FADS2 Gene Function And Transcriptional Regulation Mechanism Preliminary Study In Dairy Goats

The rich fatty acid composition of goat milk has unique nutritional value,among which polyunsaturated fatty acids can treat cancer,prevent cardiovascular disease and enhance immunity,which is of great physiological significance to human body.Fatty acid desaturase 2?FADS2?,as a rate limiting enzyme in the synthesis of polyunsaturated fatty acids such as arachidonic acid?AA?and docosahexaenoic acid?DHA?,can dehydrogenate its substrate at the 6th and 7th positions of the fatty acid chain to form double bonds.Therefore,it is of great theoretical and practical significance to study the function of FADS2 gene and the mechanism of transcription regulation for the metabolism of polyunsaturated fatty acids in goat milk.In this study,the primary mammary epithelial cells of dairy goat?Capra hircus?were used as material,and the FADS2 gene sequence of dairy goat was cloned by PCR and analyzed by bioinformatics.Using adenovirus-mediated overexpression and siRNA-mediated interference technique studied the effects of FADS2 gene on fatty acid metabolism in goat mammary gland,and cloning the 5 'flanking sequence of FADS2 gene as well as analyzing bioinformatics.The active region of FADS2 promoter was identified by deletion mutation method,and the transcription regulation mechanism of FADS2 gene was preliminarily investigated by mutating the important elements of the active region at the same time.The results of this study were as follows:1.The CDS region of FADS2 gene?Gene bank number MK292654?was 1 335 bp,encoding 444 amino acids,which was highly homologous to Ovis aries?XM₀15103138.2?and Bos taurus?NM₀01083444.1?.Tissue expression profile analysis found that FADS2 gene expression was the highest in liver,but it was the lowest in small intestine.2.Obtaining an effective siRNA sequence down-regulated the expression of FADS2 gene at the mRNA and protein levels significantly.When inhibiting the expression of FADS2 gene in GMECs,the expression of ELOVL2,ELOVL6 and FADS1 genes related to the synthesis of polyunsaturated fatty acids was up-regulated significantly.It promoted the gene expression of transcription factor SREBP1,fatty acid de novo synthesis FASN,ACACA and desaturase SCD1.However,it inhibited the expression of PPARA and FABP3 genes about fatty acid oxidation and transport.It also decreased the ratio of AA and DHA,inhibited the synthesis of polyunsaturated fatty acids?PUFA?and decreased the content of triglyceride?TAG?in cells.3.Constructing successfully the recombinant adenoviral overexpression vector of dairy goat AD-FADS2 and get high titer adenovirus.The overexpression of FADS2 gene in GMECs inhibited the expression of FADS1,ELOVL2,ELOVL5 and ELOVL6 genes associated with PUFAs synthesis,inhibited the expression of transcription factor SREBP1 gene and inhibited the de novo synthesis of fatty acids,but activated the expression of PPAR ? gene and promoted the oxidative metabolism of fatty acids.At the same time,the expression of fatty acid transport gene FABP3 was promoted.The overexpression of FADS2 gene in GMECs increased the contents of AA and EPA to promote the synthesis of PUFAs.It also promoted the expression of genes related to triglyceride synthesis and the accumulation of triglyceride in cells.4.The full-length 2 226 bp of FADS2 promoter sequence of dairy goat was cloned successfully and analyzed by bioinformatics.There were important transcription factor binding sites such as SREBP1,LXR,PPARs,NF-Y and SP1 in FADS2 promoter sequence.The core region of the promoter of FADS2 gene in dairy goats was found to be between-375 ^-26 bp by the analysis of deletion mutation of 5 'flanking sequence.At the same time,constructing the mutated recombinant luciferase vectors of SRE,SP1 and NF-Y in the core region,and the effects of over-expression of SREBP1 on the promoter activity of each deletion fragment and mutant vector were detected.SREBP1 directly regulated the promoter activity of FADS2 gene through the SRE and NF-Y sites of the active region.In conclusion,FADS2 gene increases the content AA,EPA and DHA in GMECs,which promote the synthesis of polyunsaturated fatty acids in the mammary gland of dairy goats.What's more,the transcription activity of FADS2 is directly regulated by upstream transcription factor SREBP1.