Molecular Characterization Study On Candidate Differentially Expressed Functional Gene Of Porcine Btg2 And Its Family Member Genes Of Btg1 And Btg3

BTG2(B-cell translocation gene 2),with properties of anti-proliferative and differentiation speedup,is a very important member of the B-cell translocation gene family.In our previous study,porcine BTG2 gene was found as a differentially expressed gene in different pig breeds from a forward and reverse Suppressive subtractive hybridization(SSH) cDNA libraries of longissimus dorsi muscle between 4-month Meishan and Yorkshire.BTG1 and BTG3 gene,members of BTG2 family genes,have been demonstrated to strongly inhibit myoblasts proliferation and stimulate myoblasts differentiation.Considered them as functional candidate genes associated with meat quality and muscle development,gene expression pattern in porcine muscle different developmental stages and different tissues,gene identification,genetic variation and its association with pig meat quality and production was carried out in this study.1.Using real-time PCR,BTG2 gene expression pattern in the skeletal muscle of 65dpc(day postconception),3dp(day postnatal),21dp,60dp,120dp and 180dp between Meishan and Yorkshire was detected.The expression trend of BTG2 in the six muscle developmental stages in Large white is coincident with Meishan pigs. That's firstly up-regulated and then down-regulated.The expression level of porcine BTG2 was relatively low at 65dpc in both Large white and Meishan pigs beeds,then increased to a peak at 60dp of Large white and at 120dp of Meishan,and then down-regulated in both breeds(p＜0.01).Highly significant expression differences (p＜0.01) between Large white and Meishan at all the stages were observed.BTG1 and BTG3 gnes expression pattern in the skeletal muscle of 3dp,21dp,35dp,60dp,90dp and 120dp btween Tongeheng and Yorkshire pig were also investigated.The expression trend of BTG1 in the six muscle developmental stages between the two breeds is same:it was expressed at 3dp with a relatively high level,and were down-regulated thereafter in both Large white and Tong eheng pigs(p＜0.01).Highly significant expression differences(p＜0.01) of BTG1 between Large white and Tongcheng pig at 3dp,21dp,60dp and 90dp were observed.The expression patten of BTG3 gene in the five muscle developmental stages in Large White is also generally coincident with Tongcheng pigs.That's firstly down-regulated and then up-regulated. Significant expression differences(p＜0.05) of BTG3 between Large white and Tongcheng pig at 35dp,60dp,90dp and 120dp were observed.2.The expression pattern of BTG2,BTG1 and BTG3 gene in heart,longissimus dorsi muscle(LD),semitendinous muscle(ST),uterus,spleen,kidney,fat and liver tissues of adult Yorkshire was detected with RT-PCR.BTG2 gene was expressed predominantly in ST,LD and heart,whereas at a moderate level in uterus,spleen, kidney and at a low level in fat and liver tissues.BTG1 and BTG3 gene were found similar in expression profile in different tissues,these two genes expressed lower in heart and muscle,middle in kidney and fat,and higher in uterus,spleen,and liver.3.Base on differential expressed tags from the SSH cDNA libraries of longissimus dorsi muscle between 4-month Meishan and Yorkshire,combining bioinformatics and RACE methods,full-length cDNA and genomic DNA of porcine BTG2 gene and partial cDNA sequence near 3'-end of BTG1 gene were isolated and identified.The gene structure of BTG2 and its deduced amino acids sequence,protein structure were analyzed using bioinformatics software.From the analysis results, boxA and boxB conserved motifs of BTG/TOB(B-cell translocation gene) gene family was found,and other three protein kinase phosphorylation sites were found too.4.Detection of genetic mutations of BTG1 and BTG2 were performed and the 4 SNPs(Single nucleotide polymorphism) were detected by PCR-RFLP.Among these 4 SNPs,no changing coding amino acid mutation was detected.Two SNPs that can be detected by PCR-RFLP in BTG2 and BTG1 gene were analyzed among different pig populations.In different pig population,Genotype frequencies of BTG1 Bsh1236Ⅰ-RFLP and BTG2 Psp5Ⅱ-PCR-RFLP were significant difference among the detection pig populations.Association between BTG2 Psp5Ⅱ-PCR-RFLP and BTG1 Bsh1236Ⅰ-RFLP genotypes and meat quality traits were determined using the Least Square Analysis of Variance Procedure in a Large White and Meishan resource family F2 population.Statistic analysis revealed that highly significant associations between PCR-Psp5Ⅱ-RFLP genotype and buttock fat thickness,ratio of lean to fat were observed.Significant associations were observed with 6～7 rib fat thickness,fat meat percentage and average backfat thickness.GG genotype of BTG2 gene is positive to increase ratio of lean meat and decrease backfat thickness.Highly significant associations between Bsh1236Ⅰ-PCR-RFLP genotype and loin eye area,intramuscular water percentage were detected.And significant associations were observed with peau percentage,buttock fat thickness,lean meat percentage,Carcass length and intramuscular fat;AA genotype of BTG1 gene is positive to increase ratio of lean meat and decrease backfat thickness.