Screening And Functional Analysis Of Important Genes For Intramuscular Fat Deposition In Buffaloes

Beef is popular because of its rich nutrition.In China,beef is mainly from cattle and continues to be in short supply for a long time,which needs to be imported to meet the national consumption.In recent years,the role of buffaloes has gradually changed from the past draught power into milk and meat source.However,the intramuscular fat(IMF)content is very low due to the long-term breeding for draught power,which results in poor taste of buffalo meat.The flavor and taste are mainly infected by the IMF content,as well as the quality and the price of beef.Therefore,exploring the inherent genetic factors and molecular regulation mechanisms that affect IMF deposition of buffalo can provide an important theoretical basis for improving the quality of buffalo,and further alleviat the pressure of beef consumption in China.In order to reveal the inherent reason for the low IMF content in buffalo,genes expression patterns of the longissimus dorsi muscle and back fat tissue of Xinyang buffalo and Nanyang cattle were analyzed,functional enrichment analysis was performed for the differentially expressed genes in adipose and muscle tissues,the important pathways that affect IMF deposition were analyzed to screening the IMF deposition-related genes,and the functional identification and promoter activity of IMF-related genes were analyzed at cell level.The molecular mechanism that results in the differential expression of IMF between buffalo and cattle was preliminary explorated,which provided a theoretical basis for improvement of the meat quality in buffaloes.The main findings are as follows:(1)The longissimus dorsi muscle area(LMA),thickness of back fat and IMF content of 30-month-old Nanyang cattle were higher than those of 30-month-old Xinyang buffalo.The LMA of Nanyang cattle and Xinyang buffalo buffalo were 70.71±11.19 cm~2and59.61±8.34 cm~2,respectively.The backfat thickness of cattle(4.03±0.81 cm)was higher than that of buffalo(1.03±0.24 cm)(p<0.05),and the IMF content of cattle(2.43%)was also higher than that of buffalo(0.51%)(p<0.05).(2)Through RNA sequencing analysis,18,713 and 18,535 m RNAs were identified in buffalo and cattle,respectively.In buffalo,a total of 369 muscle-specific m RNAs,17,906muscle and adipose co-expressed m RNAs,and 438 adipose-specific m RNAs were identified.In cattle,a total of 97 muscle-specific m RNAs,18,220 muscle and adipose co-expressed m RNAs,and 218 adipose-specific m RNAs were identified.(3)Differential expression analysis was performed for the muscle and adipose co-expressed genes between buffaloes and cattle.In total,1,566 differentially expressed(DE)m RNAs were identified,including 1,143 up-regulated m RNAs in cattle muscle compared to buffalo,and 423 m RNAs were down-regulated in cattle muscle.Differential expression analysis was performed for the muscle-specific expressed genes between buffaloes and cattle.In total,70 differentially expressed genes,including 35 m RNAs were up-regulated in cattle muscle compared to buffalo,and 35 m RNAs were down-regulated in cattle muscle.(4)GO and KEGG analysis were performed by the 70 muscle-specific expressed DE m RNAs.A total of 5 GO entries were enriched,all of which were related to the development of muscle tissue.At the same time,GO and KEGG analysis were performed by the 1,566 m RNAs co-expressed in muscle and adipose tissue and differentially expressed in in buffalo and cattle.A total of 41 GO and 7 KEGG were enriched,including the glycolysis/glycolysis pathway,and 17 differentially expressed genes as PCK1(Phosphoenolpyruvate Carboxykinase 1)were included.(5)The tissue expression analysis was performed for PCK1.Results showed that PCK1 was expressed in adipose and muscle tissue,but the expression in adipose was significantly higher than that in muscle tissue.In muscle,the expression of PCK1 in cattle was higher than that in buffaloes.While in adipose,the expression of PCK1 gene in buffaloes was higher than that in cattle.In the longissimus dorsi muscle,the content of IMF was positively correlated with the expression of buffalo PCK1.(6)Effects of PCK1 on adipogenic differentiation of buffalo adipocytes were identifed.Results showed that overexpression of PCK1 promoted lipid accumulation in buffalo adipocytes and the expression of adipogenic markers FABP4(Fatty Acid Binding Protein4)(p<0.01)and triacylglycerol synthesis gene DGAT1(Diacylglycerol-O-Acyltransferase Homolog 1)(p<0.01).(7)The results of the dual-luciferase reporter gene assay system showed that the core promoter region of the PCK1 gene of buffalo(cattle)was located between 23 bp and 406bp(402 bp)upstream of the transcription start site.In C2C12 cells and bovine myoblasts,the activity of buffalo PCK1 promoter was lower than cattle;on the contrary,in 3T3-L1adipocytes and bovine adipocytes,the activity of buffalo PCK1 promoter was higher than cattle.In summary,the gene expression patterns in the muscle and adipose tissues of buffalo and cattle were detected by transcriptome sequencing analysis in this study.Differential expression analysis and functional enrichment revealed that was differences in glycolysis/gluconeogenesis pathway between buffalo and cattle muscle tissues.Further gain-of-function experiments showed that the PCK1 gene promoted adipogenic differentiation of buffalo adipocytes.The results indicate that PCK1 gene is a promising candidate factor invoving in the regulation of IMF deposition in buffaloes.These results provide a theoretical basis for further research on the IMF deposition in buffaloes.