Study On Regulation Of MiRNA-1296 And MiRNA-370 On Dairy Goat MTHFR Gene

MTHFR is a flavoprotein and plays an important role in folate metabolism. Folate is important for milk protein synthesis, it means that the expression of MTHFR may be regulate the folate metabolism and milk protein synthesis. Single nucleotide polymorphisms(SNPs) at microRNA(mi RNA) binding sites(miR-SNPs) can affect gene expression. Prior to this study in our lab, there is a c.2494G>A mutation in the dairy goat MTHFR-3’UTR. To investigate the role of MTHFR gene in milk protein synthesis, we must find the miRNAs which regulate the expression of MTHFR gene. Therefore, we found that mi RNA-1296 and mi RNA-370 can bind on the MTHFR-3’UTR(2494G) using the prediction software. In order to research the effects of c.2494G>A on microRNA binding sites, we cloned wild type(2494G) and mutant(2494A) MTHFR-3’UTR, the fragment of wild type and mutant(2494A) MTHFR-3’UTR was used to construct the Dual-luciferase reporter vector psiCHECK?-2-MTHFR-3′UTR. The recombinant reporter vectors and miRNA mimics were co-transfected into the mammary epithelial cells and examined the relative quantity of Dual-Luciferase, and then miRNA mimics were transfected into the mammary epithelial cells to detect the expression of MTHFR.1. Cloning, sequencing analysis and construction of recombinant reporter vectors of dairy goat’s MTHFR-3’UTRThe 314 bp fragments of wild type and mutant(2494A) MTHFR-3’UTR were amplified directly from dairy goat genome. The wild type and mutant(2494A) Dual-luciferase reporter vector psiCHECK?-2-MTHFR-3′UTR were constructed by double restriction digestion, and then were confirmed via sequencing analysis.2. Culture and identification of dairy goat mammary epithelial cellsPick dairy goat mammary glands of different genotypes(wild type and mutant), and then culture dairy goat mammary epithelial cells by organization adherence method. We can get the purified mammary epithelial cells after 3-5 passages. The purified mammary epithelial cells were identified by immunofluorescence.3. Effects of miRNA on the expression of wild type and mutant recombinant reporter vector luciferaseThe recombinant reporter vector psiCHECK?-2-MTHFR-3′UTR and miRNA-1296 or mi RNA-370 were co-transfected into the mammary epithelial cells by LipofectamineTM to examine the relative activity of luciferase. The results showed that psiCHECK?-2-MTHFR-3′UTR-2494 G luciferase was significantly lower than that of psiCHECK?-2-MTHFR-3′UTR-2494 A in mammary epithelial cells. The miRNA-1296 had no effect on luciferase activity. The results show that MTHFR-3’UTR c.2494G>A mutation changes the binding site of miRNA-370.4. miRNA-370 affects the expression of MTHFRTo detect the expression of MTHFR in mammary epithelial cells of different genotype by western blot, the miRNA-370 was transfected into the mammary epithelial cells. The mi RNA-370 inhibited the expression of MTHFR in wild type mammary epithelial cells, but it had no affect on mutant.