Cloning, Expression And Characterization Of The Gene Coding For The Manganese Superoxide Dismutase In Green Alga Dunaliella Salina

The first enzymes involved in the detoxification of oxygen free radicals are the SODs, which convert superoxide anion into H₂O₂ and O₂. As the oxygen free radicals being bad for cells, the superoxide dismutases (SODs) constitute the first line of defence against ROS. And the research of the baisc knowledge about molecular biology and the application is put in a very important position. So far, based on the metal co-factor used by the enzyme, SODs are classified into three groups: iron SOD (Fe SOD),manganese SOD (Mn SOD), and copper-zinc SOD (Cu-Zn SOD).Dunaliella salina is an unicellular green alga which is unique in its ability to adapt to hypersaline environment and has high economic importance. It can survive in a medium with 0.05-5.5 mol/L NaCl and live in Qinghai-Tibet Plateau where abound with extreme UV light. The MnSOD in Dunaliella salina has almost 10 isozymes, its activity is up to 105.3U/mg which is more than ten times compare to Spirulina's.The relationship between SOD activity and environment stress in Dunaliella salina has been studied in this research. In different stress (such as salt, UV light and temperature) the SOD activity shows positive relativity with stress intensity. So it is hypothesized that SOD might take part in stress resistance. The characteristic and the number of the enzyme may cause different ability to against extreme environment. So study on SOD in gene and protein levels will help to understand the mechanism of Dunaliella salina in stress resistance.In this study we got these results:1. Characterizing the DsMnSOD under stress conditions.Since Dunaliella salina have the ability of salt-, alkli- and UV- tolerance, we mesured the activity of MnSOD under stress conditions in the cell extract. The result of that the activity of MnSOD was closely related to the stress demonstrated that the MnSOD in Dunaliella salina had the function of stess resistance. This result is the base of our works.2.Cloning the full-length cDNA of MnSOD in Dunaliella salina.The 150bp EST sequence of DsMnSOD gene has been gained by using homologous clone. We aligned DsMnSOD gene of some species, and designed the degenerate primers according to their structural conservative region. The full length DsMnSOD cDNA was obtained by using the3'RACE and 5'RACE methods from CLONTECH.3. Bioinformatics analysis. We used bioinformatics tools analyzing the DsMnSOD gene and the deduced protein.We find that type of MnSOD proteins are highly conserved.4. Complementation analysis.We find that DsMnSOD is correlated to oxidative stress tolerance, such as cold, oxygen, UV and salt stress, specially its ability of saltand UV-tolerance.This suggests that MnSOD may play a very important role in stress resistance in Dunaliella salina.5. Expressing DsMnSOD gene in prokaryotic expression system. At last we expressed DsMnSOD through pET-30a prokaryotic expression system and purified the recombinant protein. Then we characterized the enzyme. The basic works will help us do farther research in future.In this paper, we cloned the MnSOD gene from Dunaliella salina, researched its functions by complementation analyses and characterized its protein by pET-30a prokaryotic expression system. These works, which are essential and sufficient, will help us do farther research well.