Cloning, Identification And Characterization Of The Differentially Expressed Genes Between Longissimus Doris Of Meishan×Yorkshire F1 Hybrids And Their Parents Meishan Pigs

Skeletal muscle is the most component of animal body. Therefore the development,biophsiology and metabolism of this tissue are relative to the muscle traits in animalbreeding. When two breeds with different traits are crossbred, F1 hybrids often showheterosis and have better muscle traits. In order to understand and exploit heterosis, manyhypotheses have been issued but none of them can explain the heterosis phenomenonperfectly. With the development of molecular biology and genetic, it was revealed thatheterosis is the results of differential gene expression.To study the molecular mechanism, forward and reverse subtracted cDNA librariesbetween longissimus dosi from F1 crossbreeds Meishan×Yorkshire and their parentsMeishan pigs were constructed by suppression subtracted hybridization (SSH) technique,and the differentially expressed genes were cloned and identified. The results are asfollows:1,G3PDH, a housekeeping gene, was used to assess the subtracted efficiency ofevery subtracted library. In each subtracted cDNA libraries, G3PDH was subtractedefficiently at more the 2¹⁰ folds and some even more than 2¹⁵ folds. It was revealed thatgenes differentially expressed were also enriched at the same folds and the subtractedlibraries were successful.2,More than 600 clones were randomly picked out from each subtracted library. ByPCR analysis, 591 and 632 positive clones were isolated from the subtracted libraries inwhich Meishan as tester, Meishan×Yorkshire as driver and Meishan×Yorkshire as tester,Meishan as driver, respectively. Most of the insert fragments were 150—750bp.3,Dot-blot was carried out by using forward subtracted cDNA and reversalunsubtracted cDNA as probes. In the subtracted libraries which Meishan as tester andMeishan×Yorkshire as driver, 27 differentially expressed clones were obtained. Furtheranalysis showed they represented 14 ESTs: 4 are known in pig, 7 are unknown in pig buthave a high similarity with human and other species genes, and 3 have no significantsimilarity with known genes. In the subtracted libraries which Meishan×Yorkshire testerand Meishan as driver, 20 differentially expressed clones were obtained. Further analysisshowed they represented 11 ESTs: 1 is known in pig, 8 are unknown in pig but have ahigh similarity with human and other species genes, and 2 have no significant similaritywith known genes. Some of these ESTs were subjected to further identification bysemi-quantitative RT-PCR analysis.4,Using in silico cloning and SMART RACE technology, we obtained 4 full-length cDNA and 1 ORF cDNA:â‘ MRLC2 (Myosin Regulatory Light Chain 2), GenBankaccession number DQ490129, full-length cDNA 990bp, ORF 519bp;â‘¡HSPC117(Hematopoietic Stem/Progenitor Cells), GenBank accession number DQ508263,full-length cDNA 2015bp, ORF 1518bp;â‘¢BIN1 (Bridging Integrator 1), GenBankaccession number EF117688, full-length cDNA 2001bp,ORF 1305bp;â‘£neuroblastomaras oncogene (H-Ras/N-Ras/K-Ras family), partial full-length cDNA 986bp,ORF 570bp,GenBank accession number DQ325522;⑤PYGM (Glycogen phosphorylase, muscleform), GenBank accession number DQ508264, full-length cDNA 2843bp,ORF2529bp.MRLC2 and HSPC117gene demonstrated high-lever expression in Meishan pigs;BIN1,N-Ras and PYGM genes demonstrated high-lever expression inMeishan×Yorkshire.5,By ORF Finder,GENSCAN,Signal P3.0,ProtParam,Prosite and MEGAsofeware, we analyzed the conserved domains,gene structures and their functions. Inaddition, the corresponding phylogenetic trees were constructed in different species.6,The tissue expression profiles were performed in different tissues (muscle, fat,heart, liver, spleen, lung, kidney, stomach, small intestine, uterus, ovary, testis)by semi-quantitative RT-PCR analysis.7,Partial genomic sequence of porcine BIN1 gene,N-Ras gene and PYGM geneswere obtained by comparing genomic sequence in different species. 3 SNPs were detectedand their polymorphisms were analysis in different pig breeds. And find the lean meatpercentage of genotype AA is 3% more than the genotype BB and the fat meat percentageis lower 5% than genotype AA, so it may be a useful locus for Molecular AssistedSelection of pig breeding.