Cloning And Functional Analysis Of Porcine PNPLA3 And PNPLA4

Meishan breed is not only one of the best local Chinese pig breeds,but also is an important breed resource in the world.Cloning,characterization and functional analysis of important genes in this breed would benefit accumulating and exploring the gene resources for further genetic improvement and breeding.Patatin is an evolutionarily conserved domain that plays an important role in the lipid metabolism.Recently a novel human family of PNPLAs(patatin-like phospholipases )have been identified by bioinformatics.Among human PNPLAs, three members(PNPLA2,PNPLA3,PNPLA4) have important effect on lipid metabolism and anabolism.Although porcine PNPLA2 was cloned recently,there is no report so far on the molecular characterization of PNPLA3 and PNPLA4 in pigs. We reported here the cDNA cloning,chromosome mapping and tissue distribution of PNPLA3 and PNPLA4 in Meishan pigs.The transcriptional control of PNPLA3 was also explored using IBrS2 cells transfected with eukaryotic transcription factor KLF15.Our findings provide the basis for clarifying the functions of porcine PNPLA3 and PNPLA4 in lipid metabolism.The main results are as follows:1.PNPLA3 and PNPLA4 were cloned in Meishan pigs using RT-PCR.Sequence analysis showed that porcine PNPLA3 contained an open reading frame of 1326 bp, which encodes 441 amino acids,and PNPLA4 contained 771 bp ORF which encoded 256 amino acids.The deduced amino acid sequence of porcine PNPLA3 showed 61.5%,55.2%,and 57.2%similarity to human,mouse and rat PNPLA3,respectively. Porcine PNPLA4 shared 61.5%amino acid identity with its human homolog.2.Reverse transcription PCR revealed two alternative splicing transcripts, PNPLA4 variantl and PNPLA4 variant2,were present in Meishan pigs.PNPLA4 variant2 contains and open reading frame of 705 bp and encodes a protein of 234 amino acids,generated by a deletion of 66 bp from the entire fouth exon of the wild type PNPLA4 variantl.Such an alternative splicing pattern may occure exclusively in pigs.PNPLA4 variant2 was detected in various tissues except lung,adipose and cereberum,although at lower levels as compared to PNPLA4 variantl. 3.Radiation hybrid(RH) technique was employed to map porcine PNPLA3 to chromosome 5p11-15.Porcine PNPLA4 was located to Xp24,as determined by search against High Throughput Genomic Sequences available at GenBank.4.Tissue expression patterns of porcine PNPLA3 and PNPLA4 were analyzed by real-time PCR in various tissues of 4-month-old Meishan pigs.The results showed that the expression of PNPLA3 mRNA was relatively higher in adipose and liver tissues,lower in heart,lung and kidney,and present at trace amounts in spleen,small intestine and cerebrum.Porcine PNPLA4 mRNA level was significantly higher in liver and lower in heart,and at marginal levels in other tissue.5.Porcine PNPLA3 was differently expressed in tissues from genetically obese pigs(Tongcheng) and lean pigs(Large White).Real-time PCR indicated that the mRNA level of PNPLA3 in liver from obese pigs was significantly lower than their lean counterparts,but such difference was not obvious in adipose tissue.6.In order to investigate the transcriptional control of KLF15 on porcine PNPLA3,IBrS2 cells were transiently transfected with pcDNA 3.1-KLF15.After 24-h transfection,the accumulation of lipid droplets in IBrS2 cells was determined by Oil Red O staining.The changes in gene expression pattem were also monitored using real-time PCR.The results showed that KLF15 facilitated the lipid droplets accumulation in IBrS2 cells and up-regulated the expression of PPARγand PNPLA3, which indicated that KLF15 might regulate the transcription of PNPLA3.