Molecular Cloning And Characterization And Expression Analysis Of The Small-Tailed Han Sheep Adipoq Gene

Adipose tissues play an important role in maintaining normal life activities and physiological metabolism of sheep. Fat trait is also an important economic trait that could influence the quality of mutton. Adiponectin is an endogenous specific-protein secreted exclusively by adipose tissue and also a kind of important adipokines. In this study, the full-length cDNA of adiponectin gene was cloned, a series of bioinformatics analysis of the gene and its deduced protein were conducted, and the expression abundance of this gene at mRNA and protein levels was analyzed. The main findings are showned as follows:1. The full length was cloned from Small-Tailed Han Sheep by methods of reverse transcription PCR and rapid amplification of cDNA ends. The sequence data has been submitted to GenBank(Accession number: KM216385). The obtained full-length c DNA of 2616 bp contained a 720 bp open reading frame(ORF), a 76 bp 5′ UTR(untranslated region), a 1820 bp 3′ UTR. The ORF encodes a polypeptide of 239 amino acids.2. Bioinformatics analysis showed that the deduced protein had a predicted molecular weight of 30 kDa and a theoretical isoelectric point of 5.88. A signal peptide was predicted to be present in the protein, indicating it could be secreted into the extracellular. Eight phosphorylation sites were predicted at serine, threonine and tyrosine. And five O-glycosylation sites but none N-glycosylation site were found in this protein encoded by Adipoq gene. Homology analysis showed that the amino acid sequence of sheep Adipoq had the highest homology with that of goat.3. It showed tissue expression differences of Adipoq gene in Small-Tailed Han Sheep and Dorper sheep. Among different organizations, the expression of Adipoq mRNA was detected in subcutaneous adipose tissue, perirenal adipose tissue, inguinal adipose tissue, biceps muscle of arm, and cardiac tube but in liver, kidney, ovarian was not. Adipoq gene had the highest expression level in the three adipose tissues and low in others, and the difference in expression of these tissues was significant(P < 0.01). But there was no significant difference between the three kinds of adipose tissues(P>0.05). In different varieties, the expression of Adipoq gene in biceps muscle of arm of Dorper sheep was significantly higher than that of Small-Tailed Han Sheep(P <0.01).4. It showed tissue expression differences of Adipoq protein in Small-Tailed Han Sheep and Dorper sheep. Among different organizations, adiponectin protein was detected in subcutaneous adipose tissue, perirenal adipose tissue, inguinal adipose tissue, biceps muscle of arm, and cardiac tube but not in liver and kidney. And the adiponectin protein expression in adipose tissues were significantly higher than other tissues(P <0.01). While, there was no significant difference in expression abundance between different adipose tissues. Among different breeds, the expression of adiponectin protein of Small-Tailed Han Sheep was higher than Dorper sheep in all the three adipose tissues. But the difference was not significant(P> 0.05). Adiponectin protein expression in biceps muscle of arm of Dorper sheep was higher than Small-Tailed Han Sheep, and it was opposite in cardiac tube. And the two difference were both not significant(P>0.05).In summary, this study successfully cloned adiponectin gene, analyzed the structural characteristics of Adipoq gene and its deduced potein and the expression abundance at transcription and translation levels in different tissues. The whole results may provide some basis for subsequent research in explaining the function of adiponectin.