碩士 / 國立臺灣海洋大學 / 水產養殖學系 / 94 / Methyl farnesoate (MF) is a sesquiterpene produced and released by the mandibular organ of crustaceans. MF, an analogue of the insect juvenile hormone III, has been implicated to play a vital role in the regulation of the growth and reproductive development in crustaceans. Farnesoic acid O-methyl transferase (FAMeT) is the key enzyme involved in catalyzing the final step in the MF biosynthetic pathway. In the present study, we report the expression of white shrimp (Lipopenaeus vannamei) FAMeT recombinant protein by employment of E. coli expression system and used it to catalyze synthesis of MF. The shrimp FAMeT mRNA is widely distributed in many tissues and the highest expression was detected in the brain and nerve by real-time quantitative PCR analysis. After eyestalk ablation (ESA), the level of FAMeT mRNA expression increased gradually when the time increased. The coding region of FAMeT cDNA fragment was cloned into pET28b(+) expression vector, then transformed into E. coli to express the FAMeT recombinant protein. After 8 hrs culture in the N-Z Amine Broth, the highest product was found after 3 hr inoculation with 0.08 mM IPTG induction. The recombinant protein mainly exists in the inclusion bodies of E. coli The recombinant protein was dissolved in a denaturing solution and purified with Ni-NTA resin column affinity chromatography. After purification and refolding process, the 36 kDa protein represented about 43% of the insoluble protein. The recombinant protein yield in the E. coli system was approximately 120.5 mg/L. When adult shrimp were injected with rFAMeT (1 µg/g body weight), resulted in MF expression increased gradually. After different rFAMeT and ESA treatment, the highest level of MF was found in the ESA+rFAMeT injection group. The MF level of rFAMeT injection group is higher than ESA group at 6th day. The recombinant protein produce in the present study had biological function and can be used to catalyze MF synthesis.
| Identifer | oai:union.ndltd.org:TW/094NTOU5086048 |
| Date | January 2006 |
| Creators | Jr-Ting Chen, 陳芝庭 |
| Contributors | Jenn-Kan Lu, 陸振岡 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 93 |
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