| Skeletal muscle is the most abundant component of animal body,which account about 40%of the body weight.And the traits associated with growth and development are most important tbr meat livestock production.On the other hand,Chinese indigenous pig breeds and exotic pig breeds are differing in growth rate,meat quality etc.It has been proved by studies that differential expression of genes is associated with such differences. In order to thrther understand the mechanism of skeletal myogenesis and development and elucidate the causes underlying the differences,forward and reverse subtracted cDNA libraries between longissimus dorsi muscle of LargeWhite and Meishan pigs were constructed by suppression subtracted hybridization(SSH) technique.Screening and molecular biological analysis of differentially expressed genes were carried out and the results are as follows:1.Two housekeeping genes,GAPDH andβ-actin,were used to assess the subtracted efficiency of the subtracted libraries.In each subtracted cDNA libraries,the reaction cycle numbers between pre-subtrated and post-subtracted were more than 10 cycles. these means that the subtracted libraries were qualified for further analysis.2.The subtracted cDNA fragments obtained were ligated to pGEM-T vectors and subtracted libraries consisting of 3000(LargeWhite) and 2500(Meishan) clones, respectively,were obtained.By PCR analysis,2739and 2109positive clones were isolated from the subtracted libraries with 91.3%and 84%as positive rate,respectively. Most of the insert fragments were 200-1200bp.3.Dot-blot analysis was carried out for verification with 25%as positive rate.Then 350 and 280 positive clones from Large White and Meishan subtracted libraries were sequenced and the obtained sequences were analyzed by BLAST software.313 high quality sequences were obtained by the subtracted library which LargeWhite as tester. Then these ESTs incorporated into 101 contigs,which representing 70 genes and 5 unknown genes and 4 new ESTs with no similarity with all the sequences of Genbank. while the other 237 sequences in Meishan-as-tester subtracted library were incorporated into 97 contigs which representing 50 known genes and llunknown genes and 5fragments with no similarity with other sequences of Genbank.4.LargeWhite subtracted library had more genes associated with glycolysis than Meishan subtracted library while Meishan library had more genes associated with mitochondrion.5.Further verification for part of the genes were carried out by Real-Time RT-PCR using SYBR Green fluorescent dye with HPRT as internal control.6.One differentially expressed gene,Pyruvate Dehydrogenase Kinase 4(PDK4) was further analyzed.It has been confirmed that PDK4 has a higher expression level in Meishan compared with LargeWhite at the age of 120d.(1) The expression levels of PDK4 at different developmental stages(65day post coitus(dpc),3d,35d,60d,120d.180d after birth) of both pig breeds share similar tendency.Both the two breeds had a highest expression at neonatal(3d after birth),then decreased while the age increased with a significant decrease at 21 d,and decreased to the lowest level at 35d.Afterwards the expression level increased in Meishan,but had a low level at both 120d and 180d.(2) The comparison revealed that PDK4 had a significant higher expression level in Meishan compared with Large White at 65dpc,3d,60d,120d while the difference was not significant at 180d.(3) Semi-quantity RT-PCR was used to analyze the spatial expression of the PDK4 gene.The mRNA of PDK4 can be detected in skeletal muscle with a high level,however, significant low level mRNA was detected in adipose tissue.(4) Using in silico cloning and SMART RACE technology,we obtained the full-length eDNA 3589bp and exon 4(162bp),exon 9(299bp) and exon 10(1696bp).Then a phylogenetic tree was constructed,which revealed that the porcine PDK4 had a closer genetic relationship with the PDK4 of Bos Taurus,than with Gallus gallus,Xenopus tropicalis.(5) Polymorphsiam analysis:A G/A substitution in intron 9 was analyzed in 311 individuals of LargeWhiter xMeishan F2 population using PCR-SSCP.The association analysis results showed that the polymorphism in PDK4 had significant(P＜0.05) or highly dominant significant(P＜0.01) effect on rate of lean to fat(RLF),drip loss rate, water holding capacity,color value of longissimus dorsal muscle(CVLD),muscle fat content and muscle water content(MWC).(6)About 2.3kb promoter sequence was cloned and analyzed.Many transcription factor binding sites associated with muscle development were found.Part of the sequence was relative conserved sequence with a 70%similarity with human sequence.It presumed that this conserved sequence had a important role in the expression of the gene.7.Transducer lof ERBB2(TOB1) was cloned based on the EST differentially expressed muscles between parental Large White and progeny Landrace×LargeWhite, and further analysis was carried out.(1) We obtained the full-length cDNA 2263bp consisting CDS 1224bp which coding 347amino acid(aa).Using in silico cloning and SMART RACE technology with Large White longissimus dorsi muscle SMART cDNA as template.Several AU motifs (ATTTA),might associate with the instability of mRNA,were found in 3’UTR.It indicated that this gene was early responding and functioned in regulation.(2) A phylogenetic tree was constructed,which revealed that the porcine TOB1 had a closer genetic relationship with the primates.(3) Comparative analysis of cDNA in four pig breeds reavealed 6 aa alterations.(4) RNA of TOB1 had a higher expression level in Meishan compared with Large White at 120d.(5) RNA of TOB1 had a higher expression level in Meishan compared with LargeWhite at various developmental stages with a similar tendency which was increasing from 65dpc until 180d,but had a lower expression at 60d.(6) Semi-quantity RT-PCR was used to analyze the spatial expression of the TOB1 gene.The mRNA of TOB1 can be detected in skeletal muscle with a high level,however, significant low level mRNA was detected in adipose tissue. |
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