Cloning, Expression And Characterization Of Fe-Superoxide Dismutase From Ceratopteris Thalictroides

Ferns are, evolutionarily, in a pivotal position between bryophytes and seed plants. The genus Ceratopteris is an aquatic fern grown in tropical and subtropical regions of the world. Superoxide dismutases are one family of important antioxidant metalloenzymes that catalyze superoxide radicals from cellular oxidative metabolism into hydrogen peroxide and molecular oxygen. As the key enzyme, it plays a central role in protecting plants against oxidative stress. Fe-sod was thought existed in the lower plants and primitive organisms, now researchers found that it is present in a number of seed plants. The purpose of this study is to obtain the full length cDNA of Fe-sod and to detect the expression of Fe-sod in Ceratopteris thalictroides under different stresses. A pair of degenerate primers designed based on the highly conserved sequences to clone the 458bp mid-fragment of SOD gene from Ceratopteris thalictroides. The obtain full-length cDNA was obtained by the rapid amplification of cDNA ends (RACE) technique. It consisted of 1212 nucleotides and an open-reading frame of 798 bp encoding 266 amino acids. Furthermore, the recombinant pET32-SOD plasmids were transformed into host E. coli BL21 for expression. The recombinant protein about 43kDa was induced by IPTG, it was expressed as an insoluble form and fusion protein mainly existed in the inclusion body. So the inclusion body was used to purify this recombinant protein, and the product was used as the antigen to immune rabbit. Fe-sod expression level under different stresses as chilling and illumination stress was detected by western-blotting. The results indicate that, the Fe-sod could be induced to express by low temperature, strong lighting or weak lighting.