Cloning And Functional Research Of Porcine CIDEs Family

Meishan breed is one of the best local porcine breeds of China,and is also an important breed resource in the world.Cloning,characterization and functional analysis of significant genes would benefit accumulating and exploring the gene resource information of important local porcine breeds in China,which would shed light on theories related to the further genetic improvement of excellent porcine breeds.Apoptosis is an evolutionarily conserved phenomenon that plays an important role in the regulation of normal cellular activities in eukaryotes.Recently a novel family of cell death-inducing DFF45-like effectors(CIDEs) have been identified. Among CIDEs,three from human(hCIDEa,hCIDEb,hCIDEc) and three from mouse (mCIDEa,mCIDEb and FSP27(fat specific protein 27,a mouse homologue of human CIDEc)) have been reported.But there are not any reports on CIDEs in pigs.Except as pro-apoptotic proteins,it has been reported that CIDEs family could be involved into lipid or fat metabolism,and are therefore candidate genes for human obesity.To clarify the characteristic and functions of porcine CIDEs family protein, and provide basic molecular information useful for the further investigation of the three genes,which will be helpful in better understanding of the role of CIDEs in lipid metabolism.Using Meishan pigs,in this study we first reported the cDNA cloning, chromosome mapping and expression analysis of CIDEa,CIDEb and CIDEc in pigs. The expression difference in mRNA levels of these genes was also compared between obese pigs(Tongcheng) and lean pigs(Large White).And then we reported the novel splice variant,the genomic DNA sequence and the core promoter of CIDEb in pig. Finally,eukaryotic and prokaryotic expression vectors were constructed through molecular cloning technologies and a target gene was overexpressed in a cell line through cell culturing and gene transfection,the expression regulation of these genes by some factors was conducted using IB cells.The main results are as follows:1.Three CIDEs were cloned using the techniques of RT-PCR.Sequence analysis shows that both of CIDEa and CIDEb contain open reading frame of 660 bp,which encode 219-amino acids,and CIDEc contains a coding region of 717 bp,which encodes 238-amino acids.Porcine CIDEa deduced amino acid sequence shows a similarity to human(80.5%) and to mouse(75.8%);Porcine CIDEb deduced amino acid sequence shows a similarity to human(85.8%) and to mouse(79.1%);Porcine CIDEc shares 82.8%and 73.2%amino acid identity with human and mouse, respectively.2.Primers designed according to the sequences of CIDEs cDNA from porcine,and the sequence of mRNA,genomic DNA from human.Genomic fragments of genes were obtained by PCR of porcine genomic DNA,two intron sequences and two genomic sequences were amplified.Then primers were designed according to the sequences of these introns to map these 3 genes using radiation hybrid(RH) technique. The 3 genes were mapped on 3 chromosomes.Among them,CIDEa,CIDEb and CIDEc were located to chromosome 6q21-26,7q21 and 13q31(all LOD＞5), respectively.And the positions of these genes are corresponding to that in human.3.Tissue expression patterns were analyzed by the method of Semi-quantative RT-PCR in 11 porcine(Meishan) tissues including adipose(subcutaneous back fat), spleen,lung,kidney,liver,small intestine,brain(cerebellum),stomach,muscle (longissimus dorsi),heart,lymph(neck lymph node).The results showed that all the members of porcine CIDEs family mRNA are highly expressed in white adipose tissue,except this,CIDEa mRNA was expressed at relatively high levels in porcine lymph(although the contribution of white blood cells cannot be excluded) and kidney, but at lower levels in spleen,liver and heart.Weak expression of CIDEa was detected in small intestine and muscle.CIDEb mRNA level was relatively high in porcine liver, small intestine and lymph,and was lower in spleen,lung,kidney and brain.But stomach,muscle and heart showed trace expression of CIDEb.CIDEc mRNA level was relatively high in porcine brain,small intestine and lymph,and was lower in kidney and stomach.Spleen,muscle and heart showed weak expression of CIDEc.4.Expression analysis of porcine CIDEs was conducted in tissues of genetically obese pigs(Tongcheng) and lean pigs(Large White).Semi-quantitative analysis indicated that the mRNA level of both CIDEa and CIDEc in white adipose from obese pigs was significantly higher than their lean counterparts,similar but less obvious difference was also present in liver tissue.However,CIDEb mRNA level in white adipose from obese pigs was lower than in their lean counterparts.5.Alternative splicing of the CIDEb gene was also identified by reverse transcription PCR,revealing that two transcripts,CIDEb and CIDEb2 were present in pigs.CIDEb comprised a full-length open reading frame with 219 amino acids. CIDEb2 encoded a protein of 143 amino acids,it is generated by a deletion of 191 bp from the wild type CIDEb.CIDEb variant 2 was detected in the above tissues except stomach,although the expression levels are lower than CIDEb.A search of human, mouse and rat EST databases and mRNA databases indicated that the novel splice event of CIDEb may occures exclusively in pigs,and have not detected in human and rodents.6.Analysed the CpG island and promoter of CIDEb genomic sequence by Bioinformatics,and forecast the transcription factor recognition sites of CIDEb core promoter,such as PPAR/RXR,NF-κB,CREB,SREBP,HNF-4α,KLF15 and HMGA family and so on.Then we constructed several vector of CIDEb's promoter,and transfected the P-CIDEb-pEGFP-N1 into IB cells,to illustrate the transcriptional activity results of CIDEb promoter from fluorescent photography.7.It is effective to overexpress CIDEb in IB cell line through transfection with lipofectamine.We constructed several expression plasmid for full length CIDEb by molecular cloning technologies(eukaryotic expression vectors CIDEb-pIRES-EGFP, CIDEb-PcDNA3.1(-),and prokaryotic fusion expression vectors CIDEb-PGEX-4T-1),and transfected the CIDEb-pIRES-EGFP into IB cells to make sure it is usable,to prepare for the last function research.