| Pork is the main source of animal protein for human beings. With the development of life level, people put forward higher requirements for the flavor. But the meat quality was declined as long-term pursuit of growth rate in the breeding. Many candidate genes of growth, backfat thickness and meat quality traits were isolated, and a large number of molecular markers were identified. But a large number of molecular markers and the effects of interactions between multiple genetic markers have yet to be studied further. In this study, we measureed growth rate, backfat thickness and meat quality traits, and screened candidate genes by analyzing the effect of candidate related to growth, rate, backfat thickness and meat quality. In addition, we isolated the porcine MUSTN1 and MASTR genes, which related to muscle development and growth by comparative genomics and modern molecular biology, and analyzed the characteristic, mapping, expression, SNP and its association with economic traits and preliminarily studied the function of MASTR. The main results were listed as follows:1. There exised large variation of the phenotypic values of age at 100 kg, average backfat and intramuscular fat content (IMF) by analysis the performance of the experimental population, it indicating that the performance could be future improved by combining traditional breeding and molecular breeding methods. The phenotypic correlation analysis indicated that backfat thickness related with the trait of age at 100kg (except for backfat at the shoulder). High phenotypic correlation was also observed between IMF and others meat quality traits.2. The effect of signal gene between candidate genes in growth rate (IGF2, JHDM1A), backfat (FTO, COPB1, CMYA2, ANK1 and PSMC5) and meat quality (H-FABP, MASTR., CAST, UCP3 and MUSTN1) and economic traits were analyzed by using generalized linear model. The results indicated that IGF2 and FTO gene have a significant association with 100 kg at age (P<0.05); JHDM1A and ANK1 gene have a significant association with backfat at the shoulder (P<0.05); FTO, PSMC5 and ANK1 gene have significant or very significant association with backfat at the last rib (P<0.05/P<0.01); PSMC5, CMYA2 and ANK1 gene have significant or very significant association with average backfat (P<0.05/P<0.01); H-FABP and MASTR gene have significant association with IMF (P<0.05); MYOD1 gene was highly significant association with muscle pH score (P<0.01) and significant association with drip loss (P<0.05); H-FABP gene have significant association with water losing percentage (P<0.05); In the present study there no significant association with meat quality about CAST and MUSTN1 gene. 3. The candidate genes for growth traits included IGF2 and JHDMIA, and for backfat thickness included FTO, COPB1, CMYA2, ANK1 and PSMC5. The results of multiple gene combination analysis indicated that CMYA2, JHDMIA and PSMC5 together affected the backfat at the loin; JHDMIA gene and ANK-Bsh1236 I site affected the backfat at the shoulder; FTO, CMYA2 and PSMC5 affected the backfat at the last rib; PSMC5, CMYA2 and COPB1 affected the average backfat. The candidate gene for meat quality traits included UCP3, H-FABP, MASTR, MUSTN1 and MYOD1, among which H-FABP and MASTR affected IMF.4. In this study, the interaction effect of genes on target traits was analyzed by gene interaction model. Association analysis revealed that PSMC5 and FTO gene were significantly associated with the age at 100 kg (P<0.05), PSMC5 and CMYA2 gene significantly associated with the average backfat (P<0.05), PSMC5, JHDMIA and IGF2 gene significantly associated with the backfat at the last rib (P<0.05), PSMC5 and ANK1-Bsh1236 I, CMYA2 and FTO gene significantly associated with backfat at the shoulder (P<0.05), FTO and COPB1 gene significantly associated with backfat at the loin (P<0.05), UCP3 and MYOD1 with drip loss (P<0.05). H-FABP, MYOD1 and MASTR gene significantly associated with muscle pH score (P<0.05).5. The porcine MUSTN1 genes was cloned by ombining bioinformatics and RT-PCR methods, and was submitted to GenBank database, the accession numbers were GU345805, the porcine MUSTN1 gene contained 3 exons, encoding 79 amino acids. The IMpRH was empolyed to determine the chromosomal locations of MUSTN1 genes, the results showed that MUSTN1 gene was assigned to SSC13q12-22 (LOD=5.85), and closely linked to SW1400; The expression level of MUSTN1 genes was detected by the RT-PCR in 6 different tissues, which include heart, liver, spleen, kidney, stomach and skeletal muscle. The result showed that MUSTN1 gene was ubiquitously expressed in all tissues and had a higher expression in skeletal muscle. The SNP of MUSTN1 gene was genotyped in the experimental population which was constructed by our lab and the animal husbandry bureau of the Tongcheng County. By analyzing the association between SNP and economic traits, the results indicated that the c.265C>T mutation of MUSTN1 gene was significantly association with the ADG, percentage of ham, proportion of lean and bone of the ham, meat color (P<0.05).6. The porcine MASTR gene was cloned in this study by using the above methods, and were submitted to GenBank database, the accession numbers were JF957602, JF957603 and JF957604, and MASTR gene including three transcripts (MASTR-a, MASTR-b and MASTR-c), encoding 416,282 and 256 amino acids respectively, MASTR-a transcript encode amino acid sequence contained MEF2 transcription factor binding domain and SAP domain. The IMpRH were empolyed to determine the chromosomal locations of MASTR gene, the results showed that MASTR gene is assigned to SSC6q11-q21 (LOD= 10.82), and linked to SW782. The expression level of MASTR gene was detected by the RT-PCR in 90d embryo stage,5 weeks and adults large white pigs which include heart, liver, spleen, kidney, stomach and skeletal muscle. The results showed that MASTR-a transcript had higher expression in skeletal muscle at all stages. The SNP of MASTR gene was genotyped in the experimental population which was constructed by our lab and the animal husbandry bureau of the Tongcheng County. By analyzing the association between these SNPs and economic traits, the results indicated that the c.18C>T of MASTR gene have significantly association with drip loss and water loosing percentage (P<0.05).7. The 2311 bp upstream promoter of the porcine MASTR gene was obtained and constructed into pGL3 reporter vectors. Eight vectors were obtained and tranfected into pk-15 cells. Through dual-luciferase assay system, the results showed that transcription factors SP1 and MYOD1 were involved in transcriptional activation of MASTR. The over-expression and siRNA experiment showed that MASTR could promote the C2C12 cell proliferation.This study provided new ideas for multiple marker-assisted selection in breeding. The discovery of MUSNT1 and MASTR gene would lay a good foundation on the seledtion of candidate genes in growth and meat quality traits. The preliminary study of MASTR gene provided the basis for the further study of its function muscle growth and development. |
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