| In pigs,fat depots are mainly constituted by the subcutaneous and visceral fat,the intermuscular fat surrounding the muscle tissue and the intramuscular fat within the muscle tissue.Fat deposition in skeletal muscle strongly influences meat quality.Many efforts have been carried out so far to identify key factors and genes affecting this trait,.pointing out the complexity of the physiological control of fatness.Intracellular lipid depots are stored in triacylglycerol-rich lipid droplets,dynamic organelles with important functions in the maintenance of cellular energy homeostasis.Adipose differentiation related protein(ADRP)is a cytosolic protein that promotes the formation and stabilization of the intracellular lipid droplets,organelles involved in the storage of lipid depots.Porcine ADRP gene represents positional candidate for fat deposition,affects carcass and meat quality.In order to study on the effect of ADRP gene of porcine IMF content,our study of adopt PCR amplifying and cloning sequences to acquire the ADRP gene,using bioinformatices method predict the structure and function of ADRP protein,and also to construct a phylogenetic tree to reveal the evolutionary relationship of various species.Then we construct deletion expression vectors of the promoter and detect the core promoter region of ADRP by the luciferrase reporter gene system.We detect the different levels of the methylation of the promoter of ADRP in the FAT and LEAN by bisulfite sequencing.The effects of ADRP overexpression on porcine preadipocytes and underlying molecular mechanism were also detected by Oil red O staining and real-time RT-PCR.The main results achieved were as follows:1.Bioinformatic analysis of porcine ADRP geneThe porcine ADRP gene is located on chromosome 1 and contains 8 exons and 7 introns.The porcine ADRP cDNA contains a 1377 bp open reading frame,encoding a deduced protein of 459 amino acids.The porcine ADRP contains no signal peptide,typical hydrophobic regions and transmembrane region,but is a nucleoprotein residing in cytoplasm.2.Porcine ADRP gene 5'flanking regulatory sequence cloning and promoter activity analysisThe ADRP gene 5' flanking of the 1719bp promoter sequence was cloned.First,the construction of five dual lucifease expression vector were built,transiently transfected C2C12 cells and PK15 cells.The result showed that the core promoter of porcine ADRP gene is located in the translation initiation site upstream of-234bp to-926 bp region.Transcription factor bingding site prediction found that the ADRP gene promoter region containing the YY1,Sp-1 and so on,among which YY1 and Sp-1 is the metylation sensitive site.3.CpG island methylation analysis of ADRP geneWe detected the core promoter of the DNA methylation status by sisulfite sequencing techonology.We analyzed the methylation rate of 21 CpG sites in CpG Island of porcine ADRP gene from the FAT and the LEAN,finding that the nucleotide sites take up 66.67%on the whole sites of the FAT,and the LEAN is 74.76%,actually there is no significant difference.In the analyzed methylation situation of the 7th?10th?12th CpG sites,there is significant difference between them,the level of methylated site in the FAT is 46.7%,however the level of the LEAN is 86.67%.The reserch results of the high methylation level of porcine ADRP gene in the LEAN with the low expression level of mRNA,which suggested that the promoter methylation may play an important role in modification of ADRP gene m RNA eapression.4.The effects of ADRP overexpression on porcine preadipocytes and underlying molecular mechanism were also detected by Oil red O staining and real-time RT-PCR.Oil red O staining showed that the preadipocytes accumulated lipid much lower compared with the pcDNA3.1 transduced controls.Real-time PCR was used to quantify adipogenic markers PPAR? and FABP4.The genes expression compared with control also increased significantly(p<0.05).Our study showed that ADRP has a regulatory role in preadipocytes differentiation. |
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