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Cloning and Functional Characterization of Δ4-Fatty Acid Desaturase Gene and Δ5-Fatty Acid Elongase Gene from Two Marine Microalgae

Author: GuoBing
Tutor: JiangMuLan
School: Chinese Academy of Agricultural Sciences
Course: Microbiology
Keywords: Polyunsaturated fatty acids Δ4- fatty acid desaturase Δ5- fatty acid elongase Ball Isochrysis Phaeodactylum tricornutum Pichia pastoris
CLC: Q943.2
Type: Master's thesis
Year: 2011
Downloads: 90
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Abstract


Ball galbana (Isochrysis sphaerica) tricornutum refers algae (Phaeodactylum tricornutum) are two very important marine microalgae, they are nature polyunsaturated fatty acids (Polyunsaturated fatty acids, PUFAs) primary producers in PUFAs The commercialization of the production has a great potential for development. Ball Isochrysis rich in docosahexaenoic acid (Docosahexaenoic acid, DHA), Phaeodactylum algae eicosapentaenoic acid (Eicosatetraenoic acid, EPA) high content. With the deepening of the positive role of DHA and EPA and other PUFAs and human health research, more and more people pay attention to both marine microalgae. In this paper, two marine microalgae for the study of materials, and the impact of environmental factors on the ball galbana growth and fatty acid composition of marine microalgae DHA synthesis gene cloning and functional verification, gene co-expression to carry out the study, the following results: (1) determination of the light intensity of 16 ~ 58 μmol / (m ~~ 2 · s) and 15 ~ 30 ℃ under the conditions of the ball galbana growth curve and the fatty acid composition and content. The results show that, in the set within the range of light intensity, illumination intensity is stronger, faster microalgae. Of light intensity on the content of the various fatty acids there are differences, the light intensity is too low and too high, are not conducive to algae total body unsaturated fatty acids (Total unsaturated fatty acids, TUFAs) of accumulation. When the light intensity of 24μmol / (m ~ 2 · s) of DHA in the highest accumulation of total fatty acids 11.77%. Ball Isochrysis optimum growth temperature is between 20 ~ 25 ℃, low temperature conditions conducive accumulation of TUFAs. Light intensity of 24μmol / (m ~ 2 · s), the temperature is under 20 ℃ TUFAs highest concentrations, up to 64.99%; same light intensity, temperature of 15 ℃ under the conditions of DHA 12.19% of the total fatty acids. (2) using degenerate PCR, genome walking, and RT-PCR method, Δ4-fatty acid desaturase gene cloned from the ball Isochrysis named isfad4. Read isfad4 full length 1284bp, encoding 427 amino acids of the deduced polypeptide sequence having a typical fatty acid desaturase conserved domains, including a cytochrome b5 binding domain and three histidine clusters. Analysis of the fatty acid composition of the transformants isfad4 transformed into Pichia yeast (Pichia pastoris) GS115 add exogenous substrate fermentation culture. The results show that isfad4 was highly expressed in Pichia pastoris, of exogenous recombinant strains substrate conversion efficiency reached 79.8%. (3) starting from the known sequence of the unknown gene fragment was cloned from Phaeodactylum tricornutum by extending the primer to the Δ5-fatty acid elongase gene, named ptfae5. 1110bp, encoding for ptfae5 total length of 369 amino acids, and the deduced polypeptide sequence having Δ5-fatty acid elongase enzyme typical histidine conserved domain and the endoplasmic reticulum binding domain. The ptfae5 transformed into Pichia pastoris exogenous addition of substrate fermentation culture analysis of the fatty acid composition of the transformants. The results show that the highly expressed in Pichia pastoris ptfae5 in recombinant strains of EPA into docosapentaenoic acid (Docosapentaenoic acid, DPA), the conversion rate was 91.8%, arachidonic acid (Arachidonic acid ARA ) is converted to 22 carbon arachidonic acid (Adrenoic acid, DTA) conversion rate of 81.1%. Truncated to ptfae5 transformed into Pichia pastoris analysis secondary to some missing enzyme activity. The results show that partial deletion of the N-end of the auxiliary areas Δ5-fatty acids does not change the extension of the specificity of the enzyme on the substrate, but the absence of the auxiliary area will reduce the enzyme activity of the enzyme. (4) to utilize isfad4 and ptfae5, built Pichia pastoris coexpression vector pAOd4e5. The conditions pAOd4e5 transformed into Pichia pastoris GS115 add exogenous substrates the isfad4 and ptfae5 co-expression studies. The results showed that the isfad4 and ptfae5 in Pichia pastoris highly expressed recombinant strains of EPA and DPA (n-3) conversion efficiency of 90.8% the ARA and DTA conversion rate of 83.2% and 90.9%, respectively.

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