Cloning And Expression Of The Manganese Superoxide Dismutase Gene Of E.Coli In Lactobacillus Bulgaricus

Objective: To enhance its tolerance to oxygen, the superoxide dismutase gene encoding the antioxidant enzyme Mn-SOD was cloned and expressed in Lactobacillus bulgaricus that lacks SOD. And this work would lay a foundation for developing SOD fermented milk.Methods and Results: 1).According to the sodA encoding sequence of E.coli MG1655,a pair of primers was designed and the sodA gene was amplified by PCR techniques. After purification, the sodA gene fragment was digested with restriction endonucleases XbaI and HindIII, and then linked with the expression vector pMG36e digested with the same enzymes to construct the recombinant plasmid pMG36e-sod. Restriction enzyme digestion, PCR identification and DNA sequencing analysis confirmed that the construction of pMG36e-sod was successful. 2).In order to optimize the electroporation procedures for Lactobacillus bulgaricus, several parameters were tested including the cell growth phase, voltage, resistance, and the composition of the wash and electroporationbuffers. In our results the maxium transformation efficiency was about 103 transformants per μg of DNA under such conditions.3).The recombinant plasmid pMG36e-sod was transformed into Lactobacillus bulgaricus by electroportion. And the expression of sodA gene in Lactobacillus bulgaricus was verified by SOD activity assay. It was about 590.5NU/mgprotein. In the experiments we also found that the expression of sodA gene is beneficial to aerobic growth of Lactobacillus bulgaricus. Conclusions: 1)The recombinant plasmid pMG36e-sod was successfully constructed.2)Developing a effective electroporation procedure for Lactobacillus bulgaricus. 3)The sodA gene was expressed in Lactobacillus bulgaricus successfully. And expression of sodA gene in Lactobacillus bulgaricus can enhance its tolerance to oxygen.