Cloning And Expression Induced By S.Sclerotiorum Of SOD Gene In Brassica Napus

Superoxide dismutase, one kind of metalloenzyme, which has been found ubiquitously among oxygen-metabolizing organisms.According to the requirement of the metal species at the active site of the enzymes, superoxide dismutase has been classified four type: Cu/Zn-SOD, Mn-SOD, Fe-SOD and Ni-SOD. It could catalyse the dismutation of superoxide anion radical to molecular oxygen and hydrogen peroxide.By comparison of varied kinds of plants' Cu/Zn-SOD and Fe-SOD amino acid sequences and nucleotide sequences in GenBank, a pair of degenerate primers that was used for RT-PCR to amplify the two SOD genes segments in Brassica napus was designed and PCR product was sequenced.The 5' end and 3' end sequence of Cu/Zn-SOD gene and the 3' end sequence of Fe-SOD gene were synthesized by RACE-PCR and sequenced. So, the cDNAs of Cu/Zn-SOD and Fe-SOD genes were gained by overlapping sequenses. The deduced Cu/Zn-SOD amino acid sequence has the similarity of 99% with the sequence of Brassica rapa ssp. pekinensis cytosolic Cu/Zn superoxide dismutase blasted in GenBank and 97% for the Fe-SOD3 with Arabidopsis thaliana.Bioinformatics tools analyzing Cu/Zn-SOD gene sequence in the ORF which was 459bp, could express a protein of 152aa. MW of Cu/Zn-SOD was 15.179kD, pI was 5.65, so it was an acidic protein.Its second structure involved some extended strand and random coil.The protein had hydrophilic character and protein site prediction showed that Cu/Zn-SOD had two of Copper/Zinc superoxide dismutase signature 1 and signature2, one of N-glycosylation site, five of N-myristoylation site and one of casein kinase II phosphorylation site.Bioinformatics tools analyzing Fe-SOD gene sequence in the ORF which was 492bp, could express a protein of 163aa. MW of Fe-SOD was 18.899kD, pI was 5.07, so it was an acidic protein.Its second structure involved some alpha helix and random coil.The protein had hydrophilic character and protein site prediction showed that Fe-SOD had one of manganese and iron superoxide dismutase signature and one of N-myristoylation site.We also designed primers according to cDNA sequence of Cu/Zn-SOD amplified its partial genome from ZhongYou821 genome. The corresponding genomic sequence (1,322...