Cloning, Chromosomal Localization And Association Analysis Of Porcine SMPX,STARS,VGLL2 And VGLL4 Genes Which Relating To Muscle Development And Growth

It has been pig breeding research priorities to enhance and accelerate the lean growth rate. But the conventional breeding method to improve these characteristics have been greatly restricted, to combine it with molecular marker assisted breeding will be the inevitable trend of pig genetic improvement.The study of pig muscle growth can help people to know the general molecular mechanisms of the animals muscle growth, also can provide good evidences for the study of animal growth model and improved pig growth characteristics by molecular marker assisted selection in future. In recent years, a number of candidate genes for muscle growth had been identified, but few of them have been applied to the practice of production, the molecular mechanisms study of pig muscle development and growth is not yet deep. By using bioinformatics method combined with molecular biology techniques, we cloned and located four porcine genes which related to muscle development and growth, and preliminary research and forecaste the gene function, and achieved the following results:1. Combining the EST data and the rapid amplification of cDNA ends (RACE) method, full-length cDNA of the pig SMPX and STARS gene were isolated and identified, part sequences of VGLL2 and VGLL4 were obtained. The sequences has been submitted the GenBank database, the accession number are DQ104414, DQ536197, DQ241740 and DQ241739 respectively. The cDNA sequences of SMPX and STARS gene were analysed by bioinformatics, mostly were the study of the protein sequence and the presumed functional domain.2. Part promoter sequences of pig SMPX gene was identified by comparative genome methods, and the sequence was analyzed for transcription factor binding sites. Then the sequence has been submitted to the GenBank database, the accession number is DQ 104415.3. The pigxrodent somatic cell hybrid panel (SCHP) was used for regional assignment of both genes and the INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel was employed to determine the precise location of the four genes. Statistical analysis revealed that SMPX was located to the pig chromosome Xp24 and closed to SW1903 with LOD scores of 7.04, STARS was located to the pig chromosome 4p13 and closed to SW871 with LOD scores of 11.98, VGGL2 was located to the pig chromosome Ip13 and closed to SW1653 with LOD scores of 8.07, VGGL4 was located to the pig chromosome q23-(l/2 q41) and closed to SW1864 with LOD scores of 5.75. All LOD scores is bigger than 5.0.4. Gene tissue distribution and stage distribution was analyzed by the semi-quantitative RT-PCR in different tissues and different stages. The result of RT-PCR displayed that SMPX and STARS genes were highly expressed in heart and skeletal muscle, low or no expression in other tissues. Moreover, SMPX and STARS have a differential expression level in skeletal muscle in different stages.5. There were a TaaI-RFLP (A-G) in SMPX 3'UTR and a Bsh1236I-RFLP (T-G) in STARS CDs by detection SNPs. Eight breeds (Duroc, Large White, Min pigs, Qingping pigs, Lantang pigs, Yushan pigs, Large Black-White and Meishan pigs) were genotyped for genetic variation analysis and x~2 test on the allele frequencies was performed. The results indicate that the genotype frequencies of all the loci were significant difference among the different populations.6. The two SNPs of SMPX and STARS were genotyped in the experimental population which constructed by our lab. The general linear model (GLM) was used to analyze the association between these SNPs and some performance traits. The results showed that all the sites have effects on some traits except. There is a highly significant association between SMPX genetic Taal polymorphism and intramuscular fat content (P＜0.01); There is a highly significant association between STARS genetic Bsh1236I polymorphism and average backfat thickness (P＜0.01).7. Recombinant prokaryotic expression vector pET28a-SMPX was constructed and it expresses the porcine SMPX protein successfully by the detection of SDS-PAGE and Western-blot. To further examine its biological characteristics and functions laid the foundation.