| Polyunsaturated fatty acids (PUFAs) are unbranched fatty acids containing more than one double bond. They have a number of important nutritional and pharmaceutical values including EPA and DHA. They are also involved in the human inflammatory response, cardiovascular events, adipogenesis, immune response, blood pressure regulation, cholesterol metabolism, development of allergies and infant retinal structures. Recently, their potential functions of reducing heart disease, improving vision sensitivity and reading ability have been verified. Currently, the principal source of PUFAs is deep-sea fish. However, it is considered insufficient and endangered due to an expanding market and environmental pollution. Marine microalgae, the primary producers of PUFAs, are being explored as an alternative source for yielding these essential elements of health.Desaturases and elongase system are critical enzymes involved in biosynthesis of PUFAs and occur in most living cells. Desaturases are iron-containing enzymes that introduce a double bond in a specific position counted from the carbonyl end of the fatty acids, aerobically. Rencently, more and more genes encoding enzymes involved in VLCPUFAs biosynthesis of microalgae, eg. desaturases and elongases, have been identified. And the genes of desaterases and elongase systems have important biotechnological appeal from genetic engineering point of view. The identification and prospecting of these genes becomes a novel method for enhanced PUFAs production.Phaeodactylum tricornutum, a marine microalga, is rich in EPA, which accumulates up to 30% of the total fatty acids. This marine diatomwas used for represents an attractive production system for PUFAs such as EPA and DHA. In this dissertation, P. tricornutum has been as a useful source for cloning fatty acid desaturase and elongases genes. The results and significance of this study is summarized as follows:1. The marine diatom Phaeodactylum tricornutum is an ideal candidate for studying the PUFA synthetic. In order to investigate the effects of different time of nitrogen starvation on the growth characteristics and chemical compositions in Phaeodactylum tricornutum , algal cell density, chlorophyll a and fatty acid composition have been examined in laboratory cultures by employing different time of nitrogen starvation(12h,24h,48h,72h,96h ). Our results indicated that the growth curveof Phaeodactylum tricornutum was not remarkable difference, but chlorophyll a and fatty acid composition were significantly affected by time of nitrogen starvation. The production of EPA with much longer time of nitrogen starvation decreased.2.Δ5 fatty acid desaturase is the key enzyme in synthesis of arachidonic acid and Eicosapentaenoic acid.Δ5 fatty acid desaturase enconding gene (abbreviated as D5) of Phaeodactylum tricornutum was amplified from the alga with the primers designed according to the sequence retrieved from GenBank and inserted into pET28a, yielding a recombinant plasmid pET28a-D5. After being transferred into E.coli BL21 (DE3), a transformant containing the recombinant plasmid was selected out with its insert confirmed through resequencing. IPTG successfully reduced the expression of D5 in this prokaryotic system as was proved by SDS-PAGE analysis.3. In this study, aΔ5-fatty acid desaturase encoding gene was isolated from P. tricornutum and expressed in yeast. The recombinant protein conversed di-homo-γ-linolenic acid (DGLA) (20:3Δ8, 11, 14) into AA at an efficiency of 24.7% and eicosatetraenoic (20:4Δ8, 11, 14, 17) into EPA at an efficiency of 59.4%, indicating that it functions in theω-6 andω-3 pathways of polyunsaturated fatty acid biosynthesis. In view of the results, elucidating and understanding the regulatory mechanisms leading to these highly selective accumulations becomes a clear prerequisite in order to implement PUFA biosynthesis in oilseed crops such as linseed or rapeseed.In a word, the identification of theΔ5 fatty acid desaturase gene of P. tricornutum involved in the EPA/DHA production is important in biotechnological applications. The gene can be used in the production of PUFA-rich transgenic plant oils for therapeutic and prophylactic use. Also, advances in understanding gene regulation in PUFA biosynthesis will also impact the single-cell oil industry, such that growth conditions of the microalgae can be manipulated to enhance the production of EPA/DHA. This in turn will impact the marine fish-farming industry which depends on microalgae for enhancing the levels of PUFAs in fish. All the above will eventually afford the public an economical source of balanced n-3/n-6 PUFA-enriched oils that will greatly impact general health and nutrition in the future. This work set the molecular basis for further studies on P. tricornutum to better understand its various cellular processes including EPA biosynthesis. |
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