SNPs And Promoter Efficiency In 5' Flanking Region Of THRSP Gene And Its Association With Backfat Thickness Of Pigs

This study was designed for TP526 sequence analysis in 5'regulatory region of THRSP porcine gene to identify the function of TP526 and its association with fat production capacity of pig breeds. All test animals contained fat-type pigs (Dingyuan pigs, Jixi Black and Wannan Spotted) and lean-type pigs (Landrace, Large White, Duroc and their hybrids). Genomic DNA was extracted from ear tissue of pig. TP526 sequence in 5'regulatory region of THRSP gene was cloned using PCR. TP526 functions were investigated using sequencing, structure prediction, SNP detection, dual-luciferase reporter gene validation, as well as its association with pig backfat thickness. The results showed that:1. The length of TP526 sequence was 484bp, and the homologies were 65.6% and 58.9% with human and mouse, respectively. The size of promoter sequence was 50bp. The DNA bending index of 20~24bp, 42~51bp and 447~450bp regions in TP526 sequence were 81.04±5.25, 77.37±4.87 and 69.00±4.59, respectively. And there were many sequences inverted repeat in TP526.2. The analysis of transcription factor binding sites showed that HSF2, C / EBPb, C / EBPa, SRY, CdxA, GR, AML-1a, CRE-BP, CREB, S8, Nkx-2, GATA- 1, MZF1, GATA-2,, MEF-2, TRE, RevREs and other regulatory elements inserted in TP526 sequence.3. SNPs in TP526 sequence was detected and found G→A mutation site at 98 bp (G98A), C→T at 229bp (C229T), A→G at 376bp (A376G), and T→C at 400 bp (T400C) upstream from the CDS start of THRSP gene, and T→G at 9bp (T+9G) downstream from the CDS start. The PCR products digested with StyI restriction enzyme produced 9 joint genotypes of A376G-G98A, and their restriction sites cleared away when G→A at G98A and A→G at A376G; 7 combined genotypes of C229T-T+9G were found in the PCR products digested with MwoI, and when C→T at C229T, its restriction site cleared away, but a new MwoI restriction point emerged when T→G at T+9G; 5 restriction points existed in the PCR products with Fnu4HI, and when T→C at T400C, the restriction site of mutation position disappeared.4. The frequency of C gene at T400C was 0.6568 in the fat-type pigs, while the frequency in lean-type pigs was 0.3595; the frequencies of A gene at A376G were 0.5571 and 0.4944 in the fat-type and lean-type pigs; the frequencie of C gene at C229T was 0.7362 in the fat-type pigs, and 0.9607 in lean-type pigs; and the frequencies of G gene at G98A were 0.6142 and 0.6629 in fat-type and lean-type pigs, respectively. 5. Using dual luciferase reporter genes assay to analyze the regulation efficiencies of different genotypes of TP526 promoter, the results showed that the promoter effect of C gene promoter at T400C was best, about 3.3088 times of the T gene (P<0.01); the promoter effect of A gene at A376G was significantly higher than that of G gene (P <0.01); the promoter effect of A gene at G98A was highest (P<0.01).6. The least squares analysis showed that the backfat thicknesses of finishing pigs with CC and CT genotype at T400C were significantly higher than that with TT genotype (P<0.05), and that between CC and CT genotypes did not significantly differ. The backfat thickness of pigs with AA genotype at A376G was significantly higher than that with AG and GG genotypes (P<0.05), but the differences among different genotypes at C229T or at G98A were no-significant.