Cloning, Tissue Expression Profile And SNP Analysis Of The Porcine SLC13A5 And Wnt10b Gene

[Objective] A recent study indicated that solute carrier family 13 (sodium-dependent citrate transporter member 5, SLC13A5) gene and WntlOb were related to the intramuscular fat content (IMF) in pigs. This study was carried out to clone the SLC13A5 and Wnt10b cDNA in pig, and analyzed its cDNA sequence and its protein by bioinformation method, and analyzed expression profile of ten tissues of 25-day-old Yorkshire and Shaziling pig, respectively.[Method] The Yorkshine pig was regarded as the research object, the full-length cDNA sequence of porcine SLC13A5 and WntlOb gene were acquired from the longissimus dorsi muscle by Rapid Amplification of cDNA of End method, and their sequences and protein functions were analyzed and predicted by bioinformatics method. Then we respectively investigated the expression level of SLC13A5 and Wnt10b in ten tissues through qPCR analysis. One SNP site in the sequence was identified by PCR-RFLP, then its allelic frequencies and genotypic frequencies were calculated, associations between different genotypy with age at 100kg and corrected back fat thickness were analyed.[Result] The full-length of SLC13A5 gene was 2118 bp, the open reading frame length was 1665 bp,5'end length was 557 bp, the length of 3'end was 1320 bp, and the coding region encoded a protein of 333 amino acids. Pig SLC13A5 gene and other species phylogenetic tree showed that pigs had the closest relationship with Ovis aries, and followed with Macaca mulatta, Xenopus laevis, Columba livia, and Pseudopodoces humilis. Amino acid sequence analysis revealed that SLC13A5 protein was a fat-soluble protein, and its relative molecular weight was 61183.7 Da, and theoretical pI was 7.45. The SLC13A5 protein predicted included 18 phosphorylation sites and 8 transmembrane regions. The secondary structure was dominated by random coil, and the tertiary structure of SLC13A5 protein showed a forniciform helix structure. SLC13A5 gene were differently expressed in the Yorkshire and the Shaziling piglet. The expression of the SLC13A5 gene in Cecum was extremely remarkable(P<0.01) in the above two breeds, and in its liver and crureus was significantly different (P<0.05). In the same breed, expression of this gene in longissimus dorsi muscle was significantly different with other 9 tissues (P<0.05). One SNP site of A251G in the sequence was identified by PCR-RFLP, and its associations of SNP with age at 100kg and corrected back fat thickness were significantly different (P<0.05).The full-length of WntlOb gene was 2075 bp, including 1170 bp open reading frame coding 389 amino acids. The homology analysis found that amino acid sequence of pig WntlOb gene share more than 94% identity with the Human, Bos taraus, Pan troglodytes, Rattus norvegicus, Equus caballus, Macaca mulatta, and Mus musculus. The amino acid sequence analysis revealed that the WntlOb gene of pig encoded water-soluble protein and its relative molecular weight was 42873.2 Da, isoelectric point was 9.46. The WntlOb protein contained 20 phosphorylation sites and one WNT1 domain. WntlOb played a significant role in fatty metabolism and transport and binding functions process. The expression profile of WntlOb gene was analyzed in different tissues by real-time PCR, results indicated that the expression of WntlOb gene in the longissimus dorsi muscle was extremely remarkable(P<0.01) between Shaziling piglet of 25 d and Yorkshine pig, and in its intestine and crureus was significantly different (P<0.05), in other tissue was no significantly different. There is one SNP detected at amplified fragment-607G>C loci by PCR-RFLP method. Its associations of SNP with age at 100kg and corrected back fat thickness were not significantly different (P>0.05).[Meaning] The above studies have laid an theotetical foundation for further study of the SLC13A5 and WntlOb gene in the pig fat deposition process and fatty acid metabolism.