Molecular Biological Analysis Of Porcine Three Candidate Genes For Fat Deposition

In this study,three candiadate genes in pig related to fat depositon and lipid metabolism were studied in molecular and celluar level.Adipose triglyceride lipase (ATGL),first identified and characterized by several laboratories in 2004,is a novel triacylglycerol lipase with the official name of PNPLA2,and alternative names of iPLA2ζ, desnutrin,TTS2.2 and PEDF-R.It was described to predominantly hydrolyze the first ester bond of triglycerides,which results in the conversion of triglyceride(TG) to diglyceride(DAG),and then hormone sensitive lipase(HSL) breaks down the DAG into FFA.ATGL and HSL coordinately catabolize stored triglycerides in adipose tissue of mammals.Adiponectin gene(Acrp30) was first reported in 1995,and it is now known to be involved in the regulation of energy homeostasis involving food intake,carbohydrate and lipid metabolism.Adiponectin circulates in blood in multiples such as trimers, hexamers and a high molecular mass species.The globular adiponectin exists independently in vivo and has the similar functions to the full-length adiponectin. Adiponectin can stimulate the fatty acid oxidation in skeletal muscle,decrease plasma triglyceride,and improve glucose metabolism by increasing insulin sensitivity.Now polymorphisms in human adiponectin gene have been recently reported to be associated with obesity,insulin sensitivity and the risk of type 2 diabetes.Resistin gene(RSTN) was first discovered in 2001,and is a hormone specially secreted from adipocytes.Resistin is an important linkage between obesity and insulin resistance.The further investigation of resistin will have significance in directing the prevention,detection and treatment of type 2 diabetes.As for its important role in lipid metabolism and fat deposition,here we describe the molecular characterization of porcine ATGL gene,including the isolation of full-length cDNA and DNA sequence,mRNA expression profile,polymorphism detection,promoter activity detection,fusion protein expression,cell clone selection,and so on.Meanwhile, porcine adiponectin gene and resistin gene were considered to be important candidate genes for fat deposition and carcass traits,and investigated for polymorphism screening and potential effective molecular markers selection.The main results about the three genes are as follows:1.Study on porcine ATGL gene1) Isolation and characterization of full-length cDNA sequence.A 2055bp full-length cDNA of porcine ATGL gene was identified by contig-assembling from EST data and the Rapid Amplification of cDNA ends(RACE) method,and submitted to GenBank database with EF648448.The full-length cDNA sequence contains a 1461bp open reading frame encoding a protein of 486 amino acids with a calculated molecular mass of 53.2 kDa and an isoelectric point of 7.90.Like other ATGL proteins,porcine ATGL is predicted to contain an N-terminal patatin-like domain where there is a predicted 'esterase of theα/βhydrolase fold' domain and a catalytic dyad(GXSXG and DXG/A) consensus sequence.The homologous patatin phospholipase A active residues of porcine ATGL correspond to Ser⁴⁷ and Asp¹⁶⁶.Predicted porcine ATGL shares high homology(＞83%) with that of cow,dog,mouse,rat and human proteins,and has the closest genetic relationship with cow.2) Isolation and characterization of full-length genomic DNA sequence.Using the genomic walking method,a 7044bp full-length genomic DNA sequence of ATGL gene was obtained.Comparison analysis with homogenes revealed that porcine ATGL gene consists of 9 coding exons.The locations of splice donor/acceptor sites in all introns conform to the GT/AG rule.3) Isolation and bioinformatics analysis of promoter.A 998bp 5'-flanking sequnence of porcine ATGL was isolated by genome walking.NNPP,MotifFinder, TFSEARCH,CPGPLOT and SignalScan softwares were used for prediction of nuclear promoter,initial position of transcription,distribution of CpG island and transfactors banding to promoter.It is predicted to contain a putative transcription start site(-164 bp from the start of translation),a core promoter region(-205 to -155bp),as well as potential CAP,TATA,CAAT and GATA motifs and CREBP1,MyoD and C/EBP binding sites.In addition,two mutations were found in the upstream region of porcine ATGL gene.4) Fine chromosomal localization.The INRA-University of Minnesota porcine radiation hybrid(IMpRH) panel was applied for detecting the chromosomal localization of porcine ATGL gene.The result showed porcine ATGL gene was located on SSC2p17, with the most significant linked marker SW2443 on SSC2 at a distance of 36cR (LOD=10.26).Interesting,it has been reported a QTL for backfat thickness exsits in SSC2p17,the distal tip of the p-arm of SSC2.5) Polymorphism detection.Overlapping PCR fragments amplified from different pig breeds(Large White,Landrace and Meishan pigs) were compared to find out polymorphisms in porcine ATGL gene.Eleven polymorphic sites were found in the coding region of porcine ATGL gene.6) Association analysis for G392A mutation.A G/A substitution at nucleotide 392 of coding sequence(G392A) leading to amino acid substitution(R131H) was found to locate in the conserved patatin domain.A rapid and sensitive assay with primer mutagenesis and PCR-RFLP was developed to detect the frequency of the allele distribution in different pig breeds.Allele A was found to be dominant in Large White and Landrace,with high frequencies of 73%and 80%,respectively;while in comparison, the other allele G was found to be major in Meishan pig with a frequency of 73%.The association analysis showed that the polymorphism was significantly associated with almost all the fat deposition and carcass traits measured,including subcutaneous fat thickness,viscera adipose tissue,lean percentage,loin eye traits and rib numbers. According to the transmemebrane regions predicted by TOPpred,R131H variation locates just right at the cytoplasmic loops(64-140aa) adjacent to the third transmembrane region (141-161aa).Since the variation in the functional region of patatin,it is probable that the change of R131H could influence the spatial structure or activation level of ATGL.7) Tissue expression profile of porcine ATGL mRNA.Real-time RT-PCR experiments showed that ATGL transcripts are expressed predominantly in backfat,mildly in muscle,small intestine and heart,and weakly in liver,spleen,lung,stomach,kidney and ovary.8) Promoter activity assay.Progressive deletion constructs in the pig ATGL promoter region were created by PCR.Seven luciferase expression constructs were constructed with and sequenced,which were pGL3-Q1(-998bp～+45bp),pGL3-Q2 (-788bp～+45bp),pGL3-Q3(-631bp～+45 bp),pGL3-Q4(-568bp～+45bp),pGL3-Q5 (-472bp～+45bp),pGL3-Q6(-367bp～+45bp) and pGL3-Q7(-198bp～+45bp).Using Lipofectimine 2000 as transfection reagent,these constructs were transfected into COS-7 cell line respectively.Forty-eight hours post-transfection,Luciferase activity was assayed with Dual-luciferase reporter system and measured by Luminometer.The results indicate the 5' flanking region including up to -367bp relative to -164bp contains all the elements necessary to achieve basal promoter activity.Moreover,important positive regulatory elements reside between -568bp and -367bp,whereas negative regulatory elements lie between -788bp and -568bp.9) Fusion protein expression.As for different domains and deletions,the recombined plasmids,pATGL/EGFP,pATGL-pat/EGFP,pATGL-Npat/EGFP, pATGL-Fab/EGFP and pHSL/EGFP,were transfected transiently into IBRS-2 cells and porcine primary pre-adipocytes through liposome-mediated method,and pEGFP-N1 was used as control.The green fluorescence protein expression and localization of plasmids were observed using Laser Scanning Microscope after 24 hours of transfection.The results showed green fluorescence signal was distributed uniformly in cytoplasm in cells transfected with pATGL/EGFP or pHSL/EGFP,however,while in cells expressed fusion protein with partial ATGL protein,the fluorescence signal showed the whole cell distribution of fusion protein,including the nucleus and cytoplasm.The results indicated that only the entire ATGL protein can specially locate in cytoplasm.10) Transfected cell clone selection with G418.We identified IBRS-2 cell clone can be selected with 800μg/mL of G418.By G418 selecting and cloned culture in limiting dilution,we successfully selected G418 resistant IBRS cell clones transfected with pATGL/EGFP and pHSL/EGFP,which displayed a stable and long-term GFP expression in vitro.2.Mutations in porcine Acrp30 gene1) Exonic mutation A178G.A mutation of A178G in exon 2 that resulted in substitution of Ile60Val of porcine adiponectin gene was identified.Acyâ… PCR-RFLP was used to detect the polymorphism of the genotypes in five different pig populations(Large White,Landrace,Duroc,Chinese breeds Meishan and Qingping).The A allele frequency was significantly higher among subjects with Chinsese lard type breeds,while G allele was the only one present in those with Western lean type breeds.To determine if there was an association of the polymorphism with phenotypic variation,the mutation was tested in 267 pigs of the "Large White×Meishan" F₂ resource population.The results of association analyses showed significant associations of the genotypes with fat deposition and carcass traits.Allele G is significantly associated with increase in loin eye height,loin eye area and lean meat percentage and bone percentage,and decrease in fat mean percentage,ratio of lean to fat,shoulder fat thickness,6～7 rib fat thickness,thorax-waist fat thickness and buttock fat thickness.The substitution of A178G(Ile60Val) happened to be located at 60 amino acid in the collagenous domain of porcine adiponectin might affect the association into higher-order structures,accordingly affect the posttranslational modifications and optimal biological activity of the multimeric forms.The identified functional polymorphism will give new evidence of adiponectin as an important candidate gene affecting fat deposition and carcass traits in pigs.2) Intronic mutation A958G.A mutation of A958G in intron 2 of porcine Acrp30 gene was identified.PCR-SSCP was used to detect the polymorphism of the genotypes in three different pig populations.The G allele was only present in subjects with Chinese lard type breed Meishan,while allele A was the only one present in those with Western lean type breeds Large White and Landrace.To determine if there was an association of the polymorphism with phenotypic variation,the mutation was tested in 273 pigs of the "Large White×Meishan" F₂ resource population.Allele A is significantly associated with increase in loin eye width,loin eye area and lean meat percentage,and decrease in shoulder fat thickness,6-7 rib fat thickness and fat mean percentage.3.Mutations in porcine RSTN geneA mutation of 14bp insertion/deletion was detected in intron 2 of porcine RSTN gene. The PCR products were separated on PAGE directly.A 14bp deletion(AA) absolutely exsits in Western lean type breeds Large White and Landrace,while Meishan possesses three genotypes.The mutation was tested in 127 pigs of the "Large White×Meishan" F₂ resource population.Genotype AA is significantly associated with decrease in fat mean percentage,shoulder fat thickness,6～7 rib fat thickness,thorax-waist fat thickness and buttock fat thickness,average backfat thickness,leaf fat weight and caul fat weight;while increase in lean meat percentage and body length.