The Cloning, Expression, Polymorphism Of ANGPTL4 Gene And Their Associations With Traits In Sheep

Angiopoietin-like protein 4 (ANGPTL4) functions in promoting both fat deposition and fatty acid oxidation. On the one hand, ANGPTL4 can inhibit fat hydrolysis by limiting the activity of lipoprotein lipase (LPL), resulting in lipid metabolism-related diseases. On the other hand, it can promote lipolysis by increasing activities of catecholamine and gluco-corticoids. To date, studies on ANGPTL4 gene and/or the coding protein have been published in various species, but no study was found in sheep.This dissertation aims to study on the structure and function, mRNA and protein expression and polymorphisms of 5’UTR of ovine ANGPTL4 gene at three levels of DNA, mRNA and protein. Besides, associations of mRNA expression and polymorphisms with traits were analyzed. Ninety six lambs in six age groups (2,4,6,8,10 and 12 month of ages) from two breeds with distinct tail type i.e. Guangling Large Tailed (GLT) and Small Tailed Han (STH) were used as experimental animsls. Adipose tissue samples from great and small omental, subcutaneous, retroperitoneal, perirenal, mesenteric and tail fats were collected for mRNA expression study. The coding sequence (CDS) of the gene was cloned by RT-PCR. The CDS and amino acid sequence were analyzed by bioinformatics methods. The mRNA abundance in the seven adipose tissues was detected by Quantitative real-time PCR, and their associations with tail type and slaughter traits were analyzed. Protein expressions in four adipose tissues of female lambs were evaluated by Western blotting. Single nucleotide polymorphisms (SNPs) in the 5’UTR were detected by PCR-SSCP, and associations between SNP genotypes and relevant traits were also analyzed. The main results were as follows:(1) The CDS of ovine ANGPTL4 gene is 1233 bp, which encodes a protein of 410 amino acids with a molecular mass 45.5 kDa. The predicted ANGPTL4 is a basic instable and weak hydrophilic protein with isoelectric point of 9.02. As a secreted protein, ANGPTL4 contains a signal peptide with 24 AA and two transmembrane domains. One N-glycosylation site, one O-glycosylation site and 18 phosphorylation sites were predicted in the protein. Evolution analysis showed that sheep and cattle share the closest genetic relationship.(2) The ANGPTL4 mRNA expresses spatiotemporally and differentially in adipose tissues of GLT and STH. Breed, month of age and adipose tissues from different depots influence the mRNA expression significantly (P<0.001). Abundance in GLT was significantly higher than that in STH. Expression at 10 months was significantly different from those at other five age groups. The gene expressed more in shallow than in deep fat depots. No significant gender effect was found, but expressions in males were slightly lower than that in females. Significant two-way interactions of breed by month of age (P<0.001), breed by tissues (P<0.01) and gender by tissues (P<0.01) were also found. Differences between breed existed in the associations between mRNA expressions and relevant traits. In GLT, mRNA expressions were positively associated with all traits considered, and the significant correlations were observed between the traits and mRNA expressions in subcutaneous, retroperitoneal, greater and small omental adipose tissues. In STH, negative correlations were observed between most of the traits and mRNA expressions in retroperitoneal and greater omental fats, and the rest correlations were positive.(3) Breed, month of age and adipose tissues influence ANGPTL4 protein expression significantly (P<0.001). Significant interaction (P<0.01) between breed and month of age was also obtained. The general trends in expressions of ANGPTL4 mRNA and its coding protein were consistent.(4) Eight SNPs were detected in 5’UTR of ANGPTL4, three of them were transversions and five were transition. Significant differences in allele frequency between breeds were detected at loci of-1691 C>G and -577 T>G Though allele frequencies were not significantly different at -2361 A>G, -2288 G>C and -1855 A>G, three genotypes were detected in GLT and only two ones in STH. Both -1691 C>G and -1625 G>A had a positive overdominance effect on tail length, slaughter weight and carcass weight, but a negative overdominance effect on dressing percentage in Guangling Large Tailed sheep.