Cloning, SNP Detection And Association Analysis With Production Traits Of Porcine Diacylglycerol Acyltransferase Genes

With the development of molecular genetics,quantitative genetics and technology of molecular biology,molecular breeding techniques such as marker-assisted selection (MAS) and marker-assisted introgression(MAI) appear and are extensively used during animal breeding.Molecular breeding techniques 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 important economic traits.The adipose tissue is not only an energy reservoir pool in animals but also the greatest endocrine organ.In addition,its related traits are of significance to porcine carcass and meat quality.So in this research,two genes (diacylglycerol acyltransferase 1 and 2) which are pertinent to fat metabolism were chosen as the candidate genes for pig economic traits and were studied on.The results are as follows:1.Genome PCR walking technique was used to clone the proximal promoter region of procine diacylglycerol acyltransferase 1(DGAT1) gene.We obtained a fragment of 2556bp.Bioinformatics approaches were adopted to analyze the proximal promoter region of procine DGAT1 gene,and the putative binding sites of transcription factors in the regulatory region of the gene were analyzed by online software.2.We constructed 16 DGAT1 promoter recombinants of progressively 5'-deleted DNA fragment linked to the pGL3 reporter.3.The Large White and Meishan diacylglycerol acyltransferase 2(DGAT2) gene cDNA had been cloned and sequenced using RT-PCR method from pig liver tissues.The obtained fragment is 1481bp(GenBank accession number:EU684958),and the ORF is 1086bp,coding 361 amino acids.Using DNAStar,CLUSTAL W,Signal P3.0, Prosite and some other related softwares,we analyzed the gene structure,protein structure,and conserved motifs of DGAT2 gene.The corresponding phylogenetic tree was constructed.In addition,we cloned the intron3,intron5,intron6 of this gene,and the spliting sites conform to GT/AG rule.4.The tissue expression profile analysis showed that DGAT1 gene was expressed in all tissues;Real-time RT-PCR analysis showed that the DGAT2 gene was expressed at a higher level in liver and adipose tissue,a very lower level in other tissues,the highest level in adipose tissue.We chose three tissues(adipose tissue,liver and small intestine) closely connected with triglyceride metabolism,comparing the expression difference between Meishan and Large White pig DGAT2 gene.The result indicated that the Meishan DGAT2 gene was expressed at a markedly higher level in these three tissues than that in Large White pigs.5.We analysed the polymorphism of DGAT1 promoter region and DGAT2 gene.The results are as follows:(1) Four transition mutations were found in DGAT1 promoter region.378bp ahead of initiation codon,a C→T transition causes the change of Pvuâ…¡restriction enzyme cutting site;(2) Seven mutations were found in DGAT2 gene.Of 2 are transversion mutations,7 are transition mutations.A G→A transition in exon7 causes the change of BssNAâ… restriction enzyme cutting site.6.Genotyping of a total of two polymorphic locus showed that there were abundant polymorphisms in various pig breeds.Association analysis was performed between polymorphisms and important production traits in Large White×Meishan F₂ offspring, and the results are as follows:(1) There are significant difference between DGAT1 Pvuâ…¡-RFLP genotypes and LMP,CL2,TFT,AFT,PH(BF),MM2,IMF;(2) Statistically significant association were found between DGAT2 BssNAâ… -RFLP genotypes in exon7 and LMP,SFT,RFT,TFT,AFT,MCV2,MM1,IMF,WM.