Cloning and RNA Interference Analysis of TRIB3 Gene in Xinong Saanen Dairy Goat (Capra hircus)
PAN Tan, LV Ming, LUO Jun*, LI Cong*
College of Animal Science and Technology, Northwest A&F University/Shaanxi Provincial Key Laboratory of Molecular Biology for Agriculture, Yangling 712100, China
Abstract：Tribbles pseudokinase 3 (TRIB3) is a serine/threonine kinase-like protein, which is involved in insulin signal pathway as a negative regulator of lipid metabolism. The present study was aimed to obtain the TRIB3 gene sequence of Xinong Saanen dairy goats (Capra hircus), elucidate its structure and investigate its function on lipid metabolism through RNA interference of TRIB3 gene in goat mammary epithelial cells. Taken mammary gland from Xinong Saanen dairy goats as experimental materials, the sequence of TRIB3 gene was cloned by RT-PCR method, bioinformatics and tissue expression analyses were conducted. The results showed that the complete CDS region of TRIB3 gene was 1 074 bp (GenBank No. MK158243), encoding 357 amino acids. The TRIB3 protein had the molecular weight of 39.6 ku, the isoelectric point of 7.8, and was an unstable protein. The TRIB3 protein had 36 phosphorylation sites without signal peptides and transmembrane spiral structure. Homology analysis showed that sequence homologies of Xinong Saanen dairy goat with sheep (Ovis aries), cattle (Bos taurus) and mouse (Mus musculus) were 99%, 96% and 71%, respectively, and the sheep was the most closest species with dairy goat. Tissue expression analysis revealed that the highest expression of TRIB3 was in mammary gland, followed by adipose tissue. In addition, siRNA of TRIB3 gene were designed and transferred into goat mammary epithelial cells, the results showed that mRNA level of peroxisome proliferator activated receptor γ (PPARG) and diacylglycerol acyltransferase (DGAT1) were up-regulated by interfering TRIB3. Above results suggest that TRIB3 might be involved in the regulation of lipid metabolism in goat mammary epithelial cells as a negative factor. The present study provides basic data for further investigation of molecular mechanism of TRIB3 on milk fat metabolism in dairy goats.
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