Characterization of a Mn-SOD from the desert beetle Microdera punctipennis and its increased resistance to cold stress in E. coli cells.

Insects have developed a complex network of enzymatic antioxidant systems for handling reactive oxygen species (ROS) generated during stress. Superoxide dismutases (SODs) play a determinant role in balancing ROS in insect. However, studies devoted to SODs functions in insects under cold stress are limited. In the present study, we attempted to identify and characterize a mitochondrial manganese SOD (mMn-SOD) from the desert beetle Micordera punctipennis (denoted as MpmMn-SOD) and explore its protective effects on bacteria cells under cold stress. MpmMn-SOD is composed of 202 amino acids with conserved domains required for metal ions binding and enzyme activity. RT-qPCR experiments revealed that the expression of MpmMn-SOD was ubiquitous but tissue-specific and was induced by cold stress. An E. coli (BL21) system was applied to study the function of MpmMn-SOD. The MpmMn-SOD gene was cloned into the prokaryotic expression vector pET-32a to generate a recombinant plasmid pET-32a(MpmMn-SOD). After transformation of the plasmid into E. coli BL21, the fusion protein Trx-His-MpmMn-SOD was overexpressed and identified by SDS-PAGE and Western blotting. Antioxidant activity assay showed that the death zones of the transformed bacteria BL21 (pET32a-mMn-SOD) were smaller in diameter than the control bacteria BL21 (pET32a). Survival curves under -4 °C showed that BL21 (pET32a-mMn-SOD) had significant enhanced cold resistance compared to BL21 (pET32a). Its SOD activity under -4 °C had a significant negative correlation (r = - 0.995) with superoxide anion O2 •- content. Accordingly, under cold stress BL21 (pET32a-mMn-SOD) had lower electric conductivity and malondialdehyde (MDA) content than BL21 (pET32a). Taken together, our results showed that cold stress stimulated the expression of MpmMn-SOD in M. punctipennis. The E. coli cells that overexpress MpmMn-SOD increase their resistance to cold stress by scavenging ROS, and mitigate potential cell damage caused by ROS under cold conditions.

Characterization of two copper/zinc superoxide dismutases (Cu/Zn-SODs) from the desert beetle Microdera punctipennis and their activities in protecting E. coli cells against cold.

Superoxide dismutases (SODs) are crucial in scavenging reactive oxygen species (ROS); however, studies regarding SOD functions in insects under cold conditions are rare. In this paper, two novel Cu/Zn-SOD genes in the desert beetle Microdera punctipennis, an extracellular copper/zinc SOD (MpecCu/Zn-SOD) and an intracellular copper/zinc SOD (MpicCu/Zn-SOD), were identified and characterized. The results of quantitative real-time PCR showed that MpecCu/Zn-SOD expression was significantly up-regulated by 4 °C exposure for 0.5 h, but MpicCu/Zn-SOD was not. Superoxide anion radical (O2•-) content in beetles under 4 °C exposure for 0.5 h showed an initial sharp increase and fluctuated during the cold treatment period, which was consistent with the relative mRNA level of MpecCu/Zn-SOD. The total SOD activity in the beetle was negatively correlated to the O2•- content with a correlation coefficient of -0.437. An E. coli system was employed to study the function of each MpCu/Zn-SOD gene. The fusion proteins Trx-His-MpCu/Zn-SODs were over expressed in E. coli BL21 using pET32a vector, and identified by SDS-PAGE and Western blotting. The transformed bacteria BL21(pET32a-MpecCu/Zn-SOD) and BL21(pET32a-MpicCu/Zn-SOD) showed increased cold tolerance to -4 °C as well as increased SOD activity compared to the control BL21(pET32a). The relative conductivity and malondialdehyde content in the two MpCu/Zn-SODs transformed bacteria under -4 °C were significantly lower than the control BL21(pET32a). Furthermore, BL21(pET32a-MpecCu/Zn-SOD) had significantly higher SOD activity and cold tolerance than BL21(pET32a-MpicCu/Zn-SOD) under -4 °C treatment, and had lower conductivity than BL21(pET32a-MpicCu/Zn-SOD). In conclusion, low temperature led to the accumulation of O2•- in M. punctipennis, which stimulated the expression of extracellular MpCu/Zn-SOD gene and the increase of total SOD activity. E. coli overexpressing Trx-His-MpCu/Zn-SODs increased resistance to cold treatment-induced oxidative stress. Our findings will be helpful in further study of Cu/Zn-SOD genes in insect cold-tolerance.