Cloning, Identification And Characterization Of The Differentially Expressed Genes Between Longissimus Doris Of F1 Large White×Meishan Pigs And Their Female Parents Meishan Pigs

Skeletal muscle comprises as much as 45%-60% of a domestic animal's body mass. Therefore the development, biophysiology and metabolism of this tissue are relative to the muscle traits in animal breeding. When two breeds with different traits are crossbred, F₁ hybrids always show heterosis and have better muscle traits. Many hypothesis have been applied to but none of them could explain the heterosis phenomenon perfectly. Further understanding on molecular genetics reveal that the trait difference and heterosis actually are the results of gene differential expression. To study the molecular mechanism, forward and reverse subtracted cDNA libraries between longissimus muscle from F₁ hybrids Large White×Meishan and female parents Meishan, F₁ hybrids Large White×Meishan and male parents Large White was constructed by suppression subtracted hybridization (SSH) technique, and the differentially expressed genes were cloned and identified.The results are as follows:1.G3PDH, a housekeeping gene, was used to assess the subtracted efficiency of every subtracted library.In each subtracted cDNA library, G3PDH was subtracted efficiently at more than 2⁵ folds and some even more than 2¹⁰ folds, revealing that differentially expressed genes were also enriched at the same folds and the subtracted libraries were successful.2.More than 500 clones were randomly picked out from each subtracted library.By PCR analysis, 610 and 580 positive clones were isolated from the subtracted libraries in which Large White×Meishan as tester, Meishan as driver and Meishan as tester, Large White×Meishan as driver, respectively. Most of the insert fragments were 150-1000 bp.3.Reverse Northern Blot was carried out by using forward subtracted cDNA and reversal unsubtracted cDNA as probes. In the subtracted library by Large White×Meishan as tester and Meishan as driver, 52 differentially expressed clones were obtained. Further analysis showed they represent 41 ESTs: 32 are known and 9 have no homology from NCBI. In the subtracted library by Meishan as tester and Large White×Meishan as driver, 47 differentially expressed clones were obtained. Further analysis showed they represent 36 ESTs: 28 are known and 8 have no homology from NCBI. Some of these ESTs were subjected to further identification by semi-quantitative RT-PCR analysis.4.Using in silico clonging and SMART technology, we obtained 7 full-length cDNA and 3 partial full-length cDNA: (1)PPP1CB (Protein phosphatase 1, catalytic subunit, beta isoform), GenBank accession number DQ396471, full-length cDNA 2759 bp, ORF 984 bp; (2)SPC12 (Signal peptidase 12 kDa subunit), full-length cDNA 818 bp, ORF 309 bp; (3)SLN (Sarcolipin), GenBank accession number DQ456976, full-length cDNA 727 bp, ORF 96 bp; (4) SCYE1 (Small inducible cytokine subfamily E, member 1), full-length cDNA 1107 bp, ORF 963 bp; (5)RPL5(Ribosomal protein L5), full-length cDNA 1059 bp, ORF 894 bp; (6)COX7C (Cytochrome c oxidase subunitâ…¦c) has 2 transcripts of different lengths: COX7C1 (Cytochrome c oxidase subunitâ…¦e transcript 1), GenBank accession number DQ456972, full-length cDNA 463 bp; COX7C2 (Cytochrome c oxidase subunitâ…¦c transcript 2), GenBank accession number DQ456973, full-length cDNA 427 bp, but they have the same 309-bp-length ORF; (7)DOC1 (Downregulated in ovarian cancer 1), partial full-length cDNA 3140 bp containing full CDS and 3'end, ORF 2682 bp; (8)EIF4E (Eukaryotic translation initiation factor 4E), full-length cDNA 918 bp containing full CDS and 5'end, ORF 738 bp; (9)ZA20D2 (Zinc finger, A20 domain containing 2), GenBank accession number DQ492686, cDNA 2346 bp containg full CDS and 5'end, ORF 642 bp. PPP1CB, SPC12, SLN, RPL5, DOC1, ZA20D2 demonstrated high-level expression in Meishan pigs. SCYE1, COX7C, EIF4E demonstrated high-level expression in Large White×Meishan pigs.5.Using ORF Finder, CLUSTAL W, CDD, Signal P3.0, Prosite and other sofeware, we analyzed the gene structure, protein structure, conserved domains of genes PPP1CB, SPC12, SLN, RPL5, DOC1, ZA20D2, SCYE1, COX7C, EIF4E. In addition, the corresponding phylogenetic trees were constructed.6.The tissue expression profile was performed in different tissues (heart, liver, spleen, lung, kidney, adipose tissue, testis, uterus, embryo, ovary, longissimus muscle) by semi-quantitative RT-PCR analysis. The expression levels of COX7C1 were much higher than that of the shorter transcript COX7C2 in almost all tissues.7. Full genomic sequences of COX7C, SLN, SPC12 and partial genomic sequences of PPP1CB, SCYE1 were obtained using PCR-Walking, and their polymorphisms were analyzed in different pig breeds. (1)SPC12, 1651bp, containing 3 introns and 4 exons. (2)COX7C, 2150bp, containing 1 introns and 2 exons. In whole genomic sequence, 7 SNPs were identified, of which 5 are transition mutations, 1 is transversion mutation and 1 is deletion mutation. (3)PPP1CB, 391-bp-length and 721-bp-length fragments at 3'UTR. 7 SNPs were identified and all of them are transition mutations. (4)SCYE1, 418bp. 7 SNPs were identified, of which 5 are transition mutations, 2 are transversion mutation. (5)SLN, 615bp, which containing the only one exon and partial 5'and 3'UTR. 2 transition mutations were identified.8. Only two genotypes of Rsa I polymorphism of PPP1CB gene were detected in Meishan, Large White, Landrace and Large WhitexMeishan groups. The frequency of allele A was 0.96 and 0.72 in Meishan group and Large White group, respectively; and the frequency of allele B was 0.87 in Large White×Meishan group.