Cloning, Tissue Expression And Genetic Polymorphism Analysis Of CAST, MC4R And BTG1 In Sheep

The purpose of this study is to find the SNPs sites of candidate genes associated with production traits by molecular biotechnology and offers a basis for molecular marker-assisted selection. In accordance with the literature reports, CAST, MC4R and BTG1 were considered as candidate genes, which were cloned and then detected for SNPs sites, and to reveal the association between SNPs and production traits. Simultaneously, we did mRNA tissue-expression profiles of candidate genes. The results were as follows:Primers were designed based on cattle sequences and the contigs from ESTs database of sheep, the partial fragments of three genes were isolated, and the corresponding protein sequences were deduced and compared with some of other species. The fragments length of CAST transcript 2 and 4 mRNA were 4538bp and 1467bp, and the GenBank accession numbers were: FJ843091 and FJ843092, respectively. The fragment length of MC4R was 1919bp, and the mRNA of 1788bp had been submitted to GenBank (EU622853). The fragment length of BTG1 mRNA was 1385bp, the GenBank accession number was FJ444829.The tissues expression profiles of CAST transcript 2, transcript 4 and MC4R genes were detected in heart, liver, spleen, lung, brain, kidney, testis, ovary, muscle and stomach of seven-month-old Dorset by semi-quantitative RT-PCR. The transcript 2 of CAST was expressed in ten tissues, and the transcript 4 of CAST was only expressed in testis tissue. The MC4R gene was expressed in ten tissues, and the expression level was higher in the brain, and lower in others. The tissues expression profile of BTG1 gene was detected in heart, liver, spleen, brain, kidney, testis, ovary, longissmus dorsi, biceps femoris and stomach of six-month-old Small Tailed Han Sheep and Dorsett by semi-quantitative RT-PCR and real-time RT-PCR. The BTG1 gene was expressed in ten tissues, and the expression level was highly in the testis and ovary. It might that BTG1 plays an important role in testis and ovary.We detected of SNPs of 3'-UTR of CAST by PCR-SSCP method. There were four SNPs in the CAST-S3 primer, which were g.G73A, lacking CA at 105 and 106 sites, g.G210A and g.G219A. There were also four SNPs in the CAST-S7 primer, which were lacking T at 80 site, g.G83A, g.G158A and g.G163T, and it was at the same time between lacking T at 80 site and g.G83A in all the individuals measured. We found four SNPs of MC4R using sequencing, which located 309, 508, 1232 and 1456 site. No mutation was detected in the BTG1 gene.Altogether three SNPs of CAST and MC4R were detected using PCR-RFLP and PIRA-PCR,which were g.G73A of CAST-S3, g.G163T of CAST-S7and g.G1232A of MC4R gene . The allele frequencies, genotype frequency and variation within and across different breeds were analyzed. The association between production traits and the SNPs was analyzed by general linear model, the results were as follows: locus g.G73A of CAST-S3 was associated with loin muscle depth and loin muscle area, the means of loin muscle depth of genotype GG and AG were higher significant than genotype AA (P<0.01), the means of loin muscle depth of genotype GG and AG were not significant different (P>0.05); the means of loin muscle area of genotype GG and AG were higher than genotype AA (P<0.05), the means of loin muscle depth of genotype GG and AG were not significant different (P>0.05). Locus g.G163T of CAST-S7 was associated with back fat (P<0.05), the means of back fat of genotype GG was higher than genotype GT. Locus g.G1232A of MC4R was associated with back fat (P<0.05), the means of back fat of genotype AG and AA was higher than genotype GG.