Cloning, Prokaryotic Expression, Promoter Regulation And Association Analysis Of The Porcine PNLIPRP2 Gene

The lipase gene family plays an important role in lipid metabolism and transport,so associate with some serious diseases such as obesity,diabetes,and atherosclerosis. Pancreatic lipase-related protein 2(PNLIPRP2) gene is a member of the lipase gene family,In human,it may be involved in intestinal lipolysis.Meanwhile,PNLIPRP2 is responsible for fat digestion as long as milk is the main food for the mouse.By facilitating fat storage,the consequence of which is hyperglycemic diabetes,expression of PNLIPRP2 decreased in TH mice(an animal model for typeâ…¡diabetes) when mice were fed a 4%fat diet.In this study,PNLIPRP2 gene is selected to be a candidate gene which may impact fat deposition in pig.It lays an important basis on further study of the PNLIPRP2 gene on the impaction of fat deposition through its cloning,expression pattern analysis,prokaryotic expression,promoter regulation,polymorphism identification and association with production traits.This work provid a molecular genetic foundation on its application in the molecular marker assisted selection(MAS) in the future.The main results are as follows:1.The full-length sequence of coding region of the porcine PNLIPRP2 gene was cloned using the porcine specific primers which were designed from the pig EST contig obtained by assembling ESTs from GenBank according to the corresponding human cDNA.Intron 1,3,and 4 of porcine PNLIPRP2 gene were also cloned,the splitting sites conform to the GT/AG rule in these introns.Other introns were identified based on bioinformatics and comparative genomics.The obtained cDNA and full genomic sequences have been summitted to GenBank database and the GenBank accession numbers are EU715062 and EU715063 respectively.With the help of CLUSTALW, Tmprep,Signal P3.0,and ProtParam software,the structure and function of the coded protein were analyzed and predicted,the phylogenetic trees were constructed as well.2.The tissue expression profile analysis showed that the PNLIPRP2 gene was expressed at a high level in intestine,a decreased in brain and fat,a lower in bone marrow and stomach and almostly had no expression in other tissues of the two month Large White porcine.Meanwhile,a high level in intestine,a decreased in ovary and a lower in other tissues of the four month Large White porcine.3.We found an L:T/M:C single nucleotide polymorphism(SNP) at the 93 bp in intron 3 and the PCR-Eco130â… -RFLP detection for this SNP was developed.The polymorphism for the mutation was analyzed among the different pig groups,and allele frequencies and the allele distribution were determined among these populations.In the 223 Large White×Meishan F₂ resouce family,the SNP was genotyped by the PCR-Eco130â… -RFLP for carcass trait and meat quality traits association analysis.We found that this SNP was significantly associated with RFT, DLR,WHC,MCV1(P＜0.05).4.Recombinant prokaryotic expression vector PGEX-KG-PLAT_PL was constructed and the porcine PLAT_PL protein was successfully expressed.The expression result was confirmed by the detection of SDS-PAGE.5.We got a consensus 3.7Kb promoter sequence up-stream to the translation initiation site by genome walking.Bioinformatics approaches were adopted to analyze the proximal promoter region of procine PNLIPRP2 genes,and the putative binding sites of transcription factors in the regulatory region were analyzed by online software NNPP,MethPrimer,and TRANSFAC.