Identification of a Potential microRNA Target Site in 3'UTR of OLR1 Gene Affecting Milk Production and Meat Traits in Cattle

The oxidized low-density lipoprotein receptor 1 (OLR1) gene plays an important role in the degradation of oxidized low-density lipoprotein, which can cause damage to the arterial endothelium. Previous studies have shown that the SNP of OLR1 gene in the 3’UTR region was associated with milk production and health traits in dairy cattle and lion eye area and marbling depth in Qinchuan cattle. MicroRNAs (miRNAs) are non-coding RNAs of about 22 nucleotides in length that have significant effect on biological processes by causing targeted mRNAs degraded and translation repressed. Because of the binding sites of miRNAs was usually on the 3’UTR region of target gene, so the hypothesis was that the SNP in OLR1 3’TUR region caused binding sites of miRNAs change, which caused different genotypes of OLR1 gene associated with different milk production and meat traits. The potential miRNAs of OLR1 3’UTR were predicted by bioinformatics method. Bta-miR-370 was identified from the 16 total potential miRNAs because the SNP in OLR1 3’UTR caused the disappearance of bta-miR-370 binding site. The objective of this study was to verify the interaction between the SNP in 3’ UTR of OLR1 gene and bta-miR-370 using a dual-luciferase reporter assay. A 360 bp fragment of OLR1 3’UTR from wild-type Qinchuan cattle and a C>A mutation individual using primers OLR-3UTR-f (with an XbaI tail) and OLR-3UTR-r (with a SpeI tail) were amplified. Then the PCR products were inserted into the pGL3-control vector. At the same time, the bta-mir-370 eukaryotic expression vector was constructed. The 3’UTR-pGL3 vector, bta-mir-370-pcDNA3.1 (+) plasmid and pRL-TK Renilla luciferase construct were cotransfected in HEK293 cells. The luciferase assays were performed 24 h later after transfection. The results showed that in the experimental group the activity of luciferase was significantly lower than that of negative and positive groups, which indicated that 3’UTR of OLR1gene was the target gene of bta-mir-370 and the SNP caused the binding site of bta-mir-370 disappear so as to affect milk production and meat traits in cattle.

Key words: OLR1 gene; bta-mir-370; SNP