碩士 / 國立屏東科技大學 / 水產養殖系 / 92 / This research is aimed to understand the different SOD types which are responsible for immunological and physiological responses, through molecular cloning of SOD sequences in different tissues, pathogenic challenge, environmental stress and expressions of different SOD types in freshwater giant prawn, Macrobrachium rosenbergii. Full lengths of cDNAs encoding cytosolic Mn-SOD (cytMn-SOD), mitochondria Mn-SOD (mtMn-SOD) and Cu,Zn-SOD were cloned in the hepatopancreas of freshwater giant prawn by PCR. The 1339 bp cytMn-SOD cDNA contains 858 bp open reading frames corresponding to 286 amino residues. Deduced peptide was estimated with pI 5.52 and molecular weight 31 kDa. Within the amino sequence, there are 4 amino residues (H110, H158, D243 and H247) concerned with manganese ion binding and 2 possible N-glycosylation sites, NXT and NXS. A highly identity (54 - 77%) was found in cytMn-SOD amino sequence comparing that of other species. The 1202 bp mtMn-SOD cDNA of prawn contains 660 bp open reading frames corresponding to 220 amino residues. Deduced peptide was estimated with pI 7.12 and molecular weight 24 kDa. Within the amino sequence, there are 4 amino residues (H32, H80, D164 and H168) concerned with manganese ion binding and 2 possible N-glycosylation sites, NXT and NXS. A highly identity (62 - 83%) was also found in mtMn-SOD amino sequence comparing that of other species. The Cu,Zn-SOD cDNA contains 843 bp nucleotides with 603 bp open reading frames corresponding to 201 amino residues. Deduced peptide was estimated with pI 4.75 and molecular weight 21 kDa. Within the amino sequence, there are 4 amino residues (H64, H67, H84 and H141) concerned with copper ion binding, 4 amino residues (H84, H92, H101 and D104) concerned with Zinc ion binding and 2 possible N-glycosylation sites, NXT and NXS. The amino sequence of Cu,Zn-SOD compared with that of other species shows poor identity (24 - 54%).
Identity between cytMn-SOD and mtMN-SOD is 54%. Comparing the amino sequences between Mn-SOD and Cu,Zn-SOD, a poor identity with only 14 amino residues identical was observed. Identities between cytMn-SOD and mtMn-SOD are higher than that compared with Cu,Zn-SOD among different species. The putative protein structures of cytMn-SOD and mtMN-SOD are primarily α-helix and random coils while that of Cu,Zn-SOD are β-strand and random coils, responsible for major difference in their secondary structures. Both cytMn-SOD and mtMN-SOD are found with the same expression quantities in all tissues in distribution survey. In contrast, Cu,Zn-SOD expresses only in hepatopancreas and hemocytes. Cu,Zn-SOD expression activities are high in hepatopancreas but only trace amount of Cu,Zn-SOD expression are found in hemocytes. Gel electrophoresis and activity staining reveal that hepatopancreas contain Mn-SOD and Cu,Zn-SOD, while hemocytes and muscle contain only Mn-SOD, in freshwater giant prawn.
To evaluate the SOD mRNA expression profiles during pathogenic challenge, Lactococcus garvieae were injected into freshwater giant prawn for 3 hrs and quantitative real-time PCR were taken place to determine the SOD mRNA amounts. The cytMn-SOD and Cu,Zn-SOD mRNA expressions in hepatopancreas were less in L. garvieae injection treatments than that in saline injection treatments. Expression of Cu,Zn-SOD mRNA in hemocytes were significantly higher in L. garvieae injection treatments than that in saline injection treatments in 3 and 6 hrs after injections. Hepatopancrea cytMn-SOD, mtMn-SOD and Cu,Zn-SOD mRNA expressions were examined after immersion of prawns in different CuSO4 concentrations for 6 days. Those mRNA expressions were significantly higher in 0.2 mg l-1 CuSO4 treatments than that in 0 and 0.3 mg l-1 CuSO4 treatments. Furthermore, Mn-SOD, Cu,Zn-SOD and total SOD protein activities were all significantly higher in 0.2 mg l-1 CuSO4 treatments than that in 0 and 0.3 mg l-1 CuSO4 treatments. The average activities of SOD in hepatopancreas decreased while the treatment of CuSO4 concentration increased.
| Identifer | oai:union.ndltd.org:TW/092NPUST086007 |
| Date | January 2004 |
| Creators | Tung, Ying-Hsiu, 董盈秀 |
| Contributors | Cheng, Winton, 鄭文騰 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 157 |
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