靳宗洛2006-07-262018-07-062006-07-262018-07-062005-07-31http://ntur.lib.ntu.edu.tw//handle/246246/17469The total superoxide dismutase (SOD) isozymes activity in green bamboo were analyzed, at least 7 isoforms of CuZnSOD and 1 MnSOD were identified. Southern blotting analysis suggested that there are at least 4 CuZnSOD and MnSOD genes. When supplying 0.05 to 0.2 mM copper or manganese ion into the bamboo leaf crude protein extract resulted in no activities band-shifting of isoforms. When denaturing and renaturing of the protein extract with 8 M urea followed by dialysis, shown the same result as copper ions treatment. The abundance of green bamboo SOD genes and the complexity of CuZnSOD isoforms suggested that green bamboo has a more complex regulation in its antioxidant system. Which may tribute to the physiologically special properties of green bamboo under oxidative stress? By alignment of the published MnSOD cDNA sequences in monocots, the degenerate primers were designed according to the conserved regions. Two full-length cDNA sequences coding for green bamboo BoMnSOD were cloned by the RACE and RT-PCR. The deduced amino acid of BoMnSOD consists of 231 amino acids, including the essential domains of MnSOD and a mitochondrial transit peptide (27 a.a.) in the N-terminus. The BoMnSOD share 75-89% identity with MnSODs from other plants. The cDNA coding regions were cloned into the pGEX-6P-1 expression vector, and expressed in the Escherichia coli BL21 (DE3) strain. Four full-length cDNA sequences coding for green bamboo BoCuZnSOD also cloned. One full-length BoCuZnSOD cDNA clone of 793 bp consists of 152 amino acids, without transit peptide was analyzed. This cytosolic BoCuZnSOD shares 81-95% identity with other plant’s. Recombinant GST-BoCuZnSOD and GST-BoMnSOD remained of the SOD activity, both of which were stable at alkaline pH and declined to 10% after incubation at 60°C for 20 min. The SOD activity of recombinant BoCuZnSOD and BoMnSOD were stable more than 3 days incubated at room temperature. BoMnSOD and BoCuZnSOD cDNA sequences from green bamboo not only overexpressed in prokaryotes but also remained stable under a broad range of pH, higher temperature, also very stable in the room temperature. These properties are beneficial for applications in commercial, such as in cosmetics for skin protection or defending un-esthetic effects caused by oxygen-containing free radicals. The promoter of BoCuZnSOD and BoMnSOD were also studied, analysis of the cis-element within the promoter is proceeding to help us understand the physiological regulation of BoCuZnSOD and BoMnSOD.application/pdf1203018 bytesapplication/pdfzh-TW國立臺灣大學植物科學研究所journal articlehttp://ntur.lib.ntu.edu.tw/bitstream/246246/17469/1/932321B002029.pdf