| Genomic imprinting refers the different expression of paternal and maternal alleles in offsprings. More specifically, some genes only express from their paternal alleles and their maternal alleles keep silence while some other genes express only from their maternal alleles and the paternal alleles keep silence. The gene which has those characters can be called imprinted gene. Imprinted genes generally locate on chromosome in clusters and form an imprinted domain. The majority of imprinted genes plays significant roles in many aspects of embryonic growth, development, adult behavior, meatyield, lean meat percentage and feed efficiency in mammals. In addition, imprinted genes have close relationship with the susceptibility and immunity of some diseases. Therefore, identifying more imprinted genes in pigs and analyzing the conservation of genomic imprinting among different species are helpful to provide the basic molecular biological information for porcine molecular breeding.Many researchs indicated that the human 7q32 and 11p15.5, in synteny with mouse chromosome 6 and chromosome 7, respectively, contain the MEST/PEG1 and CDKN1C/KCNQ1OT1 imprinted domains. Both imprinted domains have significant biological functions and have been reported to be associated with SRS and BWS. Therefore, our studies use comparative biology to speculate two novel imprinted domains in pigs which contain the candidate imprinted genes COPG2, MEST, CDKN1C and NAP1L4 according to their imprinted status and chromosome location in other mammals. We take Rongchang, Landrace and F1 hybrid pigs of Rongchang and Landrace cross as experimental animals and the porcine COPG2, MEST, CDKN1C and NAP1L4 were cloned and then its molecular features were analyzed. Mapping of the porcine COPG2, MEST, CDKN1C and NAP1L4 genes to porcine chromosome were performed with the IMpRH panel. Through the direct sequencing of the RT-PCR products, the imprinting status of the four genes in different tissues of F1 heterozygotes was analyzed. The tissue expression of the four genes was examined by real-time PCR. The main results are as follows:1, We obtained the full-length cDNA sequences of the four genes using PCR and RACE metholds. (1) The 2817 bp COPG2 sequence was cloned and this sequence contains 35 bp 5'UTR, 166 bp 3'UTR and 2 616 bp ORF, which was deposited into Genebank under accession number GU372414. Nucleotide sequence analysis showed that the porcine COPG2 shares 93% and 89% identity to the human and mice, respectively. Amino acid sequence analysis showed that the COPG2 protein shares 96% and 95.5% identity with the human and mice, respectively. (2) A 2 167 bp MEST sequence was obtained and this sequence contains 16 bp 5'UTR,1 222 bp 3'UTR and 981 bp ORF which was submitted to Genebank under accession number HM126537. Nucleotide sequence analysis showed that the porcine MEST shares 84% and 88% identity with the human and mice. (3) The 1 171bp CDKN1C sequence was cloned and this sequence includes 786bp ORF which was deposited into Genebank under accession HQ679903. (4) A 2 655 bp NAP1L4 sequence was obtained and this sequence contains 470 bp 5'UTR,1 024 bp 3'UTR and 1 161 bp ORF which was submitted to Genebank under accession number HQ679902. Nucleotide sequence and amino acid sequence analysis showed that the porcine NAP1L4 shares 84% and 88% identity with the human.2, Using direct PCR sequencing, we identified the imprinted status of the 4 genes in tissues of F1 hybrids of Landrance boar×Rongchang sow or Rongchang boar×Landrance sow. (1) Porcine COPG2 was biallelically expressed in the one-month Fi heterozygote tissues examined. (2) Porcine MEST was paternally expressed in heart, stomach, skeletal muscle, kidney, lung, bladder, tongue and fat of one-month pigs, while biallelically expressed in liver, small intestine and spleen of one-month pigs. (3) Newborn porcine CDKN1C was maternally expressed in heart, tongue, bladder, ovary, spleen, liver, skeletal muscle, stomach, small intestine and placenta, while biallelically expressed in lung and kidney. (4) Newborn porcine NAP1L4 was biallelically expressed in all the tissues examined.3, In order to investigate the tissue-specific distribution of the 4 candidate imprinted genes, real-time PCR were performed to measure gene expression. (1) Newborn porcine COPG2 showed the highest expression level in placenta (P<0.01), followed by the spleen, ovary, heart, skeletal muscle, tongue and kidney (P<0.01) and the lowest expression level was detected in liver, small intestine, stomach, lung and bladder (P<0.01). In the one-month pigs, COPG2 showed the highest expression level in kidney (P<0.01), followed by the liver and the lowest expression level was found in lung, skeletal muscle, liver, small intestine, tongue and spleen (P<0.01). (2) the expression level of newborn porcine MEST was significantly highest in placenta than other tissues (P<0.01) while the highest expression level was detected in kidney of one-month pigs (P<0.01). In addition, the expression level of MEST was gradually decreased according to the development. (3) the expression level of newborn porcine CDKN1C was significantly highest in placenta than other tissues (P<0.01) while the highest expression level was detected in lung and kidney of one-month pigs (P<0.01), followed by the skeletal muscle and heart. The lowest expression level was detected spleen, tongue, bladder, stomach, liver, fat and small intestine. In addition, the CDKN1C showed the highest expression level in placenta on days 30 of gestation than on days 60, 90 and 105 gestation. (4) the expression level of newborn porcine NAP1L4 was significantly highest in placenta than other tissues (P<0.01) while the lowest expression level was detected in liver and stomach (P<0.01). In one-month pigs, the highest expression level was detected in lung, heart and kidney (P<0.01) and the lowest expression level was detected in spleen and tongue. In addition, the NAP1L4 showed the highest expression level in placenta on days 30 of gestation than on days 60,90 and 105 gestation.4, By the IMpRH panel, porcine COPG2 and MEST were assigned to porcine chromosome SSC18ql.2-q2.2 and closely linked with CL365941 with the LOD of 14.32 and 8.5, respectively. Porcine CDKN1C and NAP1L4 were assigned to porcine chromosome SSC2 and closely linked with IMpRH06175 with the LOD of 15.78 and 17.94.Conclusions:The results of mapping and imprinting status confirm two novel imprinted domain on porcine chromosome 18 and 2. In addition, the imprinting status of COPG2, MEST, CDKN1C and NAP1L4 are conservative among mammalian species. |
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