碩士 / 國立高雄海洋科技大學 / 水產食品科學研究所 / 97 / The complete coding sequence of copper-zinc superoxide dismutase from Rachycentron canadum was cloned and characterized by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of complementary DNA ends (RACE) reactions. Analysis of nucleotide sequence revealed that the Cu-Zn SOD cDNA clone consists of 811 bp with an open reading frame of 465 bp encoding a protein of 154 amino acids. Four conserved amino acids responsible for binding copper (H47, H49, H64 and H121) and four conserved amino acids responsible for binding zinc (H64, H72, H81 and D84) were observed in the Cu-Zn SOD protein sequence. The calculated molecular mass of the proteins is 16.8 kDa with an estimated pI of 6.13. Sequence comparison showed that the Cu-Zn SOD deduced amino acid sequence of Rachycentron canadum has similarity of 85.8%, 81.5%, 77.8% and 75.3% to those of Danio rerio (NM_131294), Mus musculus (NM_011434), Homo sapiens (NM_000454) and Drosophila melanogaster (M24421), respectively. The Cu-Zn SOD was overexpressed with IPTG in E. coli BL21(DE3) and purified with immobilized metal affinity chromatography. The purified recombinant Cu-Zn SOD had a specific activity of 1820 U/mg protein and was purified 8.9-fold, with a yield of 36.8%. Electrophoresis revealed a single band of recombinant protein. The purified enzyme was stable over a pH range of 4.0-11.0. Only slight decrease of enzyme activity was identified at temperatures lower than 65 °C. The enzyme was not inhibited by DTT, NaN3, PMSF, SDS and -mercaptoethanol, but was inhibited by KCN and hydrogen peroxide. In the presence of 1% Triton X-100 and Tween 20, the enzyme showed approximately 37.6 and 64% activity loss. The real-time PCR (qPCR) analyses showed that expression of Cu-Zn SOD mRNA predominated in liver and gill, and at a lower level in the head kidney and skin. The effects of dietary fatty acids on the liver of relative antioxidant enzymes expression were evaluated in 3, 9-week growth trial, in which cobias with an initial weight of 80 g were fed diets containing 15% lipid. Among the lipids, 6% was fish oil and the remaining 9% were fish oil (rich in EPA and DHA), perilla oil (C18:3n-3), safflower oil (C18:2n-6), olive oil (C18:1n-9) or palm oil (C16:0). In the liver, among the dietary treatments, significantly higher expression levels of Cu-Zn SOD, Mn SOD, glutathione peroxidase and glutathione reductase mRNA were detected in perilla oil group, catalase mRNA in palm oil and olive oil group. Linear regression analysis showed that Cu-Zn SOD mRNA expression was positively (p＜0.05) correlated with dietary C18:3n-3 levels, Mn SOD mRNA expression was positively (p＜0.001) correlated with dietary C18:3n-3 level, catalase mRNA expression was negatively correlated with dietary C14:0, C20:1n-9, C20:5n-3 and C22:6n-3 level, glutathione reductase mRNA expression was positively correlated with dietary C18:3n-3 levels at wk 3. At wk 9, Glutathione reductase mRNA expression positively correlated with dietary C20:1n-9, C20:5n-3 and C22:6n-3 level.
| Identifer | oai:union.ndltd.org:TW/097NKIM8084011 |
| Date | January 2009 |
| Creators | Shih-Ming Hsu, 許時銘 |
| Contributors | Rey-Chang Chang, Der-Shyan Sheu, 張瑞璋, 許德賢 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 105 |
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