Fission Vibrio Delta 4 Desaturation Enzyme And Saline Bargh Algal Delta 5 Extended Double Enzyme Genes In Turn Promoting The Transformation Of Epa To Dha

Docosahexaenoic acid (DHA,22:6n3) is one of the important polyunsaturated fatty acids (PUFAs). To a certain extent, it can exert prevention and treatment on certain diseases to make us stay healthy. Because of the lack of certain enzymes, the human body doesn’t have the ability to produce DHA to maintain its growth and development, so we can only gain from the diet.At present, taking advantage of transgenic technology, the biosynthesis of DHA in plants has been realized. But the relevant reports in mammals is still very rare. Different organism is accustomed to different codon sequence, they have their particular performance. In this study, with the help of the software Codon Adaption Tool, the original gene sequence of Δ4desaturase gene from marine fungus Schizochytrium sp. and the Δ5elongase gene from marine alga Pavlova salina have been modified into the mice preferred codon. in order to be successfully expressed in mouse cells. Then.we respectively inserted the two modified target gene and its corresponding screening marking, the resistance gene of neomycin and hygro, into the expression vector pcaggs, to make the recombinant plasmids pcagga-neo-Δ4and pcaggs-hygro-Δ5.We also make the combinant plasmids pcagga-neo and pcaggs-hygro,which only inserted the resistance gene, as the negative control.In this study, we used the liposome-mediated method, to co-transfect the combinant plasmids pcagga-neo-Δ4and pcaggs-hygro-Δ5into the sub-strains of Chinese hamster ovary (chinese Hamster ovary. CHO) cell. CHOK1;meanwhile. co-transfect the combinant plasmids pcagga-neo-Δ4and pcaggs-hygro-Δ5into CHOK1as the negative control. And the condition of transfection and screening were both adjusted and optimized. Then we make the drug sensitive detection of G418and hygromycin B to CHOK1. to get the optimum screening concentration. After comparing some different kinds of transfection reagents, we selected LipofertamineM2000.which is the most conducive one to transfect our target gene into cells, and can get the most monoclonal cells.Culturing in the medium which contains dual resistance for about30d, we can obtain the monoclonal cell lines.Respectively choose40monoclonal cells which in good condition from both experimental and negative control group to expand cultivation, extract the total cellular RNA. reverse transcript it into cDNA, and finally detect and identify by RT-PCR. to see whether the monoclonal were transferred into the two target genes. Then, select the positive monoclonal No.38. which expressed the two target gene balancely. and the negative control monoclonal No.22, to expand cultivation, and add the substrate EPA. When the cell density reached90%, we extract the total cellular fatty acids and methyl ester, and then sealed with nitrogen.In this study, we use the gas chromatography-mass spectrometry (GC-MS) method to analyse and identify the total cellular fatty acid composition. The results of RT-PCR and GC-MS both show that, the gene of Δ4desaturase and Δ5elongase all successfully expressed in CHOK1, playing the dehydrogenation and extend action, that is to say, they can together catalyze the substrate EPA to convert to DHA.Analysed the GC-MS result.we know, the total content of polyunsaturated fatty acids between the two groups is not different significantly;but the content of EPA and DNA is significantly different.Thus, in the control group, the added EPA was not or rarely transverse to DHA, while the experimental group took advantage of the EPA to convert it to DHA.In summary.in this study, we successfully constructed two recombinant plasmids.which contained the modified target gene Δ4and Δ5; successfully co-transfected the plasmids in CHOK1cells,and obtained the positive monoclonal cell lines; fanally, we analysed the fatty acid content and composition of the experimental group and control group by the technology of GC-MS. which confirmed Δ4desaturase gene and Δ5elongase gene can work together to improve the content of DHA in CHOK1cells. The result has laid a solid foundation for exploring the synthesis of DHA in animals by transgenic technology, and provides a new perspective and research methods for the svnthesis of DHA.