Study On The Cloning, Expression And Genetic Variation Of Ovine UCP2 And UCP3 Genes

UCP2 and UCP3 are mitochondrial inner-membrane proteins and can inhibit the generation of ATP and reactive oxygen species by uncoupling mechanism, thus function in body protection and energy metabolism. Sheep is one of the important economic domestic animals. Studies on the structures and genetic expression profiles of UCP2 and UCP3 genes can be useful for understanding their complex metabolic mechanism in the body. However, little is known about their nucleotide sequences, molecular structures, genetic variation and expression.This dissertation aims to study the sequence structure, genetic variation, mRNA and protein expression of ovine UCP2 and UCP3 at DNA, mRNA and protein levels, respectively. RT-PCR, RACE (rapid amplification of cDNA end) and DNA sequencing techniques were used to clone the complete cDNA sequences of UCP2 and UCP3. Electronic cloning was used to obtain their genomic sequences. Taking UCP1 gene as reference, real-time PCR was used to detect the differential mRNA expressions of UCP2 and UCP3 in 13 tissues of South African Mutton Merino and Shanxi Mutton Sheep Dam Line during two time intervals of winter-spring and summer-autumn. Immunohistochemistry technique was used to study the protein expression of UCP2 and UCP3 in six adipose tissues. Various softwares were used to conduct the bioinformatics analysis on the nucleotide and amino acid sequences of the two genes. The main results are showed as follows.(1) The complete sequence of ovine UCP2 cDNA obtained by RACE was 1641 bp in length with a 927 bp open reading frame (ORF) that encoding 309 amino acids and a polyA signal. The full-length of ovine UCP3 cDNA was 1683 bp containing a 933 bp ORF that encoding 311 amino acids and three polyA signals.(2) The genomic sequence of UCP2 obtained by electronic cloning had 6264 bp in length containing eight exons and seven introns. The transcription initiation site located at 498 bp of 5’-flanking region, which contained 1 CpG island and an initial subsequence at -15～-1 bp, but no TATA box. UCP3 was 10451 bp in length containing seven exons and six introns. The transcription initiation site located at 3600 bp of 5’-flanking region, which contained a TATA box at -30～24 bp. Both UCP2 and UCP3 were localized on OVA 15 in reverse arrangement and cluster.(3) UCP1 and UCP2 mRNA expressed in all 13 tissues with higher expression in deep fat deposits than that in superficial (subcutaneous) adipose tissue (P<0.05). For nonadipose tissues, the highest UCP1 mRNA expressed in spleen and lowest in longissimus dorsi; the highest UCP2 mRNA expressed in lung and also lowest in longissimus dorsi. Basically, higher expressions during winter-spring (P<0.05) were observed.(4) The spatiotemporal mRNA expression was found for UCP3. In adipose tissues, the gene expressed higher in retroperitoneal than other adipose tissues during winter-spring. However, there was almost no expression in all adipose tissues during summer-autumn. For nonadipose tissues, the highest expression in lung, higher expressions in longissimus dorsi and heart, and lower expressions in liver and spleen, but no expression in stomach was detected. Generally, higher expressions during winter-spring (P<0.05) were also observed.(5) Immunohistochemical study showed that ovine UCP2 and UCP3 were transmembrane proteins located in the plasma membrane of fat cells. In adipose tissues, the expression trend of these proteins was agreed with those of UCP2 and UCP3 mRNAs.(6) Bioinformatics analysis showed that the polymorphism was low in cDNA sequences of UCP2 and UCP3 between and within species. Codon bias was found in their amino acid sequences in different species. Both UCP2 and UCP3 were neutral evolved. The predicted physicochemical properties, secondary and tertiary structures of ovine UCP2 and UCP3 are of significance to study the associative functions of their coding genes.In conclusion, by systematic research on the ovine UCP2 and UCP3 genes at different levels, the results provide an important scientific basis for exploring the functions and action mechanism of the two genes.