Polimorfismo e expressão do gene da UCP2 no fígado de cabras em lactação

Abstract

The uncoupling proteins of mitochondrial inner membrane uncouples the cellular respiration from ATP production thus it has an important role in the energy metabolism of the organism. Polymorphisms in genes coding for UCPs have been associated with characteristics such as fat thickness and weight gain in beef cattle, and to obesity in humans. The massive mobilization of fatty acids in early lactation can influence the expression of UCP2 showing that this protein has a role in physiological processes of this phase. Thus, the purposes of this study was to verify if the expression of UCP2 gene varies during early lactation in dairy goats liver, sequence a initial part of the goat UCP2 gene and verify the existence of polymorphism in the studied region of this gene. For this we used 40 goats (Capra hircus), all lactating animals of Saanen and Alpina breed, from which 10 mL of periferic blood were collected. Thereafter, for the analysis of gene expression, 10 of these animals were slaughtered at different stages of the early lactation (day of birth, 7 , 21, 35 days post-partum) and a sample of liver tissue was collected and stored in RNA-holder. Whole blood was used for DNA extraction and subsequent amplification of four fragments of UCP2 gene by polymerase chain reaction. Amplified fragments were then subjected to electrophoresis on 12% polyacrylamide gel (300v for 24hrs) to evaluate small size differences between samples. Subsequently the fragments were analyzed by PCR-RFLP for differences in the digestion pattern of the enzyme HaeIII. Three different samples of each fragment of the initial region of the gene were randomly selected for sequencing. From the samples of liver tissue an RNA extraction was performed, and the RNA was used for the real time PCR reactions using Syber Green fluorescent compound. To assess if the genotypes found had any influence on fat production and milk fat percentage, variance and estimation of contrasts analysis were performed. To evaluate whether the expression varied at different postpartum days, a regression analysis was made. In the promoter region fragment a fragment size polymorphism that originates two alleles was found: the alelle A, larger, and the alelle B, smaller. Then the influence of these two alelles on the percentage of fat and fat production in milk were accessed, but neither allele showed influence over these characteristics. The PCR-RFLP analysis showed a difference in enzyme digestion pattern among samples in the exon 2, originating two alleles, C and N. These were also evaluated for their influence on the percentage of fat in milk and fat production, but no relationship was found. After sequencing, the sequences were compared to each other, and a single nucleotide polymorphism was found in intron 1 but no restriction enzyme that recognizes this polymorphism was found, making impossible the genotyping of a larger number of animals by PCR-RFLP technique. The reactions of real-time PCR revealed that expression of UCP2 in the liver was higher at birth and gradually decreased with advancing lactation up to 35 days postpartum.