Enhancement Of Two Superoxide Dismutases (SODs) From The Desert Beetle Microdera Punctipennis To Cold Tolerance Of E.Coli And Drosophila Melanogaster

Temperature is one of the most important environmental factors that affects insect growth,reproduction,distribution and abundance.Insects have evolved many strategies to deal with low environmental temperatures.Micordera punctipennis is a freeze-avoiding insect.Freeze avoidance involves deep supercooling of body fluids to prevent ice formation.However,this supercooling state allows insects to continue aerobic metabolism throughout the whole winter.Constantly aerobic metabolism thought the winter,even if it slows down,is likely to be enough to induce the production of ROS in insects and cause the continuous damage of ROS to biological macromolecules.Superoxide dismutases are the main antioxidant enzyme families in organisms.They are considered as the first defense line against oxidative stresses due to their functions of converting O₂·^-to H₂O₂and H₂O.Superoxide radicals(O₂·^-)are usually the first ROS to be generated,which may trigger the formation of more reactive ROS.Therefore,SODs are considered to be the first line of defense against oxidative stress.In the present study,we identified and characterized two novel Cu/Zn-SOD genes in the cold-hardy desert beetle M.punctipennis,an extracellular copper/zinc SOD(MpecCu/Zn-SOD)and an intracellular copper/zinc SOD(Mpic Cu/Zn-SOD).The expression patterns of the two SOD genes were detected and the relationship between the superoxide anion radical(O₂·^-)content and total SOD activities were analyzed at low temperature.By overexpressing two MpCu/Zn-SOD in prokaryotic expression and transgenic drosophila,we studied the important role of Cu/Zn-SOD gene in the enhancement of cold tolerance of cells and insects.By measuring the oxidative damage indicators of lipids,proteins and DNA,we clarified that the SOD gene can effectively resist the oxidative damage to the biological macromolecules caused by ROS in cold-resistant insects by removing ROS under cold stress.Our research is helpful for in-depth understanding of the function of desert insect SOD genes to enhance cell cold tolerance,provide a new perspective for the study of cold tolerance mechanisms of cold-resistant insects,and enrich the knowlege of low temperature biology of insects.1.Cloning of two Cu Zn-SOD genes from M.punctipennis and their expression in response to low temperature.The complete c DNA sequence of the extracellular SOD(MpecCu/Zn-SOD)is 800bp in length and the open reading frame is 669 bp,encoding 222 amino acids with a putative signal peptide of 17 amino acids.The full-length c DNA sequence of the intracellular SOD,Mpic Cu/Zn-SOD is 769 bp with a 462 bp open reading frame,encoding 153 amino acids without signal peptide.For MpecCu/Zn-SOD,the sequence identity was 60%to 80%with the homologous proteins from other insect species.For Mpic Cu/Zn-SOD,the sequence identity ranged from 79%⁹3%.The bioinformatics analysis showed that the two proteins have contained conserved domains required for metal ions binding and enzyme activity.The results of quantitative real-time PCR showed that the expression of MpecCu/Zn-SOD was significantly up-regulated by 4?exposure at 0.5 h,but Mpic Cu/Zn-SOD was not induced by low temperature during the whole 11 hour.Superoxide anion radical(O₂·^-)content in the beetles under 4?exposure for 0.5 h showed an initial sharp increase and then 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 O₂·^-content with a correlation coefficient of-0.437.In summary,the results of this study indicate that MpecCu/Zn-SOD may play a role in scavenging ROS under cold conditions.2.Functional identification of the two MpCu/Zn-SOD genes in enhancing cold tolerance of E.coli cellsThe E.coli expression system has many advantages in expressing functional proteins.To study the function of each single MpCu/Zn-SOD,MpecCu/Zn-SOD and Mpic Cu/Zn-SOD gene were cloned into the prokaryotic expression vector p ET-32a to generate the recombinant plasmids p ET32a-Mpic Cu/Zn-SOD and p ET32a-MpecCu/Zn-SOD,respectively.After the transformation of the plasmids into E.coli BL21,the fusion proteins Trx-His-MpecCu/Zn-SOD and Trx-His-Mpic Cu/Zn-SOD were over-expressed respectively and identified by SDS-PAGE and Western blotting.For simplicity,in this study the two transformed bacteria BL21(p ET32?-Mpic Cu/Zn-SOD)and BL21(p ET32?-MpecCu/Zn-SOD)were defined ec SOD transformed bacteria and ic SOD transformed bacteria,respectively,and BL21(p ET32?)was defined as control bacteria.After purification of the target fusion protein by Ni-NTA purification system,the enzymatic properties of Trx-His-MpecCu/Zn-SOD and Trx-His-Mpic Cu/Zn-SOD were examined.Simultaneously,the anti-oxidative activity of the recombinant MpecCu/Zn-SOD and Mpic Cu/Zn-SOD were seperately analyzed by Oxford Cup method.The results showed that the fusion protein Trx-His-MpecCu/Zn-SOD was expressed in form of inclusion bodies,whereas the fusion protein Trx-His-Mpic Cu/Zn-SOD was existed both in the supernatant and the sediment,the proportion in the supernatant is slightly higher than that in the sediment.MpecCu/Zn-SOD had stable enzyme activity in the temperature range of 25??45?,and the highest enzyme activity was at 35?.At the same time,it exhibited a wide range of acid-base tolerance(p H3?12)and the optimum p H was 9.0.Mpic Cu/Zn-SOD had a relatively stable enzyme activity in the acid-base range of p H3?11,and the enzyme activity was the highest at p H 8.At the same time,it exhibited a wide range of temperature tolerance(15??55?),and the optimum temperature was35?.This result indicated that the enzyme activity of the two MpCu/Zn-SODs were stable.Antioxidant activity assays showed that the death zones of the two transformed bacteria ec SOD and ic SOD were smaller in diameter than the control bacteria.Survival curves under-4?showed that two transformed bacteria had significant enhanced cold resistance compared to the control bacteria.Meanwhile,they also showed increased SOD activities compared to the control.Accordingly,under cold stress ec SOD transformed bacteria and ic SOD transformed bacteria had lower relative electric conductivity and malondialdehyde(MDA)content than the control bacteria.Taken together,the E.coli cells that overexpressing Mpic Cu/Zn-SOD or MpecCu/Zn-SOD both increased their resistance to cold stress by scavenging ROS,and mitigate potential cell damage caused by ROS under cold conditions.3.Analysis of cold tolerance of the two Cu/Zn-SODs transgenic Drosophila melanogasterThe two MpCu/Zn-SOD genes,Mpic Cu/Zn-SOD and MpecCu/Zn-SOD were seperately transferred to Drosophila melanogaster via P-element-mediated transformation.By using genetic hybridization,screening and chromosome mapping,we obtained stable genetic transgenic MpecCu/Zn-SOD Drosophila lines and Mpic Cu/Zn-SOD Drosophila lines.Actin-Gal4 line was crossed with these transgenic lines to activate the expression of the transgenic Cu/Zn-SOD genes in the hybrid progenies.The adults of both lines of the MpCu/Zn-SOD-expressing fruit flies exhibited higher survival rates compared with the control flies under cold and oxidative exposure.There were higher SOD activity and lower O₂·^-accumulation in the two transgenic Drosophila lines compared to the control line under cold stress,and they also showed less oxidative damage than the control fly under low temperature conditions,indicating that the overexpression of MpecCu/Zn-SOD or Mpic Cu/Zn-SOD in the transgenic Drosophila lines enhanced their cold tolerance by decreasing O₂·^-accumulation under cold stress and resist the oxidative damage of ROS to lipid,protein and DNA.