Cloning, Polymorphism And Tissue Expression Profile Of G6PC, LCAT And Nurr1 Genes In Swine

The molecular breeding techniques such as marker-assisted selection(MAS) and marker-assisted introgression(MAI) combining with traditional breeding methods have greatly promoted the process of genetic improvement in pigs.The basis of MAS is to seek the major genes or markers which are closely connected with the significant economic traits.So in this research,pig G6PC,LCAT and Nurrl genes were isolated and identified by the method of the candidate gene and the EST information with the Comparative Genomics.We carried out the association of some traits and functional domain prediction. The main results are as follows:1.G6PC(glucose-6-phosphatase,catalytic subunit):(1) The full coding sequence and partial genomic sequences of G6PC gene from Large White,Meishan pigs were isolated and deposited Meishan's seqence into GenBank under accession number EU717834.(2) With the software CLUSTALW,DNAStar,Prosite,the structure and function of the coded protein were predicted and analyzed,the phylogenetic trees were constructed as well. (3)After sequence alignment,we found an Large White:C/Meishan:T mutation at the 657bp in exon 5,which result in polymorphism of restriction enzyme RsaI cutting site(CT↓AC),developed PCR-RsaI-RFLP genotyping method.In the 299 F₂ pigs in the Large White×Meishan resouce family,association analysis of pig's meet quality traits and carcass traits indicated that the relation between G6PC gene and SP,DP,SFT,LDpH, LDWLP,LDWHC,LDMCV are significant(p＜0.05).Compared with AA genotype,BB had a higher LMP,a lower FP and backfat thickness.(4) After sequence alignment,we found an Large White:A/Meishan:C mutation at the 460bp in intron 3,which result in polymorphism of restriction enzyme Hpaâ…¡cutting site(C↓CGG),developed PCR-Hpaâ…¡-RFLP genotyping methord.In the 257 F₂ pigs in the Large White×Meishan resouce family,association analysis with pig's meet and carcass traits found that G6PC genotype was significantly associated with BP,SFT,RFT,LDW,LDWC,BFMCV (p＜0.05),and was extremely significantly associated with LDH,LDMCV,LDIMF (p＜0.01).(5) The tissue expression profiles of G6PC gene was analysed by semi-quantity RT-PCR in pig heart,liver,spleen,lung,kidney,stomach,small intestine,uters,muscle and fat.RT-PCR results showed that G6PC gene is expressed at a high level in liver and kidney.2.LCAT(lecithin cholesterol acyltransferase):(1)The full coding sequence and partial genomic sequences of LCAT gene from Large White,Meishan pigs were isolated and deposited Meishan's seqence into GenBank under accession number EU717835, EU717836.(2) With the software CLUSTALW,DNAStar,Prosite,the structure and function of the coded protein were predicted and analyzed,the phylogenetic trees were constructed as well.(3)After sequence alignment,we found an Large White:C/ Meishan:G mutation at the 266bp in intron 1,which result in polymorphism of restriction enzyme PVuâ…¡cutting site(CAG↓CTG),developed PCR-PVuâ…¡-RFLP genotyping methord. In the 304 F₂ pigs in the Large White×Meishan resouce family,association analysis with pig's carcass traits found that the relation between LCAT gene and CL,BP are significant(p＜0.05).(4) The tissue expression profiles of LCAT gene was analysed by semi-quantity RT-PCR in pig heart,liver,spleen,lung,kidney,stomach,small intestine, uters,muscle and fat.RT-PCR results showed that the expression level of LCAT gene was the highest in liver,spleen,lung,kidney,stomach,small intestine and uters,lower in heart, muscle and fat,lowest in stomach.3.Nurrl(nuclear receptor-related factor 1):(1)The full coding sequence and partial genomic sequences of Nurrl gene from Large White,Meishan pigs were isolated and deposited Large White's seqence into GenBank under accession number EU717837, EU717838.(2) With the software CLUSTALW,DNAStar,Prosite,the structure and function of the coded protein were predicted and analyzed,the phylogenetic trees were constructed as well.(3) After sequence alignment,we found this gene is very conservative, there is not mutation.(4) The tissue expression profiles of Nurrl gene was analysed by semi-quantity RT-PCR in pig heart,liver,spleen,lung,kidney,stomach,small intestine, uters,muscle and fat.RT-PCR results showed that the Nurrl gene was expressed at a high leveal in spleen,lung and kidney,a lower in heart,liver and muscle,and had no expression in stomach,small intestine,uters and fat.