Isolation, Characterization Of Three Porcine Genes Association With Fat Deposition By Using The Positional Candidate Cloning And Comparative Genome Analysis

With the development of the science technology and the society, the traditional animal breeding is gradually combined with the molecular breeding such as the master assistant selection, transgene technology and the functional genomics, etc. They are all used as the foreland field and become the main direction for the swine breeding. As more and more bioinformatics and database are common, they afford the new method to separating and localization. In this paper, we used the method of the candidate gene and the EST information with the Comparative Genomics. We carried through the character association and functional domain prediction. The main Results are as follows:1. Based on the method of candidate gene, we found the porcine homologous EST from the ESTs. The special primers were designed through the contigs by the ESTs. PECI, WNT10B, DKK1 gene were isolated from tongcheng genome. The sequences were submitted to the NCBI GenBank and assigned the accession numbers of DQ291159, DQ518413, DQ517932.2. The full length cDNA of PECI gene was obtained through in silico cloning together with RACE. Together with the EST information, the comparatively completely full length porcine DKK1 gene had been gained. Then the whole amino acid sequencing, the protein structure and its function were all analyzed. The similarity between the porcine PECI and human were 81%, while to DKK1 gene, it reached to 84%.3. Expression profiles of PECI, WNT10B and DKK1 genes were analysis by semi-quantitative RT-PCR assay. PECI gene is widely expressed in tissues of heart, liver, spleen, lung, kidney, muscle and fat. Compared with the tongcheng and changbai pigs, we could see there was a significant difference in the tissue of lung. Carrying through the spartial profile analysis in the early embryo stage, we found its expression was going up. The expression of WNT10B and DKK1 genes were all expressly highly in the fat tissue. While the DKK1 gene was only expressed in the tissue of lung.4. The profiles of functional domain, transmembrane region, hydrophobicity and hydrophilicity of the both domains in PECI and DKK1 protein were estimated and displayed primarily. The multiple sequence alignments were carried out by the CLUSTAL W program, the results of polygenetic tree of PECI and DKK1 genes in diverse species were analyzed by the CLUSTAL W method in DNAStar software.5. The pig X rodent somatic cell hybrid panel (SCHP) and the INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel were all used for chromosome assignment. The results were: PECI gene was localized to SSC7p13, which was closely linked to S0383, with LOD score threshold of 12,84; WNT10B was mapped to SSC5p11-15, and the DKK1 gene was to SSC14q25-q26, which were separately closely linked to SW439 and SW1552, with LOD score threshold of 5,09 and 6,74.6. Studied on polymorphie sites of PECI and WNTIOB gene in 3'-untranslated region by PCR-RFLP, Genotype and allele frequencies among different pig populations were determined. Association with genotypes and phenotypes of economic traits, The study revealed that①PvulI-RFLP site of PECI gene was significant associations with Buttock Backfat, average backfat thickness, Muscle color (P＜0.05). The Cfr42I—RFLP for the porcine WNTIOB was significant association with the Proportion of lean + bone of the ham (%) and Loin eye area (P＜0.05).②The difference was highly significant (P＜0.01) between CT genotype and TT genotype of PvuⅡ-RFLP site of PECI gene in the phenotype of Muscle color. The difference was also highly significant (P＜0.01) between CC genotype and TT genotype in the phenotype of Buttock baekfat thickness. The difference between the genotype CC and TT in the phenotypes of Average baekfat at 3 points and Muscle color, CC and CT in the phenotype of Average backfat at 3 points are all significant (P＜0.05) according to the study.③The difference was highly significant (P＜0.01) between CT genotype and TT genotype of Cfr42I—RFLP for the porcine WNT10B in the Loin eye area. The difference between the genotypes of CC and CT in the proportion of lean+bone of the ham and Loin eye area, CC and TT in the proportion of lean+bone of the ham and Loin eye area were all significant association (P＜0.05) with the study.