Isolation,SNPs Detection Of Four Genes Related To Porcine Muscle Growth And Genetics Effect Analysis

The combination of molecular marker-assisted selection(MAS) and traditional breeding techniques is one of the main methods of the swine genetic improvement engineering.As the basis of molecular MAS,fingding the major gene or molecular marker which contributed genetically much to the economic traits is very important With the development of genetic map,QTL mapping and functional genome research of human,mouse and swine,we can use the information of QTL about important economic traits and choose new useful genes associated with the traits among the chromosome region which homologous to human and mouse.Based on this,this study includes two sections. The section one includes two genes coming from Doctor Xie hong tao,by F1 crossbreeds Meishan×Yorkshire and their parents Meishan pigs that were constructed by suppression subtracted hybridization(SSH) technique,and the differentially expressed genes were cloned and identified.The section two is two new genes with important economic performance which were cloned and separated by bioinformatics and biotechnology, which could have the basic information for searching the molecular assistant marker.The main results are as follows:1.Myosin light chain regulatory protein gene(MRLC2)MRLC2 can activited by MAPK,it plays an imporant role in muscle contraction and fibrosis of actin in non-Muscle Cells.1742 bp intron sequence was cloned,and 18 SNPs were detected by sequence comparison.PCR-Hin6-RFLP was developed to detect the C/T polymorphism at 178 site among 170 F₂ generations of Large White×MeiShan.The result shows that extremely significant associations between Hin6-RFLP genotypes and Dressing Percentage(P＜0.01),and sigificant association with marble profiling score BF (P＜0.05).2.Glycogen phosphorylase gene(Glycogen Phosphorylase,Muscle form,PYGM) Glycogen synthase and phosphorylase are two important enzymes to glycogen synthesis and decomposition,which activities can determine glycogen metabolism.Three introns of PYGM were isolated and sequence sizes were 828bp(intron7),480bp(intron8) and351 bp(intron9) respectively.The G/T mutation in exon near the intron7 was detected. The A/G mutation in intron 8 was detected.PCR-TaaI-RFLP was developed to detect the G/T polymorphism among 173 F₂ generations of Large White×MeiShan.The result shows that significant associations between TaaI-RFLP genotypes and lean meat percentage(P＜0.05).PCR-MvaI-RFLP was developed to detect the A/G polymorphism among 163 F₂ generations of Large White×MeiShan and extremely significant associations between MvaI-RFLP genotypes and total skin weight(P＜0.01).total bone weight(P＜0.01) and bone Percentage(P＜0.01).while significantly correlated with skin percentage(P＜0.05).3.Muscle growth factor gene(Myocyte enhancer factor 2——MEF2D)MEF2 which has a specific sequence that can bind to DNA,can bind induction growth factor and on the muscle special promoter conservative part.It is the muscle special gene activation important transcription factor.5102bp of MEF2D sequence was isolated,and a G/A mutation near 5'UTR was found.A PCR-BSP120I-RFLP was developed to detect the G/A polymorphism among 172 F₂ generations of Large White×MeiShan The result shows that significant associations between Bsp120I-RFLP genotypes and meat marbling(m.longissimus Dorsi,LD,P＜0.05),Meat Marbling(m.Biceps Femoris,BF,P＜0.05).4.Serine/arginine-Rich Protein specific Kinase 3(SRPK3) geneAs the target gene of MEF2,SRPK3 was specificly exspressed among different tissues.with the pig SRPK3 Gene function unknown,the related research is very necessary.Electronic cloning and RACE technology were used to clone the full-length cDNA,founding that the full-length cDNA is 2011 bp,ORF 1701bp,which encodes 566 amino acids,the 5'UTR 32 bp and 3'UTR 243bp.5.Swine Serine/arginine-Rich Protein specific Kinase 3(SRPK3) gene bioinformatics analysis using software Clustal X,ORF Finder,SignalP2.0,ANTHEPORT,PSORTâ…¡, TMprep to analyze and predict the SRPK3 structure,the protein SRPK3 structure and function,and system evolution features.At the same time,a molecular phylogenetic tree was built.6.The semi-quantitative RT-PCR was performed to detect the expression pattern of Swine Serine/arginine-Rich Protein specific Kinase 3(SRPK3) gene in different tissues(muscle, fat,heart,liver,spleen,lung,kidney,stomach,small intestine,the uterus,ovary and testis). It is expressed specially in muscle and heart.