Sequencing of lipoprotein lipase gene in the Mediterranean river buffalo identified novel variants affecting gene expression

摘要

Lipoprotein lipase (LPL) is a key enzyme for lipidmetabolism, playing a fundamental role in the compositionof fat in adipose tissue and milk. The LPL genehas been seldom investigated in dairy ruminants andbarely studied in river buffalo (Bubalus bubalis). Theaim of this work was to explore the genetic diversity ofLPL and its promoter and to identify functional mutations,using a combined approach based on sequencing,dual-color electrophoretic mobility shift assay, andquantitative PCR. Thirteen consensus sequences fortranscription factors were found in the promoter. ElevenSNP were detected, and the attention was focused onthe SNP with potential functional effects: g.-446A＞G,because the presence of G created a consensus motif forthe transcription factor Sp1, and g.107A＞G, which wasthe only exonic SNP. We developed PCR-RFLP methodsfor genotyping the 2 SNP and calculated the allelefrequencies. A strong linkage disequilibrium (D′ = 1; r~2= 0.903) was found between the 2 SNP. The dual-colorelectrophoretic mobility shift assay demonstrated thatonly genotype g.-446GG allowed the binding of the Sp1transcription factor, resulting in overexpression of thegene (～2.5 fold), as confirmed by the quantitative PCRresults. Haploinsufficiency is proposed as a regulationmechanism. This study adds further knowledge on thestructure of the LPL gene and its expression in riverbuffalo, with potential effects on milk qualitative andquantitative production.