The Function And Mechanism Of PPARγ Gene In Regulating Intramuscular Fat Deposition And Metabolism Using Transgenic Mouse And Pig Model

Many factors affect the pork quality traits,but the intramuscular fat content is one of the most important indicators,and it can influence tenderness,juiciness and the flavor of pork meat,it is an important economic characters.But because the inversely proportional of intramuscular fat and lean meat,traditional breeding is very difficult to improve both of them or improving intramuscular fat content on the premise of high lean.In the current days using transgenic technology to produce high intramuscular fat content of lean hogs becomes possible.PPARγis a key regulatory factor of induce white and brown fat,our research is to validation PPARγ gene function in intramuscular fat deposition and the influence of meat quality traits use genetically modified mice and pig models,to find out how PPARγ genes involved in fat metabolism regulating system,etc.We are looking forward to breeding a high lean hogs of intramuscular fat varieties,improved to provide theoretical support for genetically modified animal breeding.The main research results as follows:1.Based on PPARγ transgenic mice showed strong brown fat features in subcutaneous fat,we employed a serious of method such as morphology,real time quantitative PCR and Western Blot to do the research,we found that there was no significant difference in body wight between transgenic mice and negative controls,but the transgenicmice showed strong brown fat features in subcutaneous fat in morphology and histology.To providemolecular evidences on browning characteristics of the adipose tissue,we employed quantitativereal-time PCR to determine BAT-specific gene expressions.The transgenic mice had remarkably elevatedm RNA level of UCP1,Elovl3,PGC1 a and Cebpa in subcutaneous fat.ATP concentration was slightly decreasedin the subcutaneous fat of transgenic mice.Western blotting analysis also confirmed that phosphorylated AMPK and ACC proteins were significantly increased in the transgenic mice.Therefore,thisstudy demonstrated that over-expression of PPARγ2 in skeletal muscle can promote conversion of subcutaneousfat to brown fat formation.After the preparation of PPARγ/PGC1 alpha double transgenic mice,morphological observation muscles color turned into deep red,free fatty acid content in muscle are much higher.2.Set up 30 days Large white fetal pig cell lines,using somatic cell nuclear transfer technology preparation the PPARγ transgenic pigs,by PCR and Southern blot method to identify positive transgenic pigs,use absolute quantitative PCR method for determination of copy number,detection and analysis of the integration sites.Organizations expressed PPARγ spectrum analysis in transgenic pigs showed expression levels were raised in four kinds of muscles and three kinds of adipose tissues.Heart,kidney,intestine and brain’s expression levels also increased,PPARγ expression levels increased by 4.24 times in brain,and reached a significant level.But in the spleen,lung and stomach tissues expression quantity is much lower.3.Slaughter determination of 7 months brood PPARγ transgenic pig and negative groups.Compared with the negative pigs,we found the carcass lean meat has little difference between transgenic group and negative control group,but the content of intramuscular fat content increased by 0.8% than control group,tenderness improved 30.1N,also increased,but it reduced the drip loss and the brightness of meat.Morphological observation and skeletal muscle biopsy results also confirmed the marbling score and intramuscular fat content increased.Free fatty acid and biochemical index determination in PPARγ transgenic pig muscle and blood samples,the results showed that the free fatty acid levels increased in muscles,reduced in the blood samples,muscle triglyceride content increased.4.We detected the longest back muscle,soleus and gastrocnemius three kinds of skeletal muscle in RNA and protein levels,found a type II muscle fibers transition to type I muscle fiber type,red muscle fiber related genes such as My HC1,Myoglobin,Tnni I and My HC2 A relative expression levels are raised.