| Muscle and fat are two important factors affecting the pig muscle quality. To improve muscle quality is one of the important goals of breeders. With the development of molecular biology, swine breeding techniques have been changing gradually from conventional breeding to the molecular breeding. Therefore, understanding the impact of pig muscle and adipose tissue transcriptional regulation mechanism can help to lay a good foundation for molecular breeding. To this end, we have done the following research:(1) The transcription initiation site of BMP-3b gene in the swine was identified by5’RACE, and we found the TSS of swine BMP-3b gene was a guanine located in278bp upstream of the translation start sit, be set to+1.(2) By NCBI and EBI for pig BMP-3b gene, we get the5’upstream regulatory sequences of pig BMP-3b gene, We cloned the gene5’end of the1458bp upstream regulation sequence, and using the method of bioinformatics is forecasted, found the TATA-box and GC-box, predicted more fat related C/EBP transcription factor binding sites. Compared the proximal end of this gene promoter region among different species, we found a high conservative region at the-51--36bp which contains two GC-box.(3) Constructing eight5’end of the series of deletion fragments-dual luciferase reporter gene, and detecting the difference fluorescence activity in three kinds of eukaryotic cell, next the futher five deletion fragments was construct in PF2, we find the core promoter be likely to-46--39.(4) By using the method of point mutations to validation the above results, found that the fluorescence activity decreased significantly after mutation, further to determine the core of the gene locus.(5) Swine BMP-3b have two CpG in5’promoter and the first exon, respectively located in-354～+229and+308～+596bp, the length is585bp and288bp; Methylation analysis of the core promoter region(-277～18bp) of the gene in nine different organizations. The region exist37CG site. Found the pig BMP-3b gene methylation level is very low, One months longissimus muscle methylation degree is the highest, but only3.2%; The lowest in the stomach at six months of age, Only0.3%. (6) Using RT-PCR method to detect the BMP-3b gene expression level in Sixteen different organizations of June age large white. The results showed that the gene is Highest expressed in brain and lung, higher expressed in fat, liver, heart, and ovarian, Seldom expressed in the spleen, muscle, stomach, small intestine and kidney longest, in other tissues not found the express.(7) Respectively for the large white and meishan pigs BMP-3b gene complete CDS, total length is1428bp, encoding475amino acids. Contrast found that in546bp spot, meishan is A, whereas large white is G. But due to the rolling of codon, CTA(G) encoding the same amino acid. This site may be a potential SNP loci, and PcDNA3.1-CDS eukaryotic expression vector was constructed.(8) Using bioinformatics methods compared the different species of BMP-3b gene structure and sequence of amino acids, found that BMP-3b is a very conservative gene, Pigs, cattle, mice and humans of the gene both has three two exons and introns, and at the junction of introns and exons are conform to the typical rules of GT-AG; The amino acid similarity of swine with Cattle, sheep, horses, cats, chimpanzees, giant panda, and rat is92%、93%、94%、89%、90%、92%、90%、90%。(9) The pig PK cells were used to research over-expression analysis with the constructed eukaryotic vector PcDNA3.1-CDS. Over-expressed swine BMP-3b gene group showed significant difference (p<0.01) in compared with control group by fluorescence quantitative PCR detection, the fat related genes PPAR γ gene expression was dramatic increased. |
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