Cloning and in vitro function analysis of codon-optimized FatI gene

• Published in: Biotechnology and applied biochemistry 2014-05, Vol.61 (3), p.256-263
• Main Authors: Sun, Xiao-Feng, Sun, Xing-Hong, Teng, Mei-Li, Liu, Huan-Qi, Min, Ling-Jiang, Pan, Qing-Jie, Qin, Guo-Qing, Shen, Wei, Li, Lan
• Format: Article
• Language: English
• Subjects: Animals; Caenorhabditis elegans - enzymology; Caenorhabditis elegans - genetics; Cloning, Molecular; Codon - genetics; Deoxyribonucleic acid; DNA; FatI; Fatty acids; Fatty Acids - metabolism; Fatty Acids, Omega-3 - biosynthesis; Fatty Acids, Omega-3 - metabolism; Fatty Acids, Omega-6 - metabolism; Gene Transfer Techniques; Genetic Vectors - genetics; goat fibroblasts; Goats; Mammals; n-3 PUFAs; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; transgene; Transgenic animals
• ISSN: 0885-4513; 1470-8744
• DOI: 10.1002/bab.1166

Currently, n‐3 polyunsaturated fatty acids (n‐3 PUFAs) have attracted great attention because of their biological significance to organisms. In addition, PUFAs show an obvious impact on prevention and treatment of various diseases. Because n‐3 PUFAs cannot be endogenously synthesized by mammals, mammals have to rely on a dietary supplement for sufficient supply. The finding and application of the fatty acid dehydrogenase I (FatI) gene are expected to change the current situation because it can convert n‐6 polyunsaturated fatty acids (n‐6 PUFAs) to n‐3 PUFAs. Meanwhile, the gradual maturation of transgenic technology makes it possible to produce transgenic animals that can synthesize n‐3 PUFAs by themselves. In this study, the DNA coding sequence of FatI was synthesized by a chemical method after codon optimization according to the mammal's codon bias. The synthesized DNA sequence was introduced into Boer goat fetal fibroblasts by the constructed recombinant eukaryotic expression vector pcDNA3.1(+)‐FatI. Boer goat fetal fibroblasts were transfected by electroporation, and the stable transfected cell lines were obtained by G418 selection. Genomic DNA PCR and Southern blot were applied to verify that the foreign gene FatI was integrated into the genome of the Boer goat fibroblasts. RT‐PCR results showed the expression of FatI gene at the mRNA level. The fatty acid profile of cells carrying the FatI gene revealed an increase in total n‐3 PUFAs (from 0.61 to 0.95), but a decrease in n‐6 PUFAs (from 10.34 to 9.85), resulting in a remarkable increase in the n‐3:n‐6 ratio (from 0.059 to 0.096). The n‐3:n‐6 ratio had a 63.49 percent increase, which is a precursor of the response of n‐3 desaturase activity of the FatI gene. The study may provide a practical tool for producing transgenic animals that can produce n‐3 PUFAs by themselves, and we hope that the application will lay the foundation for animals producing n‐3 PUFAs, which will benefit human nutrition and wellness.