| Allium mongolicum Regel and its extract can improve the quality and taste of mutton.In order to explore its mechanism of action,this study used Dorperxthin-tailed Han crossbred sheep as animal model,by adding Allium mongolicum Regelextract to the diet to studiedthe muscle and adipose organizational changes.Firstly,the changes of fatty acid composition in sheep muscle and adipose tissue were measured and compared,and then the two tissues were subjected to whole-genome methylation and transcriptome sequencing,respectively.The bioinformatics method was used to analyze and compare the epigenetic effects of allium mongolicum extract on muscle and adipose of sheep.Finally,combined with some verification results,the epigenetic regulatory network of muscle and adipose tissue of mutton sheep was constructed and systematically analyzed to reveal the mechanism of the influence of allium mongolicum on the quality of mutton.At the same time,it also provides complete gene data for the difference between muscle and adipose tissue of mutton sheep,which was of great significance for the research of mutton quality and molecular breeding in the future.This study mainly involved in five experiments,In the early stage of the experiment,the water soluble extract from Allium mongolicum Regel was obtained by water extraction and freeze-drying,and then the animal experiment was carried out.The experiment selected 30 Dorper×thin-tailed Han crossbred sheep with good body condition,4.5 months old and weighing about 35 kg,which were randomly divided into 2 test groups,10 in each group.The control group was fed with the basic diet,and the experimental group added a water-soluble extract of allium mongolicum(3.4 g/day/piece)to the basic diet.The feeding experiment lasted for 75 days in the test period,including a 15-day pre-test period and a 60-day formal test period.At the end of the experiment,the initial body weight and pre slaughter body weight of each sheep were measured and compared.The fatty acid composition of longissimus dorsiand subcutaneous adipose were determined from two tissues of longissimus dorsi and subcutaneous adipose that was collected form 6 slaughtered sheepsin each group.Muscle and adipose tissue of 6 sheep(3 in the experimental group and 3 in the control group)were selected,and 12 samples were used for transcriptome sequencing,8 samples for whole genome methylation sequencing.The results are as follows.1.The results showed that the water soluble extract from Allium mongolicum Regel has no significant effect on the growth performance of Dorper×thin-tailed Han crossbred sheep,but it can reduce the content of c18:2n6c in the longissimus dorsi muscle and affect the contents of c15:1,c17:0,c18:0,c18:3n3,C23:0,c18:ln9c,PUFA,c18:2n6c and c18:3n6 in subcutaneous adipose tissue of mutton sheep.2.The analysis of the effects on the muscles showed that the water soluble extract from Allium mongolicum Regel significantly reduced the methylation level of the muscle tissue from Dorper×thin-tailed Han crossbred sheep,resulting in a total of 5,019 differential methylation regions(DMR)in the muscle tissue of meat sheep.The enrichment analysis of these DMRs related genes revealed that the allium mongolicum extract can promote the muscle tissue Gene functions related to cellular and metabolic processes were involved in signaling pathways such as MAPK and calcium signaling;on the transcriptome,allium mongolicum extract has produced 4,694 differentially expressed mRNAs in muscle tissue,of which 2,703 were up-regulated and 1,991 were down-regulated,A total of 3,092 differentially expressed genes(DEG)were involved;1412 differentially expressed lncRNAs were identified,of which 687 were up-regulated and 725 were down-regulated;these differentially expressed transcripts were enriched and it was found that allium mongolicum extract enhanced sheep Gene functions in muscle tissue,such as metabolic processes,protein binding,and ion binding,participate in fatty acid metabolism,tricarboxylic acid cycle,and some signal pathways related to metabolism and signal transduction.3.The results of the effect on adipose showed that the water extracts of allium mongolicum increased the methylation level of the whole genome DNA of the adipose tissue of meat sheep,and produced 19,983 DMRs in the adipose tissue.These DMR-related genes promoted the function of genes related to binding,protein binding and metabolism in adipose tissue.They participated in Rapl,P13K-Akt and Some other signaling pathways;In the transcriptome,4694 differentially expressed mRNAs were generated,of which 2703 were up-regulated,1991 were down-regulated,and were related to 2842 DEGs;1412 differentially expressed lncRNAs were identified,of which 842 were up-regulated,725 expressions were down-regulated;the enrichment results of DEGs and differentially expressed lncRNA all showed that allium mongolicum extracts enhanced the function of genes related to binding and metabolism in adipose tissue and participated in fatty acid metabolism pathways.4.The results showed that the water soluble extract from Allium mongolicum Regel reduced the difference between muscle and adipose tissue,increased the number of DMR genes from 2826 to 6226,reduced the number of differentially expressed mRNA from 107 to 3,and reduced the number of differentially expressed lncRNA from 9 to 1.Further enrichment analysis of this phenomenon showed that the tissue differences caused by allium mongolicum were mainly concentrated in ubiquitin mediated proteolysis,Rap1 signaling pathway,MAPK signaling pathway,adhesion spot,actin cytoskeleton regulation and cell proliferation.5.The whole genome methylation and transcriptome were analyzed to construct an epigenetic regulatory network co regulated by methylation and lncRNA,the results showed that the target genes of methylation and lncRNA between two organizations were significantly enriched in ATP binding,ubiquitin binding,protein kinase binding,cell proliferation regulation and various signaling pathways;The target of different methylation regions were mainly involved in the related functions of dsRNA,while the target genes of different expression lncRNA were significantly enriched in tissue-specific annotation,such as Golgi membrane and endoplasmic reticulum of muscle and oxidative phosphorylation pathway of adipose.In the muscle epigenetic regulatory network,MAPKAP1,FOXP2,STXBP5,WDR17 and other genes are regulated by the same lncRNA.In the fat epigenetic regulatory network,similar results also appear,different genes are regulated by the same lncRNA,and the involved co regulatory genes are related to insulin secretion,such as PDPK1,ATP1A2,CACNA1S and CAMK2D.Based on the above analysis,combined with the test results of key functional genes myoz1,PDLIM5,LMO7 and TFRC,the results of this study showed that allium mongolicum water extract can affect the epigenetic characteristics of sheep muscle and adipose tissue,which may affect the development of mutton sheep muscle,change the type of muscle fiber,and reduce the differences between tissues through the regulation of various differential genes,enhance the connection between muscle and adipose cells,affect the differentiation and migration of cells,and thereby change the distribution of adipose and its deposition in muscles,then through the insulin pathway to adjust the composition and content of fatty acids in different tissues,the quality and flavor of the mutton were finally changed. |
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