Cloning Of The Four Candidate Imprinted Genes In The Pig DLK1-DIO3 Domain And Their Monallelic Expression Analysis

Genomic imprinting in mammals refers to a form of epigenetic gene regulation that results in gene expression from a single allele in a parent-of-origin-dependent manner.Evidence is mounting that the majority of imprinted genes in mammals play a significant role in many aspects of embryonic growth and development,adult behavior,and postnatal development by means of affecting suckling and metabolism.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.The evolutionarily conserved imprinted domain,DLK1-DIO3,has become the focus of particular interest because the callipyge(CLPG) mutation in sheep causes muscular hypertrophy, and exhibits polar overdominance.The polar overdominance mode of inheritance is also present in the pig chromosomal region that is homologous to the CLPG locus in sheep.It is reported that genes in DLK1-DIO3 imprinted domain have important roles in muscle growth and fat deposit.In this study,we cloned and analyzed the imprinted status of 4 candidate imprinted genes in DLK1-DIO3 domain with RT-PCR-RFLP or sequencing directly in the F1 hybrids generated with reciprocal crosses between Large White and Meishan breeds,or Landrance and Rongchang pigs.Secondly, relative RT-PCR was adopted to analyze the tissue-specific distribution of the 4 candidate imprinted genes.Additionally,a three-generation resource population of the Large White and Meishan breeds, along with 3 genetic markers on chromosome 7(SSC7),were used to map RTL1 and DIO3 by two-point and multipoint linkage analyses.The main results are as follows:1.We obtained the cDNA sequences of the 3 genes with electronic cloning and comparative genomic technology.(1)Two alternative splicing isoforms pDLK1-B(1044bp) and pDLK1-C2 (978bp),were found expressed in one-month-old pigs,of which pDLK1-C2 was deposited into Genebank under accession number EU597631.Amino acid sequence analysis showed that the DLK1 protein shares 90%,86%,81%and 82%identity to the cattle,human,mice and rat homologies, respectively.(2) A 1383 bp MEG3 sequence was obtained and submitted to GeneBank under the accession number EF468461.Putative open reading frames(ORFs) predicated by six frames at NCBI for the sequence revealed there was no more than 282 nucleotides(74 amino acid residues) and no Kozak consensus sequence in initial ATG region.Sequence analysis demonstrated that the MEG3 shared 78%,80%and 81%identity to the human,sheep and mice homologies,respectively. (3) The genomic DNA and cDNA of complete coding sequence of 4293 bp were obtained for porcine RTL1.The sequences have been submitted to the NCBI database under the accession numbers EU781030 and EU781029.The cDNA contains a 4074 bp ORF,encoding a putative polypeptide of 1357 amino acids,and shares 83%,82%,77%and 82%identity to the human,cattle, mice and sheep homologies,respectively.These results indicate that DLK1,MEG3 and RTL1 are conserved in mammalians.2.Using the model of F1 hybrids generated with reciprocal crosses between Large White and Meishan breeds,or Landrance and Rongchang breeds,we identified the imprinted status of the 4 genes through RT-PCR-RFLP analysis and sequencing.(1) The two alternative splicing isoforms of DLK1 gene were all paternally expressed in the liver,heart,tongue,muscle,lung,spleen,fat,small intestine,stomach,and kidney in two-month-old pigs.(2) The MEG3 gene was maternally expressed in the liver,heart,tongue,muscle,lung,spleen,fat,small intestine,stomach,and kidney in one-day-old pigs.(3) The RTL1 gene was paternally expressed in skeletal muscle,heart,spleen,liver, kidney,lung,stomach,fat,small intestine and brain in 1-,30-,60-day-old pigs.(4) RT-PCR showed that the DIO3 gene was only expressed in the skeletal muscle,heart,spleen,lung,stomach,and brain of two-month-old pigs.Imprinting analysis demonstrated that only paternal allele of the DIO3 gene was expressed in the tissues detected.These observations suggest that the imprinting status of the 4 candidate genes is well kept during development in pigs,and confirm the conservation of genomic imprinting in DLK1-DIO3 cluster across mammals.3.In order to investigate the tissue-specific distribution of the 4 candidate imprinted genes, relative RT-PCRs were performed to measure gene expression.(1) The expression levels of DLK1 in one-month-old pigs across tissues were significantly different(P＜0.01).The lung and skeletal muscle had the highest DLK1 transcript abundance,followed by the tongue,spleen,and fat,which had significantly greater transcript abundance than the heart and liver did(P＜0.05).(2) The expression of MEG3 was significantly different(P＜0.01) among different tissues in one-month-old pigs.Brain and lung had the highest MEG3 transcript abundance(P＜0.01),followed by tongue and spleen,which had significantly greater transcript abundance than small intestine(P＜0.01 and 0.05, respectively).(3) The porcine RTL1 was expressed in the skeletal muscle,liver,heart,lung,spleen, kidney,small intestine,stomach,and brain of 1-day-old piglets regardless of expression levels; however the expression levels did vary by tissues.The liver and spleen express much lower levels relative to other tissues.(4) In 15-day-old F1 pigs,the transcript abundance of DIO3 was greatest in the brain,definitely detectable in the muscle,stomach,lung and spleen,rather low in the heart,and undetectable in the liver,kidney,and small intestine.These results demonstrate that the expression levels of the 4 candidate genes in porcine DLK1-DIO3 domain are significantly different among different tissues.4.For the purpose of gene mapping,the two-point and multipoint linkage analyses with CRIMAP were performed to map RTL1 and DIO3 genes.The results showed that the two genes were linked(recombination fraction=0.29 and LOD=9.71),and were mapped with microsatellites SW252,SW581 and S0212 on SSC7 in the following order(Kosambi cM;sex-average values): SW252-38.2-SW581-16.3-DIO3-15.2-S0212-17.9-RTL1.