Molecular Cloning and Biochemical Characterization ofGreen Bamboo (Bambusa oldhamii) MnSOD and CuZnSOD

• Main Authors: Tsung-Han Wu, 吳宗翰
• Other Authors: Tsung-Luo Jinn
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/18376232623131903241

碩士 === 國立臺灣大學 === 植物科學研究所 === 93 === The total superoxide dismutase (SOD) isozymes activity in green bamboo were analyzed, at least seven isoforms of CuZnSOD and one MnSOD were identified. The pattern of SOD activities were not affected when the crude protein extracts prepared from bamboo leaves were incubated with 0.05-0.2 mM copper ions, 8 M urea or dialyzed after the urea treatment. The results revealed that the seven CuZnSOD activity bands were not caused by the different number of copper ions in the proteins or different number of subunits in the native form of CuZnSOD. Southern blotting analyses revealed that there were at least four to five CuZnSOD and MnSOD genes in green bamboo. The abundance of SOD genes and the complexity of CuZnSOD isoforms suggested that the green bamboo might contained a more complex regulation system for the detoxification of reactive oxygen species (ROS)to cope with the . oxidative stresses. The published MnSOD and CuZnSOD cDNA sequences in monocots were aligned and the degenerated primers were designed according to the conserved regions. These primers were used for RT-PCR, 5’- and 3’-RACE experiments and two BoMnSOD cDNA were cloned from the green bamboo. The deduced amino acid sequences of the two BoMnSOD consist of 231 amino acids, including a putative mitochondrial transit peptide (27 a.a.) in the N-terminus and the essential domains of MnSOD. The identity of the two BoMnSOD genes and other MnSODs genes monocots was 75-89%. Four BoCuZnSOD cDNA were also cloned. The deduced amino acid sequences of the BoCuZnSOD consists of 152 amino acids and it is predicted as a cytosolic form protein. These BoCuZnSOD shares 81-95% identity with other monocot CuZnSODs genes. The coding region of BoMnSOD and BoCuZnSOD were cloned into the pGEX-6P-1 expression vector. the GST-fusion proteins were expressed in the Escherichia coli and purified by the GST affinity column. The purified GST-BoCuZnSOD and GST-BoMnSOD remained the SOD activity. Both fusion proteins were stable at alkaline pH and declined to 10% after incubation at 60°C for 20 minutes. The activity of the fusion GST-BoCuZnSOD and GST-BoMnSOD were also stable under room temperature for 3 days. BoMnSOD and BoCuZnSOD cDNA from green bamboo can be expressed in prokaryote (E. coli) and remain stable activity 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 of defending unesthetic effects caused by oxygen-containing free radicals.