Study On The Cloning, Expression And Polymorphism Of UCP4and UCP5Gene And Their Associations With Traits In Sheep

Uncoupling protein4(UCP4) and5(UCP5) were mitochondrial carrier proteins which were highly expressed in the brain tissues of animals. They not only function in thermoregulation and energy balance, but also play important roles in fat metabolism. However, up to now, no literature was found on the structure, mRNA expression and polymorphism of ovine UCP4and UCP5.In this study, the coding sequences (CDS) of ovine UCP4and UCP5were cloned by RT-PCR and sequenced. The CDS and corresponding amino acid sequences were analyzed using bioinformatics methods. Two breeds with significant differences in tail type (Guangling Large Tailed Sheep and Small Tailed Han Sheep) were used to study the ontogenetic mRNA expression of UCP4and UCP5by real-time quantitative PCR in eight tissues (cerebrum, cerebellum, hypothalamus, pituitary, and subcutaneous, perirenal, mesenteric and tail fats) from2to12months of age with interval of two months. Associations between mRNA expression and tail and slaughter traits were studied. The polymorphism of the5’ regulatory region of UCP4and UCP5in96animals from the two breeds was detected using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. Associations of the poly-morphism with tail and slaughter traits were also analyzed. The main results of the studies were as follows:(1) The length of complete CDS of UCP4was972bp encoding a protein of323amino acids. This fat-soluble membrane protein was stable and had no signal peptide but with two predicted glycosylation sites and15potential phosphorylation sites. The percentages of helix, strand and loop were56.04%,7.12%,36.84%, respectively, in the secondary structure. For UCP5, complete CDS was978bp in length encoding a protein of325amino acids. This stable protein had signal peptide, two predicted glycosylation sites and eight potential phosphorylation sites. The secondary structure was composed of51.08%a-helix,8.92%β-strand and40%loops. High similarities were discovered between sheep and most of the species studied both in nucleotide and amino acid sequences of UCP4and UCP5.(2) The UCP4and UCP5mRNA expressed in all eight tissues, but expressed significantly higher in brain tissues than in adipose tissues. Significant difference in mRNA expression was found between breeds with STH higher than GLT. The difference was also significant between months of age with the highest expression in12months of age. The tissue by breed and tissue by age interactions significantly affected the expression of UCP4(P<0.05). Only one interaction, i.e. sex by age, affected the expression of UCP5significantly (P<0.05). Different association degrees between mRNA expressions in various tissues and traits considered differed considerablely. However, positive relations were found between mRNA expressions in tail fat for the two genes and tail and slaughter traits in the two breeds. These results imply that the genetic expression of UCP4and UCP5may function in fat deposition in sheep.(3) Six SNPs were identified in the5’ regulatory region of UCP4gene, i.e.-1073T>C,-1029T>C,-898C>T,-853G>C,-552C>G and-544T>G in order of upstream to downstream the regulatory region. Two SNPs,-2133T>C and-1148G>C, were detected in the5’ regulatory region of UCP5. The SNP polymorphism of UCP4and UCP5had some but not significant effects on tail and slaughter traits in sheep.