Porphyra Yezoensis Apoplastic Manganese Superoxide Dismutase Gene Expression Analysis

Porphyra yezoensis is one of the most important economical species of the world in the genus and is considered as the model algae of the intertidal seaweeds. It is also an ideal material for genetics, molecular physiology research of anti-stress and genomics. To study the molecular mechanism of Porphyra yezoensis accommodating to environmental stresses, Porphyra yezoensis being selected as experimental materials , gene expression and enzyme activity of superoxide dismutase is chosen to be the researching object.Characteristic amino acids belonging to manganese superoxide dismutase (Mn SOD; EC 1.15.1.1) were found by analyzing deduced amino acid sequence of Mn SOD from Porphyra yezoensis. According native-PAGE and the sensitivity of different SODs to inhibitors, only one type of SOD, Mn SOD was confirmed.A cDNA corresponding to manganese superoxide dismutase from Porphyra yezoensis Ueda was overexpressed in the Escherichia coli BL21 (DE3) using the expression vector pET-30c(+). The molecular weight of the recombinant PyMnSOD is about 30 KDa and part of it formed inclusion bodies (IBs). The best inducing conditions of recombinant enzyme are 25 oC and 4 hours. With the increasing concentration of Mn2+, the activity of the recombinant PyMnSOD is enhanced by inducement of Mn2+. It is shown that Mn2+ can enhance the enzyme activity effectually.Two generations, the leafy gametophyte and the sporophyte, have different MnSOD activity. The former is 2.68 times as the later one, which shows the acclimatization of the two generations. The Native PAGE activity stain displays only one active band, MnSOD. This result is analogous to some cyanobacterium and anaerobes and can be concluded that the anti-oxidation system is still in an original state. The recombinant PyMnSOD was used to generate a polyclonal antibody in New Zealand white rabbits that recognized PyMnSOD. The value of the polyclonal antibody is 1:8000 according to indirect ELISA. The polyclonal antibody can be used in the western blotting effectively. In order to study the cellular localization of PyMn SOD, immuno-gold labeling was used in this research. The results indicate that the PyMnSODs are located in the apoplast and there are no differences between two generations. This localizing results are analogous to the apoplast MnSOD from many high plants.To study the role of PyMn SOD playing when the leafy gametophytes were in the drought stress, they were treated in water deficit and rehydration. The enzyme activity and expression amount of PyMn SOD were assayed. The enzyme activity of PyMn SOD were increased to eliminate the toxicity of superoxide radical during the course of water deficit stress and following recovery from drought. The final enzyme activity was 2.67 times as the initial control, indicating the important role the PyMn SOD playing in protecting the membrane from superoxide radical damages. The concentration of malondialdehyde (MDA) was established as the index of water deficit stress of the leafy gametophyte.