Study On Differentially Expressed Genes In The Longissimus Dorsi Of Northeastern Indigenous And Large White Pigs

The main goal of porcine breeding work has been raising growth velocity and feeder transformation efficiency, increasing carcass lean content in the last 30-40 years. However, selection toward leaner carcasses has resulted in a decrease on pork quality. Geneticists are working to improve meat quality traits in pigs using genetic manipulation to control important genes in vivo. It is very important to study RNA expression mode for approaching molecular mechanism regulating pork quality. Traditionary RNA level detection fails to analyse systematicly gene expression. Gene chip technique possesses the advantage of large scale,high-flux,demagnification and parallel handling, which has an important role in analyzing alterations in gene expression across the whole genome.The lean-type Large White breed has lower IMF content and inferior eating quality than the fat-type Northeastern Indigenous. They are ideal experimental animals used to study the difference of meat quality. Previous studies have indicated that characteristics of longissimus dorsi play a key role in determining meat quality. Therefore, the present study constructed differences in the expression profiles of genes in the longissimus dorsi of the two pig breeds by use of high-throughput microarray. Differentially expressed genes associated with meat quality were screened. These genes were investigated by western blot,RNA interference and single-strand conformational polymorphism. It helps to elucidate roundly the biological mechanism of different meat quality.The results are as follows:meat quality traits of the two pig breeds were determined according to evaluation method of pork quality. The result indicated that meat from Northeastern Indigenous pigs was of superior quality to that of Large White animals. Differentially expressed genes in the longissimus dorsi of the two pig breeds were detected by the gene chip technique. Microarray analysis demonstrated the differential expression of 1134 genes of which 401 have a known function. A total of 136 were up-regulated and 998 down-regulated in Northeastern Indigenous compared to Large White pigs. We screened 22 genes that were candidate genes associated with pork quality. Cluster analysis of differentially expressed genes was performed by Cluster 3.0 and Treeview software. The result indicated the transcriptome levels in the longissimus dorsi of 3 Northeastern Indigenous and 3 Large White pigs are similar, respectively. GO analysis of differentially expressed genes was performed by MAS. The result indicated these genes involved in skeletal muscle regeneration,positive regulation of striated muscle development,fatty acid oxidation,actin filament binding and fatty-acid synthase activity. KEGG Pathway analysis was performed using MAS. Five significant pathways were detected, I.E. adipocytokine signaling pathway,fatty acid metabolism,regulation of actin cytoskeleton,glutamate metabolism and MAPK signaling pathway.The gene SCD and FASN that were screened through microarray are associated with fatty metabolism. Protein expressions of SCD and FASN in longissimus dorsi of the Northeastern Indigenous and Large White pigs were detected by western blot. The result indicated protein expression of them were both observed. Protein expression levels of them were obtained compared with the expression ofβ-actin, respectively. The analysis indicated protein expression levels of SCD and FASN between the two pig breeds were both different significantly. The expression of them were both markedly reduced when the Northeastern Indigenous pig was compared with Large White animal (P<0.05). It helps to investigate the contribution to fatty deposition from the two genes.Porcine preadipocype culture system in vitro was constructed. SCD-siRNA was transfected into cell. SCD gene was silenced specificly. SCD mRNA and protein expression level in porcine preadipocytes transfected by siRNA were detected by real-time PCR and western blot. The result indicated SCD mRNA and protein expression was markedly reduced when transfected was compared with non-transfected (P<0.01). It is thus clear that expression of SCD gene in porcine preadipocytes was silenced effectively. It helps to reveal the contribution to fatty depositon from SCD and comprehend molecular mechanism regulating pork quality.Microarray experiment indicated the gene A-FABP involed in fatty acid metabolism,lipid biosynthesis and lipid metabolism. The gene L-FABP contributes to uptake and conveying of fatty acid. It maintains the relative balance of fatty acid metabolism in vivo. Genetic polymorphisms of the two genes were investigated by SSCP and sequencing. The results showed there were three kinds of genotypes. The sequencing results found that the polymorphisms of the gene A-FABP and L-FABP were due to a point mutation in position 3481bp and 1740bp, respectively. The x test showed that the pig populations were in Hardy-Weinberg equilibrium for the polymorphism both in the A-FABP and L-FABP gene (P>0.05). The association analysis between different genotypes and meat quality traits was performed by SPSS 13.0. The results revealed that the polymorphisms of the two genes had both a significant effect on IMF content and marbling (P<0.05). However, no significant conclusion can be drawn concerning other traits (P>0.05). Western blot was carried out to detect protein expression levels of A-FABP in three kinds of genotypes individuals'longissimus dorsi. The result found the expression of A-FABP was markedly reduced when genotype DD was compared with genotype CC (P<0.05). The results ascertained the effect on meat quality traits from the two genes. It helps to establish theoretical basis for selection and genetic improvement of pork quality.