Effects Of PPAR? On Intramuscular Fat Content And Muscle Fiber Type Transformation In Transgenic Pig Model

Pork is the main source of human animal protein.There are many factors that affect the quality of pork,such as marble,water holding capacity,color,tenderness and so on.Intramuscular fat content is one of the most important muscle quality traits,and it can influence muscle flavor and juicy.So,it is an important economic trait.Studying on the regulation mechanism of fat metabolism is of great significance to improve the muscle quality traits.Peroxisome proliferator-activated receptor?(PPAR?)plays an important role in adipocyte differentiation and is a key regulator of adipose tissue formation.In this study,PPAR?transgenic pig model was used to validate the mechanism of PPAR?in lipid metabolism.The main results are as follows:1.On the basis of founder randomly integrated PPAR?transgenic pigs,the transgenic herds were expanded and identified.Traits measurement showed that the content of intramuscular fat was significantly increased(40%),and other carcass and meat quality traits,such as lean meat rate,were not significant.2.The expression of PPAR?in transgenic pigs was significantly up-regulated in the longissimus dorsi,gastrocnemius muscle,soleus muscle and brain.The levels of transcription and protein were detected in the longissimus dorsi and gastrocnemius.The genes related to fat development such as C/EBP?,Fox O1,LPL and UCP1 were up-regulated.At the same time,the expression of genes related to oxidized myofibers was up-regulated,while the expression of genes related to the type?fibroin was down-regulated,indicating that PPAR?had influence on the transformation of fiber type and promoting the fat formation.3.We used CRISPR/cas9 site-specific integration technology to obtain the site-specific integration PPAR?transgenic pigs at Rosa26 site.The detection of integration site,copy number and Southern blot of each generation of transgenic pigs showed that the foreign genes were stably integrated and inherited in the genome.The expression of PPAR?gene in muscle tissue of F0 and F1 transgenic pigs was significantly higher than that of wild type pigs,indicating that the target gene was stable in mRNA and protein expression levels.4.HE,BPDIPY and oil red O staining showed that intramuscular fat deposition increased in site-specific integration PPAR?transgenic pigs.Traits measurement showed that intramuscular fat content(20%)and marbling(18%)increased significantly,while lean meat percentage remained unchanged.5.We detected the genes involved in adipogenesis regulated by PPAR?,and the results showed that they were all up-regulated.We have verified this in porcine muscle preadipocytes.6.Through proteomic sequencing analysis,we found that the oxidative metabolism in muscle cells of site-specific integrated transgenic pigs was significantly enhanced,and the number of oxidized muscle fibers was significantly increased.By detecting the expression of calcium related pathway proteins,we preliminarily verified the mechanism of a neural calcium regulatory pathway in the muscle overexpression of PPAR?and promoting the increase of oxidized muscle fibers.The results were verified in the isolated porcine muscle satellite cells.7.We evaluated the safety of site-specific integrated transgenic pigs in terms of behavior,survival competitiveness,blood physiological and biochemical indicators,semen quality,HE staining of major tissues,and off-target detection.In conclusion,in this study we used site-specific integrated and random integrated PPAR?transgenic pig models to study the effects of skeletal muscle-specific overexpression of PPAR?on pig muscles.Overexpression of PPAR?promotes intramuscular fat deposition by activating the high expression of downstream genes,and regulates the increase of oxidized muscle fibers through Ca²⁺-mediated phosphatase and kinase.The research results provide a new and effective way to improve meat quality traits in pig breeding.