| In order to investigate the fundamental property and lay the foundation for further research of the newly discovered Large White with glabrous phenotype. In this paper, the Normal Large White was as control, the growth characters, body size indexes, reproductive performance, carcass traits and pork quality of the Hairless were respectively detected;Moreover, genetic mechanism of glabrous trait and histological properties of the skin were studied. In the meantime, using the candidate gene approach, functional genes which is strongly associated with hair follicle development and follicle density was screened, then bioinformatics methods and biology software are used to analyze the sequence features of candidate genes, in the meantime, mutation analysis was detected by the direct DNA sequencing of PCR product, and provide a theoretical basis for the further study of the formation mechanism of glabrous trait in Large White.These results suggested that under the same feeding conditions, the individual birth weight, age of body weight at 100 kg, body size indexes, backfat thickness of body weight at 100 kg and reproductive performance had no significant difference between the Hairless and the Normal(P>0.05). There were no statistical differences in live weight, carcass weight, dressing percentage, backfat thickness, lean percentage, fat percentage, bone percentage and skin percentage of the Hairless(P>0.05). In meat quality traits, the meat color score, p H1, marbling score and intramuscular fat were not differ significantly from normal controls(P>0.05). Above all, it was indicated that the main performance of the Hairless were performed normally, that is, glabrous traits had no significant effect on the growth, reproduction ability and meat performance.This experiment was conducted by setting the crossing tests include that between same phenotypes and between different phenotypes. The offspring phenotypes were recorded as well as dominant-recessive relationship was deduced to determine the genetic mechanism of glabrous traits. The results showed that glabrous trait in Large White pigs may be controlled by a complex interplay of more genes, rather than just by genes in single or two locus.The histological structure of skin and hair fiber were observed using the methods of paraffin section and Microscopic observation. These results suggest that the hair shaft scales were like mackerel scale from the hairless pig. The proportion of medulla less in Hairless than in Normal, but the occupied proportions of cortex were higher relatively. At different stages, the average fineness of fiber in the Hairless was dramatically smaller than in the Normal(P<0.01). Furthermore, the Hairless showed a thinner dermis and a lower hair follicle density at different locations.This study showed that a total of 4 polymorphic sites(-1833C/T,-1808C/G,-1628ins/del and-12688ins/del) were detected from DKK1 promoter, which 7 haplotypes were sorted. However, there was extremely significant(P<0.01) in various genotypes between the Hairless and the Normal individual by Chi-square test. Furthermore, there was significant correlation between DKK1 promoter polymorphism and glabrous trait of Large White(P<0.01). The polymorphism at-1833C>T and-1268(ins/del) sites in promoter region were not destroyed transcription factors binding sites, however, polymorphism at-1808C>G was destroyed HNF-1C binding site and at-1628(ins/del) was not inclusive for the TATA-box through bioinformatics analysis. Moreover, this study also found that 3 polymorphic sites(-1288G>T,-1182 ins/del and-401ins/del) were detected from β-catenin promoter, from which 5 haplotypes were sorted, and significant correlation between β-catenin promoter polymorphism and glabrous trait of Large White(P<0.01). The polymorphism at-1288,-1182(ins/del) and-401(ins/del) sites in promoter region were destroyed transcription factors binding sites, moreover, we identified a four-nucleotide deletion(-1182 4N del) variant in the β-catenin promoter associated with classical TATA-box through bioinformatics analysis. Therefore, we hypothesized that these polymorphic sites might have influenced the transcriptional activity of DKK1 and β-catenin.Based on sequence features of DKK1 gene, then recombined into p GL3-basic plasmids with promoter segments with different lengths were transfected into 293 T cells and Hela cells, respectively, and its transcriptional activity were evaluated through the detection of relative luciferase activity. Results showed that it had a good specificity on the 239 T cells, the relative luciferase activity of recombinant expression vectors PGL3-DKK1 was significantly higher than negative control(P<0.01), and sequence from +292 to-1679 had maximal promoter activity, which was obviously higher than other fragments(P<0.01). Further analysis revealed that there were promoter core region(-215/+219), which maintain the basal transcription of DKK1, negative(-586/-953) and positive(-953/-1679) regulatory regions, respectively. And then the base deletion of polymorphic sites expression plasmid were constructed, and transfected into 293 T cells. The result of the relative activity of luciferase indicated that promoter activity of PGL3-4(-1628 4N del and-1268 2N del), PGL3-2(-1628 4N del) and PGL3-3(-1268 2N del) were significantly lower than that of PGL3-1(P<0.01), which means these variable sites had a marked effect on transcriptional activity of DKK1 gene.By predicting the sequence features of β-catenin gene, p GL3- β-catenin dual luciferase expression vectors with promoter segments with different lengths were constructed, then the three plasmids were transferred into 293 T and Hela cells, respectively, and its transcriptional activity were measured. Results show that the relative luciferase activity of the trial groups were very low. Then the expression level of β-catenin in different cells was analyzed by RT-PCR and real-time quantitative PCR, which showed that relative quantitative expression from high to low was 293 T, Hep G2, PC3, LNCap and MCF7 cell, respectively. So we presumed that the expression of β-catenin gene is specific to the type of cell. |
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