| Polyunsaturated fatty acids(PUFAs) are important components of cell membrane,which plays important functions in body's hormone metabolism and the regulation of enzyme activity. The lack of PUFAs will cause airframe physiological disorders and lead to many diseases. Among which,ω- 3PUFAs andω- 6 PUFAs have important biological functions. More and more studies shows thatω-6 PUFAs is not crucial for human beings.Contrarily,their excess will bring many healthy problems. Butω-3 PUFAs are beneficial PUFAs,which havebeen proved to exert many preventive and therapeutic actions on many diseases such as cardiovascular diseases,arthritis,cancer and neuropathic diseases.Human and mammals can't synthesizeω-3 PUFAs in their bodies(so as toω-6 PUFAs), so they obtain PUFAs by dietary intake. However the source ofω-6PUFAs is abundant but that ofω-3 PUFAs is very limited, which lead to the level ofω-3 PUFAs in mammals is relatively too low compared withω-6 PUFAs, the ratio ofω-6/ω-3 PUFAs as high as 16 or more. according to the related research, the appropriate ratio ofω-6/ω-3 should be 1,so human beings usually short inω-3 PUFAs supply.It is necessary to maintain the appropriate proportion of n-3PUFAs physiological and it can serve to prevent cardiovascular disease, neurodegenerative diseases and even cancer role.The balance ofω-6 andω-3 PUFAs are essential in body's normal growth and environmental stability, how to increase the intake ofω-3 PUFAs became the key.ω-3 fatty acids desaturase play important role in the synthesis ofω-3 PUFAs, which can catalyze the conversion ofω-6 PUFAs toω-3 PUFAs by introducing anω-3 double bond into their hydrocarbon chains. Fat-1 gene is Widely exists in fungi, plants and some lower animals, and it can convert PUFAs fromω-6 toω-3 form by translating intoω-3 PUFA desaturase. Researchers have used the method of gene transfer to the fat-1 gene expression in transgenic animals, such as: mice,pigs for the study ofω-3 polyunsaturated fatty acids to provide the functions of an efficient and accurate medical, nutritional animal model. Linoleic acid is the substrate for mammals to synthesize other polyunsaturated fatty acids. Mammals can synthesisω-3PUFAs only when the linoleic acid is enough.This study adopts a series of technical means such as molecular biology to research transgenic sheep ofω-3 fatty acid desaturase gene. According to the sequences reported in Genbank about Caenorhabditis Briggsae'sω-3 fatty acid desaturase gene- sFat-1, the codons of the fatty acid desaturase genes were optimized . First, the coding sequences of the genes sFat-1(inserting enhancer SP163 in sFat-1's upstream (named SP163-sFat-1)) was artificially synthesized. Then cloned the sequences and the polony sequencing results showed that the sequences were correct. Using molecular biology technology, the eukaryotic expression vector pcDNA3.1 (+)-sFat-1-SP163 was constructed, in which SP163-sFat-1 was inserted behind the pcDNA3.(+)'s promoter CMV.Using cationic lipid-transfer method, the CHO cell lines were transferred respectively by pCDNA3.1(+)-sFat-SP163 and pCDNA3.1(+)(as the control group). The three stable cell lines were obtained after the selection of G418, the ratio of the positive cells was greater than 90%. The two Stable cell lines were named respectively by sFat-1-CHO, and pCDNA3.1-CHO. The three stable cell lines were tested by fluorescence quantitative RT - PCR and Western bolt. The Q-PCR results showed that the two target genes transcribed and about 105 times over the control group. The Western-bolt results showed that SFat-1-CHO successful expressed sFat-1 protein. Gas chromatography was taken to analysis the fatty acids of the two cell lines, the results confirmed that the contents ofω-3 PUFAs in sFat-1-CHO and was higher than pCDNA3.1-CHO, meanwhile, the ratio ofω-6 /ω-3 droped.This study provides experimental data to the further research on animals'productions rich inω-3 PUFAs by transgenic techniques.
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