Isolation, Imprinting Identification And Methylation Analysis Of Imprinted Genes In Pigs

Imprinted genes are the genes which expression is parent-dependent that is only one allele from the father or mother is expressed and the other allele from the other parent is not expressed or litter expressed.The researches on genomic imprinting in mammals show that imprinted genes have important roles in the regulation of fetal growth, development,function of the placenta and postnatal behavior.At present,most studies on genomic imprinting are in human and mouse but little in livestock.Therefore,it is of interest to identify more imprinted genes in pigs for analyzing the conservation of genomic imprinting among different species or studying the function of imprinted genes.In this study,we analyze the imprinted status of 4 candidate imprinted genes with RT-PCR-RFLP or sequencing directly in the F₁ hybrids of Large White boar×Meishan and Meishan boar×Large White pigs.Secondly,PCR-RFLP analysis is used to detect the association of the polymorphism in 2 candidate imprinted genes and the carcass traits in the pigs of Large White×Meishan F₂ hybrids.Thirdly,methylation analysis was applied in the CpG islands of 2 candidate imprinted genes in various tissues.At last,we choose candidate imprinted genes with the method of AMD(allelic message display) in the skeletal muscle of 2-month-old Large White,Meishan,F₁ hybrid of Large White boar×Meishan and Meishan boar×Large White pigs,imprinting analysis of the displayed bands was applied with the "expressed SNP".The main results are as follows:1.We choose ASCL2,NNAT,DIRAS3 and DLX5 genes as candidate imprinted genes in pigs according to the imprinted status of the genes in human and mouse and their biological functions.We obtained the complete open reading frame(ORF) of the 4 genes with electronic cloning and comparative genomic technology.(1) ASCL2,obtained cDNA sequence 1654 bp,the ORF is 582 bp,GenBank accession number DQ666420.(2) NNAT, obtained DNA sequence 2234 bp,GenBank accession number DQ666422,the gene has two alternative transcripts and the difference of the two transcripts is the exon 2.(3) DIRAS3,obtained cDNA sequence 1227 bp,the ORF is 687 bp,GenBank accession number DQ666421.(4) DLX5,obtained DNA sequence 4108b,the ORF is 687 bp, GenBank accession number EU168272.The porcine cDNA sequence of the 4 genes has high sequence similarity with human than with mouse.From the phylogenetic relationship,the distance between pigs and human is much shorter than the distance between pigs and mouse.(2) Using the model of F₁ hybrids of Large White boar×Meishan sow and Meishan boar×Large White sow,we identified the imprinted status of the 4 genes in 13 different tissues of 12 2-month-old pigs in the model through RT-PCR-RFLP analysis and sequencing directly.(1) ASCL2 gene escapes genomic imprinting in skeletal muscle,fat, heart,liver,spleen,lung,kidney,stomach,small intestine,uterus,ovary and pituitary.(2) Both of the transcripts of NNAT gene is paternally expressed in skeletal muscle,fat,heart, liver,spleen,lung,kidney,stomach,small intestine,uterus,ovary and pituitary.(3) DIRAS3 gene is paternally expressed in skeletal muscle,fat,heart,liver,spleen,lung, kidney,stomach,small intestine,uterus,ovary and pituitary.(4) DLX5 gene is maternally expressed in skeletal muscle,fat,spleen,lung,stomach,small intestine,but escapes imprinting in heart,liver,kidney,uterus,ovary,testicle and pituitary.(3) Using PCR-RFLP,we detected SNPs of 2 genes in different pig populations and observed associations with carcass and meat quality traits in Large White and Meishan F₂ hybrids.The results showed:(1) ASCL2,C1556G-MspI-RFLP is higher significant association with lean meat percentage,and ratio of lean meat vs.fat meat(p＜0.01),high significant association with internal fat rate,LD pH and BF Ph(p＜0.05).(2)DLX5, C300T-HhaI-RFLP is higher significant association with 6～7 rib fat thickness, thorax-waist fat thickness,buttock fat thickness,average backfat thickness,fat percentage, carcass length and bone percentage(p＜0.01).4.The paternal allele methylation of all the CG sites in exon 1 of DIRAS3 gene are all low,but each tissue has its character:for example,the specifical CG sites are methylated in skeletal muscle and liver,CG¹⁹ sites and CG³⁵ sites are methylated in skeletal muscle,CG²⁵ sites and CG³⁴ sites are methylated in liver.The 41 CG sites are not methylated in testicle.The methylation level of all the clones is much different in uterus, some clones have much high methylation level,but other clones have much low methylation level.The CG sites are stochastic methylation in spleen and kidney.All the CG sites are not methylated in the CpG island of DLX5 gene.5.We obtained 9 displayed bands which are expressed in a parent and a filial generation but not expressed in the other parent and the other filial generation with compined amplification of 24 arbitrary primers and 12 anchor primers.Imprinting analysis with "expressed SNP" of the displayed EST from the AMD showed that ACTN3 gene was not imprinted in pigs.For CA3 gene,the expression patterns showed 4 aspects: CA3 gene may be imprinted in ovary,the hybrid pigs of Large White boar×Meishan expressed the allele G which is originated the mother,the hybrid pigs of Meishan boar×Large White expressed the allele A which is also originated the mother.CA3 gene are bialleic expressed that is escaping imprinting in lung and liver.It is expressed the allele of Large White pigs in skeletal muscle,fat,uterus and testicle,the hybrid pigs of Large White boar×Meishan expressed the allele A which is originated the father,whereas the hybrid pigs of Meishan boar×Large White also expressed the allele A which is also originated the mother.The expression pattern is different in liver and spleen,the hybrid pigs of Meishan boar×Large White expressed allele A in liver whereas the hybrid pigs of Large White boar×Meishan are biallelic expressed in liver,the hybrid pigs of Meishan boar×Large White are biallelic expressed in spleen whereas the hybrid pigs of Large White boar×Meishan expressed the allele A in spleen.