Molecular Cloning And Function Analysis Of BTG/TOB, A New Porcine Gene Family Related With Skeletal Muscle Development

Improving production traits of pig is one of the objectives breeders pursuiting.The lean yield and quality is mainly decided by increasing numbers of fibroblast infetal development and postnatal hypertrophy of muscle fibres. Postnatal growth rate ispositively correlated with muscle fiber number forming in fetal and influenced by thebalance between protein synthesis and degradation of muscle proteins. However, themuscle fiber numbers forming in embryo myogenesis are the important key factors foranimal meat production. On the other side, the hypertrophy of muscle fibre relatedwith muscle stress and meat quality decreasing, etc. Therefore, understanding themuscle fiber development is important to efficiently balancing muscle growth rate andmeat quality.Many differentially expressed genes were detected from LongSAGE musclecDNA library constructed with three fetal stages of two swine breeds (Landrace andChinese Tongcheng) in our previous study. We chose BTG/TOB gene family whichsignificantly influenced early muscle fiber proliferation to do cloning, physicalmapping and functional study. The main results are as follows:1. Based on differentially expressed tags of LongSAGE muscle cDNA library,combining bioinformatics and experimental methods, full-length cDNA of the porcineBTG1, BTG2 and BTG3 gene, partial DNA sequence of BTG4 gene and partialcDNA near 3'-end of TOB2 gene were isolated and identified.2. The amino acids of porcine BTG1, BTG2 and BTG3 gene were deduced andthe motifs were predicted with bioinformatics tools. Besides boxA and boxBconserved motifs, several protein kinase phosphorylation sites were also found. Thephylogenetic tree was constructed as well as homology alignment. The results showedthat BTG1, BTG2 and BTG3 obviously clustered three different clades and BTG1 andBTG2 had closer relationship.3. Using RH panel, we assigned BTG1, BTG2, BTG3, BTG4 and TOB2 genes toporcine 5q24-q25, 9p11-q11, 13q47, 9p21 and 5p15 respectively, which are inagreement with comparative mapping results. There are several putative quantitativetrait loci (QTL) for pig backfat thickness, muscle color, muscle pH value, diameter ofmuscle fiber, average daily gain, market weight, percentage of fat, dressing percent,etc on the chromosome regions where these genes mapped.4. The expression level of BTG1, BTG2 and BTG3 genes in heart, stomach, spleen, lung, kidney and muscle of adult Landrace female were detected with RT-PCR.BTG1 and BTG3 genes were found similar in expression profile and expressed low inheart and muscle in Landrace while high in Tongcheng pigs. However, BTG2 geneexpressed high in heart and muscle in both breeds.5. The BTG1, BTG2 and BTG3 gene expression in the fetal skeletal muscle from33dpc (day postconception), 65dpc and 90dpc of Landrace and Tongcheng pig wasdetected with real-time RT-PCR method. The expression trend of BTG1 and BTG3gene were found the same and expressed higher in Tongcheng pig than Landraceamong three fetal stages. Their expression peaked at 33 dpc and significantlydecreased at 65 dpc and at 90 dpc (p＜0.01) in Chinese Tongcheng and no significantdifference in the three stage in Landrace. Porcine BTG2 was expressed at 33 dpc witha relatively low level then increased to a peak at 65 dpc, and was down-regulatedthereafter in Tongcheng pig (p＜0.01). The same expression trend was found from 33dpc to 65dpc, but no significant expression differences in Landrace between 65 dpcand 90 dpc were observed. The relative expression level of BTG2 at 33 and 90 dpcare both significantly higher (p＜0.05) in Landrace than Tongcheng pigs but showedno significant difference at 65 dpc between the two breeds.6. To investigate the role of BTG1, BTG2 and BTG3 in myoblasts differentiation,we assayed the mRNA expression level of these two genes in proliferated and fivedifferent differential stage murine C2C12 cells with real-time RT-PCR method.Myogenin was used as molecular marker to confirme the cells underwentdifferentiation. The result showed that the expression trends of BTG1 and BTG3genes were completely coincide with myogenin while BTG2 gene expressiondisplayed wave trend.7. Identification for mutations in the amplified fragments of BTG1, BTG2 andBTG3 genes were performed and the 4 SNPs were detected by PCR-RFLP. Two SNPslocated in 3'-UTR of BTG1 gene, one synonymous SNP located in the first exon ofBTG2 gene. The SNP located in the fifth exon of BTG3 gene which mutating from Cto T caused amino acid change.8. Allele frequencies of 4 SNPs that can be detected by PCR-RFLP wereanalyzed among different pig populations and the results showed that the genotypefrequencies of all the loci were significantly different among the different populations.9. The PsuI-RFLP and Bsh1236I-RFLP of BTG1 gene, MvaI-RFLP of BTG2and BTG3 gene were genotyped in a population which constructed by our lab. The analysis of least-square variance was used to employ association between these SNPsand some production traits. The results showed that the PsuI-RFLP genotypes ofBTG1 gene have significant difference on the shoulder backfat, the 6th and 7th ribsbackfat, dressing percent and muscle color. The Bsh1236I-RFLP genotypes of BTG1gene have the effects on the muscle color and intramuscular fat at different extent(P＜0.05). The different genotypes of MvaI-RFLP for BTG2 gene showed significantdifference on the shoulder backfat, muscle color and muscle shear force (P＜0.05) andthe different genotypes of MvaI-RFLP for BTG3 have the significant difference onthe muscle shear force (P＜0.05).10. The promoter sequence region upstream of porcine BTG1 gene wassuccessfully cloned and four recombinant pGL3 vectors were constructed. Wepredicted the structure and probable transcriptional factor sites (TFS) of the promotersequence. The TFS influence cell cycle or myoblast development such as Foreheadfamily, AP-1, SP1, Myod, Pax family were found, which indicated porcine BTG1gene acting important role in muscle fiber development.Results in this study suggest that BTG gene family influence significantly onearly skeletal muscle development. Further study on the mechanism of thesecandidate genes associated with skeletal muscle development will contribute toporcine molecular breeding and make help to important meat production.