Characterization Of The Cow ELOVLl5 Promoter And Variants In The Promoter Region Associated With Milk Quality Traits In Holstein Cattle

Long fatty acid elongase 5(ELOVL5)is an enzyme mainly involved in biosynthesis of C20–22 polyunsaturated fatty acids and plays an important role in a negative feedback regulation of lipogenesis.The expression of ELOVL5 in breast tissue of high milk fat cow was significantly lower than that of low milk fat cow.Core promoter is a minimal DNA sequence acted as the priming stage for transcription initiation.Rarely study has comprehensively characterized the cow ELOVL5 core promoter,besides the association between 5' transcriptional region variants and milk quality traits is currently poorly understood.Here the core promoter of cow ELOVL5,a 107 bp nucleotides region without TATA-box,was identified by testing their transcriptional activity of the deleted end fragments in a luciferase reporter system in bovine mammary epithelial cells(b MECs).Using direct DNA sequencing,seven singlenucleotidepolymorphisms(SNPs),1-bp del polymorphism and 1-bp ins polymorphism were identified within the promoter region of ELOVL5 gene from 230 individuals.According to the statistical analysis,1-bp del polymorphism(p<0.01)and SNP7 g.-9C>G(p<0.05)were significantly associated with somatic cell count(SCC),in addition we found that the SNP7 g.-9C>G was located in the core promoter.And 1-bp ins polymorphism was significantly associated with milk production(p<0.05).Then the transcriptional activity of different genotypes were determined using a dual-luciferase reporter assay system in bovine mammary epithelial cells(MECs),the homozygous 1-bp del mutation genotype had a 1.59-fold(p<0.01)higher activity of luciferase comparing with the wild type,the homozygous 1-bp ins mutation genotype had a 1.88-fold(p<0.01)higher activity of luciferase comparing with the wild type,the TT genotype promoter activity in SNP6 g.-110G>A was significantly higher than that in CC genotype(p< 0.01).The DNA-protein interaction between the variants and nuclear protein in b MECs was performed by electrophoretic mobility shift assays(EMSAs),the result showed that SNP6 g.-110G>A was located in the nuclear protein binding site,but other polymorphisms were not.And the binding affinity of TT allele probe was higher than that of the CC allele probe.