碩士 / 國立臺灣海洋大學 / 水產養殖學系 / 107 / The fat-1 gene encoding the omega-3 desaturase was identified in C. elegan and was proved to be able to convert omega-6 PUFAs to omega-3 PUFAs in transgenic mice and zebrafish. Because the fatty acid synthesis is mainly in the liver, spleen and intestine, we would like to establish the transgenic fish expressing fat-1 in the liver or intestine. By establishment of a transgenic fish with liver-specific or intestine-specific expression of fish-codon optimized fat-1 and AcGFP, we investigated whether the omega-3 PUFAs in whole-body could be increased. We focused on the fatty acid metabolism regulation in transgenic fat-1 transgenic fish, and further explored the changes in endogenously produced omega-3 PUFAs on inflammation-related genes. Firstly, the results showed that the liver-specific expression of fat-1 transgenic zebrafish driven by Drlfabp 2.9 kb promoter increased the omega-3 PUFAs contents to 4-fold (23.85±2.70 % vs. 5.63±4.51 %). Omega-3 PUFAs including LA（18：3n-3）, EPA（20：5n-3）, DPA（22：5n-3）and DHA（22：6n-3） had an increasing trend in liver-specific fat-1 transgenic zebrafish, especially DHA was significantly increased to 3-fold (15.70±4.10 % vs. 5.42±4.86 %) compared with control transgenic zebrafish with AcGFP expression. EPA was not detected in the control group, and the liver-specific fat-1 transgenic zebrafish with the contents of 2.49±1.46%. The fatty acid metabolism was toward fatty acid oxidation, because the expression of ppar-alpha, acox3 and cyp4t8 were activated. In addition, IL-1β, IL-6, IL-12α, IFN-γ and TNF-a, which regulate the genes involved in the promotion of inflammation, are significantly inhibited in the liver-specific expression of fat-1 transgenic zebrafish. In summary, the liver-specific expression of fat-1 in transgenic zebrafish enhanced the EPA and DHA, and inhibited pro-inflammatory cytokines and downstream genes after fed with homemade omega-6 PUFA feed. Secondly, predicted transcription factor binding sites of the intestine-specific fabp2 promoter revealed two enhancer regions in fabp2 2.5 kb promoter of Nile tilapia. The 1.5 kb fabp2 promoter containing enhancer 1 had little promoter activity whereas 2.5 kb fabp2 promoter containing enhancer 1 & 2 were active in both zebrafish and tilapia. Based on the in vivo promoter activity, we are establishing intestine-specific transgenic tilapia driven by either zebrafish Drfabp2 1.0 kb promoter or tilapia Onfabp2 2.5 kb promoter to express fat-1 and AcGFP. Furthermore, we will also explore the effects of endogenously produced omega-3 PUFAs on inflammation-related genes and protection against bacterial pathogens in transgenic tilapia.
| Identifer | oai:union.ndltd.org:TW/107NTOU5086051 |
| Date | January 2019 |
| Creators | Lin, Shi-Ting, 林詩庭 |
| Contributors | Gong, Hong-Yi, 龔紘毅 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 59 |
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