碩士 / 國立臺灣海洋大學 / 食品科學系 / 101 / Farmed grouper is a high economic value species, the yield of farmed grouper is accounting for 95% of total fishery production in Taiwan. Fresh raw grouper is chopped as fish steak, and its appearance features might be removed, making difficult in identifying the species. The DNA-based method for the identification of grouper species has been established, but distinguished by protein method is still lack. This study is to identify farming grouper species by protein-based method.
We prepared the used 8M Urea, water-soluble and salt-soluble protein extractions and then analyzed by SDS-PAGE, Urea-IEF and IPG-2DE electrophoresis. The six common farmed groupers (Epinephelus lanceolatus, E. bruneus, E. fuscoguttatus, E. malabaricus, E. coioides and E. tukula) in Taiwan were examined.
Observing the SDS-PAGE patterns of the three extraction methods are almost located in the low molecular weight (&;lt; 30 kD) region showed species differences. The salt-soluble extract method is the best way in SDS-PAGE model. The salt-soluble protein pattern, showed E. bruneus (K) in the molecular weights of 61.1, 20.4 and 18.2 kD, E. fuscoguttatus (B) at 22.7 kD and E. malabaricus (M) at 25.5 kD appeared species-specific protein bands. Use Dice coefficient to calculate the lane similarity of six species with individual other species, we can see the overall similarity of among 73.7%~92.3%, and the largest difference between E. fuscoguttatus (B) and E. coioides (C).
Urea-IEF electrophoresis patterns showed all species have species-specific protein bands in the acidic range (pH 2.5-4.6 and pH 5-7), and the water-soluble extract method is the best way in Urea-IEF model. Observing the pH 5-7 pattern showed species-specific protein bands of E. lanceolatus (G) at pI 6.74, 6.41 and 6.34, E. malabaricus (M) at pI 5.78, and E. coioides (C) at pI 5.15 and 4.64. The similarity bamong the species was 27.8%~90.5%, and the largest difference was between the E. lanceolatus (G) and E. coioides (C).
The water-soluble protein showed abundant species-specific protein spots in the region of pH 4-7 and MW &;lt; 72 kD when using IPG-2DE method. Among all the test species, more species-specific protein spots were shown in E. bruneus (K) and E. tukula (P), but E. malabaricus (M) showed less spot.
The species-specific water-soluble protein bands in the Urea-IEF (pH 5-7) were cut and digested by trypsin then further analyzed by LC-MS/MS. The species-specific proteins were of lactate dehydrogenase A chain (pI 6.74, GM1); Triosephosphate isomerase A (pI 6.41, GM2); Actin (pI 6.34, GM3; pI 5.78, MM1; pI 5.15, CM1). Parvalbumin (KA1, BA1, PA1) was identified from Urea-IEF (pH 2.5-5) gel. Therefore, the species-specific protein were identified following Urea-IEF and LC-ESI-Q-TOF approach.
The SDS-PAGE, Urea-IEF and IPG-2DE coupled with LC-MS/MS were successfully applied in identifying farmed grouper species in Taiwan.
| Identifer | oai:union.ndltd.org:TW/101NTOU5253047 |
| Date | January 2013 |
| Creators | Shih-Hao Tu, 涂士豪 |
| Contributors | Tai-Yuan Chen, 陳泰源 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 92 |
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