012.06.025

Superoxide dismutases (SODs) are metalloenzymes that represent one important line of defense against reactive oxygen species (ROS). In this paper, two novel SOD genes, MdSOD1 and MdSOD2, which putatively encode 261 and 214 amino acid residues respectively were identified and characterized from the housefly Musca domestica. The high similarity of MdSOD1 and MdSOD2 with SODs from other organisms indicated that they should be two new members of the SOD family. qPCR exhibited a universal expression of MdSOD1 and MdSOD2 detected in various tissues of housefly larva, including the fat body, gut, hemocyte and epidermis. Expression profiling reveals that MdSOD1 and MdSOD2 can be induced significantly via not only heat shock and cadmium (Cd) stress but also Escherichia coli and Staphylococcus aureus challenge. The two genes were cloned into the prokaryotic expression vector pET-28a to obtain the fusion proteins rMdSOD1 and rMdSOD2. Between them, the activity of rMdSOD2 was found by visual assay methods. ESI-LC–MS/MS analysis showed that three peptide fragments of the protein rMdSOD2 were identical to the corresponding sequence of M. domestica MdSOD2. MdSOD1 and MdSOD2 in housefly larvae were abrogated by feeding bacteria expressing dsRNA. High mortalities were observed in the larvae treated with dsRNA of SODs at heat shock, Cd stress and bacterial invasion. This phenomenon indicated that MdSOD1 and MdSOD2 are related to the survival of M. domestica under stress. This may provide new insights into the role of the two SOD genes in protecting M. domestica against both stress and bacterial invasion. ► The mRNA expressions of two SOD genes were up regulated after different stressors. ► The two genes are cloned into pET-28a vector, and the activity of rMdSOD2 is found. ► High mortality happened in the larvae when treated with RNAi at different stressors.