Preliminary Study On The Molecular Mechanism Of Intramuscular Fat Deposition In The Longissimus Dorsi Muscle Of Sheep

Since the new century,the consumption form of lamb has undergone a fundamental change,from the original seasonal,national,and regional consumption status to perennial,universal,and national consumption.The main reason for this is its excellent quality,which is highly favored.Intramuscular fat content is an important indicator reflecting the quality of lamb,which directly affects the flavor and tenderness of lamb.The intramuscular fat content of livestock and poultry is closely related to genetic,nutritional,and environmental factors,among which key regulatory factors play a crucial role in the process of intramuscular fat deposition,but there are relatively few studies on the screening and function of key genes for intramuscular fat deposition in sheep.Therefore,in this study,we used the dorsal longest muscle of 1-,2-,3-,6-,and 12-month-old sheep as the study target and used a combination of transcriptomic and bioinformatics approaches to screen and validate the key genes regulating intramuscular fat deposition in sheep in order to further reveal the pattern and regulation of intramuscular fat deposition in the dorsal longest muscle of sheep.Theoretical basis for further revealing the regulation of intramuscular fat deposition in the longest muscle of sheep The research is as follows:1.Histological differences of longissimus dorsi muscle in different growth stages of sheep,through routine tissue sections and HE staining of longissimus dorsi muscle of small-tailed Han sheep at different ages,it was found that the diameter of muscle fibers increased significantly with the increase of age(P < 0.05).The number of muscle fibers(root / mm2)decreased significantly with the increase of age(P <0.05).2.intramuscular fat deposition in the longest dorsal muscle of sheep at different growth stages,by measuring the intramuscular fat content in the longest dorsal muscle of small-tailed cold sheep at different months of age.It was found that the intramuscular fat content of the longest dorsal muscle varied with the age of the month.At 1 month of age,the intramuscular fat content of the longest dorsal muscle was significantly lower than at 2 and 6 months of age(P < 0.05),but not significantly different from at 3 and 12 months of age(P > 0.05).at 6 months of age,the intramuscular fat content reached the maximum value in 5 periods,and at 12 months of age,the intramuscular fat content reached the minimum value in 5 periods.3.Transcriptome sequencing and bioinformatics analysis of longissimus dorsi muscle of Small Tail Han sheep at different growth stages.The results showed that 159 differentially expressed genes(42 up-regulated genes and 117 down-regulated genes),1812 differentially expressed genes(923 up-regulated genes and 889down-regulated genes),2574 differentially expressed genes(1084 up-regulated genes and 1490 down-regulated genes)and 2475 differentially expressed genes(1044 up-regulated genes and 1431 down-regulated genes)were screened at 2,3,6and 12 months old,respectively.Through GO enrichment of differential genes,6,12,30 and 22 GO entries were enriched respectively.KEGG enrichment of differential genes was enriched in 12,32,61 and 60 significantly enriched pathways,respectively.The AMPK signaling pathway was selected as the pathway related to intramuscular fat,and the genes related to intramuscular fat deposition were PPARGC1A(peroxisome proliferator-activated receptor coactivator)and ADIPOQ(adiponectin).4.Immunohistochemical studies showed that PPARGC1 A and ADIPOQ proteins were expressed in the connective tissue,cell membrane and a small amount of cytoplasm of the longissimus dorsi muscle of Small Tail Han sheep at different months of age.5.Real-time PCR and Western Blot were used to verify PPARGC1 A and ADIPOQ,and 1 month old was used as the control group.There are differences between different months of age :5.Real-time PCR and Western Blot were performed to validate PPARGC1 A and ADIPOQ,and the results showed that both PPARGC1 A and ADIPOQ were expressed at 1 month of age as the control group.Differences were observed between different months of age:(1)The m RNA expression of ADIPOQ gene at 1 month of age was different from the other 4 months of age,and the ADIPOQ protein expression at 1 month of age was significantly lower than that at 3 and 12 months of age and significantly higher than that at 2 months of age(P < 0.05).(2)PPARGC1A gene m RNA expression at 1 month of age was significantly different from that at 2 and 12 months of age(P < 0.05),and PPARGC1 A protein expression at1 month of age was significantly different from that at 2,6 and 12 months of age(P <0.05).From the above results,it was inferred that ADIPOQ gene was negatively correlated with the intramuscular fat content of the longest dorsal muscle(r =-0.793,P < 0.05);PPARGC1A gene was positively correlated with the intramuscular fat content of the longest dorsal muscle r = 0.923,P < 0.05).