The Study On Transgenic Chicken Expressing ω-3 Fatty Acid Desaturase Gene

Objective: ω-3 fatty acid desaturase(Fat-1 gene) can convert ω-6 PUFAs to ω-3 PUFAs, which include EPA and DHA, play a vital role in the development of the fetus brain and optic nerve, meanwhile they have certain effect on the treatment of senile dementia、cardiovascular、cancer、schizophrenia and other diseases, however, ω-6 PUFAs are closely related to cancer occurrence. Lacking of Fat-1 gene in vivo and the uneven distribution of ω-3 PUFAs in diets, lead to the ω-6 PUFAs seriously accumulated in vivo, make the ratio of ω-6/ω-3 far exceed the perfect condition of 1:1, therefore, we must through rational meals to replenish the demand of ω-3 PUFAs invivo. Alpha linolenic acid contained in the plant can synthesis of EPA and DHA, but the synthetic efficiency is extremely low, humans can also obtain ω-3 PUFAs by feeding seafood, whereas it costs too high firstly, and also easily accumulated marine pollutant invivo. The purpose of this study was that by reforming Fat-1 gene which refers to Caenorhabditis elegans, so it could express efficiently in chicken cells and converted ω-6 to ω-3 efficiently at the same time and also optimized the tatio of ω-6/ω-3, on this basis, the transgenic chicken riched in ω-3 PUFAs could be obtained by making use of the transgenic technology.Methdos: 1. Codon optimization of Fat-1 gene、the construction of the expression vector and the analysis of the transgenic activityPancreatic enzyme digestion method was used to obtain the chicken embryo fibroblast; Based on the codon usage frequency of poultry, nucleotide sequence of Fat-1 gene of Caenorhabditis elegans was optimized and synthesized; The modified Fat-1 gene(c Fat-1) was subcloned into p LL3.7 vector, constructed the recombinant plasmid p LL3.7-c Fat-1; PCR、enzyme digestion and sequencing was used to identify the recombinant vector; Using Turbo Fect transfection reagents to transfect cultured fibroblasts in vitro with p LL3.7-c Fat-1; The expression of c Fat-1 gene in the cells was verified by RT-PCR analysis and green fluorescent detection of the transfected cells; Gas chromatograph was used to analyze the fatty acid composition and content of the transfected cells then.2. Generation of the transgenic chicken(1) Subgerminal cavity microinjection: A window was made on the top of the equator of the fresh unhatched eggs, exposed the blastoderm, 2μL recombinant plasmid was injected into the area pellucida of the central blastoderm, sealed with plastic wrap, incubated on conventional conditions;(2)Peripheral vascular microinjection: Put chicken embryos which have hatched to 13-15 periods to new standby eggshells, injected the foreign plasmid in the embryonic vessel, sealed with the plastic wrap, and turned over the eggs gently for hatch.The embryo from different incubation period were collected, and the tissue DNA, RNA, and fatty acid were extracted. After that the frozen section detection was conducted, followed by the PCR, RT-PCR, and gas chromatography analysis for the tissue of positive individuals.Results: Through the sequence alignment of the recombinant vector, it was found that the optimized sequence encoding amino acid is consistent with the Fat-1 gene; c Fat-1 gene was effectively expressed in the chicken fibroblasts. The specific expression vector that could express c Fat-1 gene in the chicken was successfully constructed. The content of ω-3 PUFAs in the transgenic cell line growed from 2.33522 to 5.21512, while ω-6 PUFAs decreased by about 1.3 times and the ω-6PUFAs / ω-3PUFAs ratio was reduced from 4.7 to 1.7. The fluorescence signal could be detected in the frozen section of the liver, chest, leg muscle and other tissues of the transgenic chicken obtained with the vascular injection method. The PCR and RT-PCR results also verified the expression of foreign gene. According to the muscle fatty acid analysis, the content of ω-3 PUFAs in the chest tissue of positive transgenic cases significantly increased, and the ratio of ω-6 PUFAs/ω-3 PUFAs falled from 5.1 in the control group to 2.2.Conclusion: The eukaryotic expression vector p LL3.7-c Fat-1 which was suitable to express in the chicken was successfully constructed by this research. The chicken fibroblasts transfected with p LL3.7-c Fat-1 could express foreign gene effectively, and ω-6 PUFAs could be converted to ω-3 PUFAs effectively. The transgenic positive cases were generated through microinjection, and the chromatographic analysis result proved that transgenic cases are capable of expressing ω-3 fatty acid desaturase. The ω-6 / ω-3 ratio in the chest muscle decreased significantly.