Isolation And Characterization Of Genes Involved In The Regulation Of Intramuscular Fat Deposition In Pigs

The molecular mechanisms controlling the intramuscular fat deposition remain poorly understood.Such knowledge is crucial to develop strategies to enhance intramuscular fat accumulation in pigs.In the present study,DDRT-PCR was used to reveal differentiaUy expressed genes between two groups with significant different IMF content.The aim of this study is to find out the genes which participate in the regulation of IMF deposition.Experiment 1:Longissimus Dorsi(LD) muscle samples were collected from 90 castrated pigs aged 180 d and evaluated for intramuscular fat(IMF) content(2.72%±0.13 %).Two groups from 90 pigs,each consisted of 6 samples,of highest(5.79%±0.35%)and lowest(0.98%±0.09%) IMF content(p＜0.01),were selected to compare the differentially expressed genes between the groups.DDRT-PCR reaction was performed by using 36 primers combinations of 3 anchored primers coupled with 12 arbitrary primers.Fourteen Expressed Sequence Tags(ESTs) wcrc gained and sequenced.The sequences were compared with GenBank database.Four out of fourteen EST seauences are known genes. They were designated as Clone1(691 bp),Clone2(160 bp),Clone3(351 bp) and Clone4 (597 bp).Clonel expression level is positivly correlated with IMF content,while Clone 2,3, 4 ncgativly with IMF contents.The clone 1 sequence shows high homology(99%) with human splicing factor,serinc-arginine rich protein(SRrpl30).Clone 2 is identity with pig MHC classⅠantigen(SLA1)(100%),clone3 is highly homologous(94%) to human p160 co-activators family member steroid receptor coactivator 1(SRC-1).Clone 4 was identity with pig myosin heavy chain 2b(MYH2b)(100%).Although SRrp130 complete coding sequences(CDS) were identified in human,the corresponding sequence in pigs still remains unknown.We initially used the human SRrp130 sequence to obtain porcine ESTs from GenBank Two contigs were established for 5′and 3′end, respectively.There was no overlap of these two contigs.Then,the interval sequence between these two contigs was obtained by sequencing.Finally,a 3234 nt sequence was gained which contained the open reading frame(ORF) encoding 805 deduced amino acids.To further confirm the relation of these 4 genes with IMF content,15 samples with highest IMF content and 15 with lowest wore evaluated for mRNA level by quantitive real-time PCR.The mRNA levels showed a significant inter-group difference for both Steroid receptor coactivator 1(SRC-1) and splicing factor serine-arginine rich protein (SRrp130) gene(p＜0.05).SRrp130 mRNA level increased as IMF content increased (r=0.54,p＜0.01),while SRC-1 reversed(r=-0.38,p＜0.05).The result suggested that both genes may be implicated in IMF deposition in pig LD muscle.Experiment 2:SRC-1/NCOA1 gene was identified as a promising gene which might be involved in IMF deposition.It is one of the three members of p160 co-activator family, which are NCOA1,NCOA2 and NCOA3.The lose-of-function studies of these three genes in mouse demonstrate their important roles in energy metabolisms.In this part,we cloned these three genes in pigs,identified their transcript variants and analyzed their expression level in relation to IMF content in Longissimus Dorsi(LD) muscle.Both in silico cloning and PCR amplification revealed a full length cDNA sequence of 6,591 bp for NCOA1 (EU346671),7,628 bp for NCOA2(EU346672) and 5,005 bp for NCOA3(EU346674) in pigs,respectively.Interestingly,three transcript variants wore identified for the porcine NCOA1 and two for the porcine NCOA2 gene.In addition,the deduced amino acid sequences indicated that isoform 2 of NCOA2 lacks the fourth LXXLL motif,the number of which has been shown to influence the selectivity and affinity for different nuclear receptors.Finally,fifteen animals with high IMF content and fifteen animals with low IMF content(p＜0.05) selected from 60 individuals wore used to investigate how the family members and their variants affect the IMF deposition in pigs using real-time PCR.Our results showed that both NCOA1 transcript variant 2(r=-0.554,p＜0.01) and total NCOA1 (r=-0.516,p＜0.01) expression levels were negatively correlated with IMF contents,while NCOA2 transcript variant 1(r=0.605,p＜0.01) and NCOA3(r=0.435,p＜0.05) wore positively associated with IMF content in LD muscle.Overall,the present study provides evidence for the first time that the p160 co-activator family might have a concordant effect on lipid metabolism in mammals.Based on the current comparative maps,NCOA2 gene is located around 65 cM on pig chromosome 4 which is occasionally a peak region of a reported IMF QTL.Interestingly, the same situation occurs in cattle that NCOA2 lies in a region which habors a QTL for marbling.Our data implies that NCOA2 might be a promising candidate gene for IMF deposition in pigs.