| Animal blood loss and hypoxia caused by slaughter will lead to a series of biochemical changes,and the storage temperature and time are the direct factors affecting muscle metabolism and meat quality.In order to fundamentally clarify the molecular mechanisms that regulate and identify meat quality,we used the Trizol method to extract total RNA of longissimus dorsi muscle of Hengshan goat meat under different three cryopreservation conditions that were freezing,ice-temperature and cooling conditions.Then,the total RNA digested with DNaseI was used for transcrip to mics library construction and detection,and the Illumina HiSeq was used for high-throughput sequencing.Clean reads filtered from raw data were compared with goat genome sequences to predict new transcripts and analyze known and new genes.The differential genes selected need to be further explored and analyzed,such as clustering analysis,GO annotation and KEGG enrichment.Thus,different genes and important pathways related to meat quality under different time and temperature were explored,and the marker genes that can monitor length of cryopreservation time were identified.The specific research results were as follows:(1)Transcriptome study of mutton during frozen storage for 4 days.There were 39 continuously changing genes identified during frozen storage for 0,2 and 4 days,which including 3 differentially up-regulated genes and 36 down-regulated genes.The three up-regulated genes were LOC102176897,LOC102191753 and a new gene BGI_novel_G001185.The FPKM value and storage time of LOC 102176897 and LOC102191753 can be fitted into the mathematical equations:Y=0.32 X-0.323(R~2=0.9999)and Y=0.154X~2-0.135 X-0.01(R~2=1),respectively.Two genes expected to be used as a potential biomarker to monitor the length of frozen storage time.The 36 differentially down-regulated genes mainly caused the changes of lamb tenderness,pH value,water retention and other qualities by participating in oxidative phosphorylation and cell apoptosis.Gene PRKCH,PRKD2,RIPK1,CHP2,and PLK3 were positively regulated the phosphorylation process,while Gene SMAD7 and WTIP inhibited phosphorylation process.Gene SLC7A6,ADGRG1,CMTM3,NOS3,PIEZO 1 and CX3CL1 were associated with cell membrane function.Besides,genes related to meat tenderness were identified,including MYH8,MYH9,SEC14L1 and KIF3C.GO annotation results showed that mitochondrial function,cell membrane function,cellular respiration and cytochrome all changed after lamb was slaughtered.KEGG enrichment results showed that the differentially expressed were higher enrichment in oxidative phosphorylation,peroxidosome,nucleotide shear repair and disease-related pathways.In summary,postmortem muscle and meat quality were regulated by oxidative phosphorylation,cell apoptosis and death.(2)Transcriptome study of mutton during iced storage for 7 days.There were none continuously changing genes identified during iced storage for 0,2,4 and 7 days.GO annotation results shown that NADH dehydrogenase associated with glycolysis was found during iced storage,which played an important role in color stability.And the actin cytoskeleton pathway associated with post-mortem muscle tenderness had also been identified which was different from frozen storage.KEGG enrichment results showed that protein digestion,fat decomposition and lipid metabolism pathways appeared,and the intermediate metabolites produced in this process affected the color,tenderness and water retention of muscle.(3)Transcriptome study of mutton during during cold storage for 7 days.There were 5 continuously changing genes were identified during cold storage for 0,2,4 and 7 days.Gene LOC102172960 can regulate meat quality by changing heme conformation.The FPKM value and storage time of gene LOC102172960,LOC102178051,FAM110D,SEC14L1 and LOC108633460 can be fitted as the mathematical equation Y=0.6757 X~2-8.789 X+29.038(R~2=0.999),Y=0.3844 X~2-4.1977 X+13.949(R~2=0.9871),Y=0.2545 X~2-3.4425 X+12.266(R~2=0.0.9951),Y=0.2881 X~2-3.198 X+8.5374(R~2=0.9766),Y=0.184 X~2-2.5306 X+8.7309(R~2=0.9988),respectively.Five gene can be used as marker gene for monitoring the length of cold storage time of mutton.GO annotation results of different genes in cold storage were basically consistent with frozen and ice storage.KEGG enrichment showed that a series of pathways related to bacterial infection were appeared,which was different from frozen and iced storage.(4)Transcriptome study of mutton at different storage temperatures and the same storage time.There were 31 differentially up-regulated genes and 38 differentially down-regulated genes identified during frozen,iced and cold storage for 2 days,and the up-regulated genes are closely related to phosphorylation and apoptosis.Genes ITSN2,IQGAP2,CSMD2 and SUSHI were involved in the phosphorylation process,and genes ITSN2 and FAT1 were related to apoptosis.Besides,genes linked to meat quality have also been found.By interacting with myo fibrin,TGM5 can improve the gelatability of protein and improve the color,water retention and viscoelasticity of meat products.In the down-regulated gene,gene TPP1 was related to apoptosis,and gene ALAS 1 and CPOX regulated muscle color by affecting heme synthesis.Protein Pexp3,encoded by PEX3,affecting the oxidation of myoglobin by regulating the fat oxidation process,thereby indirectly affected muscle color.There were 5 differentially up-regulated genes and 4 differentially down-regulated genes identified during frozen,iced and cold storage for 4 days.The differential genes were associated with the proliferation,apoptosis and death of muscle cells.The up-regulated gene TFRC encoded a cell surface transferrin receptor associated with cell death.The down-regulated gene ECM1 was related to proliferation,and the down-regulated gene ARMCX1 can inhibit cell cycle and promote cell apoptosis by knocking down in cells.At the same time and at different temperatures,the GO annotation and KEGG enrichment of different genes were similar to the results of frozen,iced and cold storage.(5)Correlation analysis of mutton with three kinds of different cryopreservation.The cor function in R software was used to analyze genetic correlations.We found that FS2 group and FS4 group had a low correlation with CT group(R~2=0.74),but FS2 group had a high correlation with FS4 group(R~2=0.99).CS group,IS2 group and IS7 group all had a high correlation with CT group(R~2?0.92).The correlation between CS7 group and CT group was the lowest(R~2=0.55),followed by CS4 group and CT group(R~2=0.65).Among the three treatments,the freezing treatment had a great influence on the gene expression of muscle,but the gene remained almost unchanged during storaging for 2-4 days.The cooling treatment had lesser influence on the gene expression,but the gene expression was change significant during storaging for 2-7 days.Compared with frozen storage and cold storage,iced treatment and iced storage had less effect on muscle.In general,the iced storage was the best method for mutton storaging 7 days. |
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