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Constructionn Expression and Functional Study of Mammal Expression Vector of ω-3Fatty Acid Desaturase Gene△17

Author: FuYong
Tutor: ChenHongJu; WangHui
School: Shandong Agricultural University
Course: Animal Genetic Breeding and Reproduction
Keywords: fatty acid desaturase gene construction recombinant plasmid CHO cells eukaryotic expression functional study
CLC: Q78
Type: Master's thesis
Year: 2012
Downloads: 87
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Abstract


Polyunsaturated fatty acids (PUFAs) are essential nutrient for human body. The ω-3polyunsaturated fatty acids (ω-3PUFAs) have broad biological functions, and exert manypreventive and therapeutic actions on cardiovascular diseases and other diseases. The balancebetween ω-6polyunsaturated fatty acids (ω-6PUFAs) and ω-3polyunsaturated fatty acids isimportant for homeostasis and normal development. Mammals can’t synthesize the precursorof ω-3PUFAs or convert ω-6PUFAs to ω-3PUFAs because of lacking necessary enzymaticactivities. So far, mammals rely on intake of PUFAs rich in ω-3PUFAs to elevating bodyconcentrations of ω-3PUFAs, and the source is quit limited.In this study, we optimized the coding sequence (CDS) of fatty acid desaturase genefrom Phytophthora infestans according mammals codons and named as△17desaturase gene.The full-length of the sequence is1086bp, it encodes362amino acid residues. Sequencealignment of the two genes revealed that85.18%gene sequence identity and100%aminoacids similarity. The Δ17desaturase gene could be functionally expressed in mammalian cells,convert arachidonic acid (ARA,20:4n-6)into eicosapentenoic acid (EPA,20:5n-3)specifically.△17desaturase gene was digested by SalⅠand EcoRⅠrestrictive enzyme and inserted intothe sequence of eukaryotic expression vector pIRES2-AcGFP1. Digestion experiments andsequencing results proved that the insertion sequence was correct, and recombinant eukaryoticexpression vector pIRES2-AcGFP1-△17was successfully constructed. The two eukaryoticexpression vectors pIRES2-AcGFP1and pIRES2-AcGFP1-△17were introduced into CHOcells by lipid mediated transfection and then obtained the stable expression cell lines throughG418selection.RT-PCR and GC analysis of cellular lipids of the stably selected cells suggest△17desaturase gene could be heterologous expression in CHO cell lines and play the role of ω-3fatty acid desaturase. Total cellular lipid analysis of transformed cells fed with ARA as asubstrate showed that the amount of ARA dropped from15.44%(in AcGFP1cells) to11.72%(in△17transferred cells), whereas the amount of EPA increased from3.77%to5.92%(in△17transferred cells). The expression of Δ17gene resulted in an86.5–246%(p <0.05)increase of EPA compared with that in the control cells. The ratio of ARA and EPA wasreduced from approximately4.09:1in control cells to1.98:1in Δ17transformed cells (p <0.05). These data demonstrate that Δ17desaturase gene from Phytophthora infestans wasoptimized synthesized successfully and could be used for further study. It also provided another resource to structure mammalian animal model and research this desaturase geneusing in generate transgenic livestock.

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