The single nucleotide polymorphism g.133A＞C in the stearoyl CoA desaturase gene (SCD) promoter affects gene expression and quali-quantitative properties of river buffalo milk

摘要

The stearoyl-CoA desaturase (SCD) gene has beeninvestigated in depth in ruminants because of its effecton milk fat composition. In river buffalo, the singlenucleotide polymorphism (SNP) g.133A＞C in the genepromoter has been associated with milk quality andyield. However, the biological reason for such effectsremains unexplored. In this study, we combined mRNAprofile analysis, an electromobility shift assay, andquantitative PCR to elucidate the role of this SNP ongene transcription and its effects on milk fat traits. Apreliminary genotyping of g.133A＞C was carried outon a group of 303 river buffaloes to choose individualsfor the downstream applications. Analysis of allelefrequencies showed an increase in the minor allele C(0.25) compared with previous findings (0.16). Six animals(2 for each genotype) were chosen for cloning and216 positive cDNA recombinant clones for SCD (72 pergenotype) were analyzed by PCR. All clones showed thesame length on agarose gel; therefore, random cloneswere chosen for sequencing. No qualitative differenceswere found and all gene transcripts assembled correctly.An electrophoretic mobility shift assay was performedto evaluate the binding of the transcription factor Sp1to DNA sequences including g.133A＞C. Genotype CCshowed a higher binding (mean ± standard error ofthe mean) than genotype AA in 2 different conditions[Enzo buffer (EB), Enzo Life Science Inc., Farmingdale,NY: 201.77 ± 4.06 vs. 141.65 ± 3.77 band intensityvalues and Poletto buffer (PB): 95.90 ± 1.15 vs. 67.30± 2.14 band intensity values]. The subsequent quantitativePCR confirmed the upregulation of the CCgenotype compared with the AA and AC genotypes.The association study with milk fat traits revealed afavorable effect of allele C. The heterozygous genotypehad the highest values for monounsaturated fatty acids,oleic acid (C18:1 cis-9), polyunsaturated fatty acids,and odd- and branched-chain fatty acids, and the lowestvalues for saturated fatty acids and atherogenicand thrombogenic indices; the heterozygous genotypediffered significantly from the AA genotype. The ACgenotype has previously been associated with highermilk yield. Therefore, the g.133A＞C SNP is a markerwith dual effects and is an interesting candidate forassisted selection programs in river buffalo. These dataclarified the biological role of the SNP g.133A＞C in theSCD promoter and how it affects gene function, providingimportant knowledge on the genetic backgroundof lipid metabolism, including the future possibilityof selecting alleles with quantitatively or qualitativelyfavorable effects.