ABSTRACT
As an important triglyceride hydrolase in mammalian cells, patatin-like phospholipase domain-containing 2 (PNPLA2) predominantly performs the first step in triglyceride hydrolysis. The objective of this study was to detect and evaluate the effects of mutations in the 5' upstream region of porcine PNPLA2 gene with fat deposition and carcass traits. Four single nuclear polymorphisms were identified, including g.161969 T>C, g.161962 A>G, g.161953 C>G and g.161904 G>T, and subsequently genotyped in five pure breeds. Three haplotypes were constructed, including H1(CGGT), H2(TACG) and H3(CACT), which were the most abundant haplotypes in Duroc (0.75), Landrace (0.78) and Chinese indigenous breeds (>0.73), respectively. Duroc individuals with the H1H1 diplotype always exhibited the lowest feed conversion ratio (FCR) (P < 0.05), while H2H2 had the thickest backfat thickness (P < 0.05). Landrace individuals with H2H3 had lower backfat thickness (P < 0.05), higher muscle thickness (P < 0.05) and estimated lean meat percentage (P < 0.05) than those with diplotype H2H2 and H3H3. Luciferase assay indicated pGL3-basic-H2 had the highest activity and pGL3-basic-H1 had the lowest activity in driving reporter gene transcription in HEK293 cells in vitro. In H1 haplotype, two GR binding sites and an ERα binding site were predicted to be introduced. While in H2 and H3, there were other transcriptional factor binding sites predicted in H2 and H3, such as Sp1, AP-2 and CAC-binding proteins, which were broadly expressed transcription factors and capable of contributing to basal promoter activity. The reduced basal promoter activity of H1 may be due to the lack of inducement for GR and ERα binding sites in HEK293 cells. The identified functional polymorphisms provide new evidence of PNPLA2 as an important candidate gene for fat deposition and carcass traits in pigs.
Subject(s)
Lipase/genetics , Phospholipases A2/genetics , Polymorphism, Single Nucleotide , Quantitative Trait Loci , Sus scrofa/genetics , Animals , Genetic Association Studies , HEK293 Cells , Haplotypes , Humans , Meat/standards , Muscle, Skeletal/growth & development , Promoter Regions, Genetic , Quantitative Trait, Heritable , Sus scrofa/growth & development , SwineABSTRACT
5-AZA-2'-deoxycytidine (5-AZA-CdR) is a demethylating, teratogenic agent and a mutagen, which causes defects in the developing mouse and rat after implantation. Our previous data indicated that 5-AZA-CdR (0.2 and 1.0 muM) inhibited the development of mouse preimplantation embryos. Pronuclear embryos exposed to 5-AZA-CdR at the pronuclear stage were unable to form 8-cell embryos, while 2-cell-stage embryos exposed to 5-AZA-CdR only developed into uncompacted 8-cell-stage embryos. And there was no formation of blastocysts when 4-cell embryos cultured in 5-AZA-CdR. In our present study, we detected Dnmt1o protein and some developmental gene expression in order to find the reasons for the developmental arrest. Dnmt1o could not traffic to 8-cell nuclei as control when embryos were exposed to 5-AZA-CdR. Dnmt1o was in cytoplasm at 2-cell and 4-cell stages before and after treated with 5-AZA-CdR. Gene expression changes were also detected in this research. Our data indicated that connexin 31 (Cx31), connexin 43 (Cx43), connexin 45 (Cx45), E-cadherin (Cdh1) and beta-catenin (Ctnnb1) were all downregulated by 5-AZA-CdR. Cx31, Cx43 and Cx45 are members of connexins family, which have a central role in gap junctions. Cdh1 and Ctnnb1 are necessary for the foundation of tight junctions. Therefore, developmental arrest induced by 5-AZA-CdR may be caused by the failure of Dnmt1o cytoplasmic-nuclear traffic and the down-regulation of developmental gene expression. Normal compaction and blastocoel cavitation need Dnmt1o traffic to 8-cell nuclei and the right gene expression, especially the correlative genes in gap junctions and tight junctions.
Subject(s)
Azacitidine/analogs & derivatives , Blastocyst/drug effects , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , Embryonic Development/drug effects , Mutagens/toxicity , Teratogens/toxicity , Animals , Azacitidine/toxicity , Blastocyst/physiology , Cadherins/antagonists & inhibitors , Cadherins/metabolism , Connexins/antagonists & inhibitors , Connexins/metabolism , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation , Decitabine , Down-Regulation , Female , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/physiology , Mice , beta Catenin/antagonists & inhibitors , beta Catenin/metabolismABSTRACT
Porcine circovirus type 2 (PCV2) has been linked to several disease syndromes during the last decade. A deficiency in selenium has also been associated with the increases of virulence of some viruses and severity of infectious disease. In order to evaluate the effect of different selenium sources and levels on PCV2 replication in PK-15 cells, three selenium sources, i.e. sodium selenite, kappa-selenocarrageenan and dl-selenomethionine at concentrations of 0, 2, 4, 8, and 16 micromol/L were used throughout this experiment. PCV2 loads in PK-15 cells were measured by a newly developed real-time quantitative PCR. A significantly inhibitive effect of dl-selenomethionine on PCV2 replication in vitro was demonstrated and the inhibition was concentration dependent within the range of 2-16 micromol/L. The inhibitive effect of dl-selenomethionine on PCV2 replication may be caused by enhanced activity of glutathione peroxidase. Our results may serve as a basis for further studies of the biological function of selenium and control of PCV2 infection.
