Meat Performance And Integrated Analysis Of The Muscle Tissue M RNA–miRna In Qianhua Mutton Merino And Small Tail Han Sheep

Qianhua Mutton Merino(tentative name)is a new sheep breed used for both mutton and wool in Jilin Province.This new sheep breed has outstanding meat production performance and produces fine wool,whereas Small Tail Han Sheep is widely used as the maternal variety in Jilin Province and Northern China.Therefore,it is of great theoretical and practical significance to reveal differences in meat production performance,especially regarding the mechanism of meat production,between new mutton sheep breed and widely used local sheep breed.This paper evaluated meat performance differences between a newly bred sheep variety,Qianhua Mutton Merino,and a local variety,Small Tail Han Sheep,based on mRNA and microRNA(miRNA)expression profiles for sheep longissimus dorsi muscles,and explored the mRNA and mi RNA regulatory mechanisms of sheep muscle growth and development by integrative analysis of microRNA-mRNA.First,slaughter performance,mutton quality traits,muscle nutrition,and muscle histological characteristics were studied for Qianhua Mutton Merino and Small Tail Han Sheep.Then,mRNA and miRNA libraries of longissimus dorsi muscle were constructed for the two sheep breeds,expression patterns of mRNAs and miRNAs in muscle tissue were identified using high-throughput sequencing and bioinformatics analysis,and differences in mRNA and miRNA expression profiles were analyzed.Subsequently,integrated analysis of mRNA and miRNA was conducted,and an miRNA–mRNA network was constructed.Finally,the high-throughput sequencing results were validated by qRT-PCR,and a double luciferase reporter gene detection system was used to verify the targeting effect of miRNAs on target genes.The main results are as follows: 1.The Qianhua Mutton Merino and Small Tail Han Sheep were selected for mutton performance research.The results showed that Qianhua Mutton Merino was better than Small Tail Han Sheep with regard to slaughter performance,including live body weight,carcass weight,slaughter rate,net mutton rate,eye muscle area;mutton quality traits,including tenderness,water loss rate,cooking rate,and pH value;muscle nutrition compositions,including protein,fat,water,and amino acid and fatty acid contents;and muscle histological characteristics,including muscle fiber diameter.Mutton performance significantly differed between the two sheep breeds.2.The mRNA libraries of muscle tissue were successfully constructed for the two sheep breeds.A combined approach of high-throughput sequencing and bioinformatics analysis was used to identify the mRNA expression profiles for sheep muscle tissues,with a total of 960 differentially expressed genes were identified,including 405 up-regulated and 555 down-regulated genes.GO functional annotation and KEGG pathway analysis showed that the differentially expressed genes were significantly enriched in 252 biological processes and 53 metabolic pathways.GO tree and pathway networks were successfully constructed to identify the different genes related to muscle growth and development for the sheep,such as MRFs,GXP1,and STAC genes,and related pathways,such as metabolic,tricarboxylic acid cycle,and glycolysis/gluconeogenesis pathways.3.A gene-act network and co-expression network were built,which were closely related to sheep muscle growth and development.4.The expression levels of nine differentially expressed genes in the muscle tissues of Qianhua Mutton Merino and Small Tail Han Sheep were verified by q RT-PCR.The quantitative and sequencing results were highly consistent,which confirms the accuracy of the mRNA sequencing results.5.The miRNA libraries of muscle tissue were successfully constructed for the two sheep breeds.A combined approach using high-throughput sequencing and bioinformatics was used to identify the muscle tissue miRNA expression profiles of sheep.A total of 153 known sheep miRNAs were identified,of which 152 miRNAs were co-expressed in the two sheep breeds,and one miRNA was only expressed in Small Tail Han Sheep.A total of 8067 novel mi RNAs were identified.Differential expression analysis of miRNAs revealed four known miRNAs and 146 novel miRNAs.6.Combining mRNA library data,revealed 26 candidate target genes of four known miRNAs and 8067 candidate genes of 146 novel miRNAs that were selected,GO functional annotations and KEGG pathway annotations showed that 26 candidate genes,which are the target genes of four known sheep miRNAs,were significantly enriched in 86 biological processes,including muscle organogenesis,myoblast migration,cell proliferation,and adipose tissue development,and nine metabolic pathways,including carbohydrate,nucleotide,and amino acid metabolic pathways.7.The expression levels of eight differentially expressed miRNAs in the muscle tissue of Qianhua Mutton Merino and Small Tail Han Sheep were verified by stem-loop qRT-PCR.The quantitative and sequencing results were highly consistent,which confirms the accuracy of the miRNA sequencing data.8.The mi RNA–mRNA network of sheep muscle tissue was successfully built,and oar-miR-655-3p and target gene ACSM3,and oar-miR-381-5p and target gene ABAT were selected for subsequent analysis based on GO and KEGG analyses.Target gene verification was conducted for oar-miR-655-3p and oar-miR-381-5p using double luciferase reporter gene detection techniques.The results showed that oar-mi R-655-3p can specifically target the ACSM3 gene,and oar-miR-381-5p can specifically target the ABAT gene,which indicates that the ACSM3 gene is the target gene of oar-miR-655-3p and the ABAT gene is the target gene of oar-miR-381-5p.