The Molecular Mechanism Of Regulating Tenderness Of Longissimus Dorsi Muscle Of Donkey Was Studied Based On Transcriptome And Metabolome

Tenderness is one of the important indicators of meat quality and is considered to directly affect the quality of meat.Guangling donkey is one of the five fine donkey breeds in China,with the characteristics of good meat quality and rich protein content.At present,there are few reports on the quality of donkey meat,especially in the molecular regulation mechanism of donkey meat tenderness.Therefore,this experiment took Guangling donkey as the research object.Transcriptome sequencing technology and metabonomic sequencing technology were used to analyze the longissimus dorsi muscle of Guangling donkey with different meat tenderness,and preliminary showed the internal material change network of Guangling donkey with different meat tenderness,which provided a theoretical basis for the further study of molecular breeding of Guangling donkey.The main results were as follows:(1)The WBS and IMF content of longissimus dorsi muscle of 30 Guangling donkeys were measured.According to the comprehensive data of WBS and IMF content,the longissimus dorsi muscle of 8 donkeys was selected and divided into two groups,high tenderness group(HT,n=4)and low tenderness group(LT,n=4).These samples were used for transcriptome sequencing and metabolome analysis.(2)A total of 1253 DEGs were detected by RNA-Seq between the HT group and LT group,among which832 genes were up-regulated and 421 genes were down-regulated.The results of GO function enrichment showed that myofibril,contractile fiber contractile fibers,contractile fiber part,icosatetraenoic acid binding,arachidonic acid binding,cytoskeletal protein binding,long-chain fatty acid binding,Hsp70 protein binding,regulation of response to stress,muscle system process,muscle contraction and negative regulation of cellular protein metabolic process and negative regulation of protein metabolic process were involved in the regulation of meat tenderness.KEGG pathway analysis results showed that the s glycolysis/ gluconeogenesis,glucagon signaling pathway,insulin signaling pathway,HIF-1 signaling pathway,pentose phosphate pathway,AMPK signaling pathway,fructose and mannose metabolism,insulin resistance,glycerolipid metabolism,glycerophospholipid metabolism and focal adhesion may affect the meat tenderness of Guangling donkeys.Combined with GO function enrichment and KEGG pathway enrichment,a total of 17 candidate genes related to meat tenderness were screened,including 8 genes related to muscle,namely ANKRD1,ASB2,BDH1,CRYAB,TNNT3,MYL1;11 genes related to intramuscular fat,namely DGAT2,ELOVL6,IGF1,PPARα,PPARγ,LPL,FABP4,SCD,GPAM,PCK1 and HOXC10.The expression of 8 genes was verified by q RT-PCR.(3)A total of 225 DAMs were detected by LC-MC between the HT group and LT group,among which154 metabolites were up-regulated and 71 metabolites were down-regulated.KEGG pathway analysis of DAMs,there were 11 main metabolic pathways,including the purine metabolism,pentose phosphate pathway,histidine metabolism,glycine,serine and threonine metabolism,glycerophospholipid metabolism,glutathione metabolism,Fox O signaling pathways,D-Glutamine and D-glutamate metabolism,citrate cycle,arginine biosynthesis,alanine,aspartate and glutamate metabolism,the results suggest that these pathways may be related to meat quality.In addition,combined with database annotation and KEGG enrichment pathway,43 metabolites related to meat tenderness were screened,including 33 up-regulated metabolites and10 down regulated metabolites.(4)Transcriptome and metabolome combined KEGG analysis showed that there were 57 common pathways enriched to KEGG pathway,among which glycerophospholipid metabolism,pentose phosphate pathway,alanine,aspartate and glutamate metabolism,arginine and proline metabolism and glucagon signaling pathway might be involved in the tenderness regulation mechanism of Guangling donkey(P < 0.1).Through correlation analysis,we found that there were DEGs and DAMs with Spearman correlation coefficient greater than 0.8 in the above five pathways.In the glycerophospholipid metabolism,the metabolites lyso PC(18:1(9Z)),choline,lyso PC(17:0),lyso PC(18:0),glycerophosphocholine and glycerylphosphorylethanolamine were closely related to the PLA2G12 A,DGKE,DGKH and GPAM genes.In the pentose phosphate pathway,the metabolites ribose-1-phosphate and sedoheptulose-7-phosphate were closely related to PFKM,PGM1,GPI,LOC106841113 and LOC106828083 genes.In the alanine,aspartate and glutamate metabolism pathway,the metabolites adenylosuccinate and citric acid were closely related to RIMKLB and DDO genes.In the arginine and proline metabolism pathway,the metabolite creatine was closely related to LOC106837908 and GAMT gene.In the glucagon signaling pathway,the metabolite citric acid is closely related to the PRKAB2 gene.In addition,the top 15 DAMs that had a greater impact on transcriptome data were mainly concentrated in lipids and lipid-like molecules,amino acids and its derivatives,and nucleotide and its metabolites;while the top 15 DEGs that had a greater impact on metabolome data were mainly concentrated in genes related to muscle development and energy metabolism.These results were helpful to further explore the molecular mechanism of meat tenderness regulation,and provided theoretical basis for molecular breeding of Guangling donkey.