| The content of intramuscular fat has a great influence on tenderness,juiciness,flavor and aroma of beef,so improving the content of intramuscular fat can improve the quality and value of beef.Animal transgenic technology can transform the genome structure and genetic composition of animals,and cultivate new animal varieties quickly and efficiently.The most important upstream technology of animal transgenes is to select the ideal target gene and lay the foundation for the directional gene breeding.Diacylglycerol acyltransferase 1(DGAT1)is an important enzyme that controls the final rate limiting step of triacylglycerol biosynthesis.Fatty acid binding protein 4(FABP4)is responsible for the binding and transportation of fatty acids by combining with fatty acids.Peroxisome proliferator activated receptor?2(PPAR?2)is a key transcription factor in adipogenesis and regulates adipogenesis.In this study,according to the needs of the national transgenic high-quality beef breeding project undertaken by the laboratory,three fat deposition genes DGAT1,FABP4 and PPAR?2 were selected to carry out a comparative study on the ability of fat deposition in Qinchuan beef muscle satellite cells,so as to provide technical basis for beef transgenic breeding.The main results of this study are as follows:1.Construction of bovine DGAT1,FABP4,PPAR?2 over expression vector(1)The coding sequence of DGAT1,FABP4 and PPAR?2 of Qinchuan cattle were cloned by using the c DNA of Qinchuan cattle adipose tissue as template.(2)pEGFP-N1-DGAT1?pEGFP-N1-FABP4?pEGFP-N1-PPAR?2 were constructed with pgfp-n1 as the skeleton vector.The results of double enzyme digestion and Sanger sequencing showed that the construction of the overexpression vector was correct.2.Isolation and identification of muscle satellite cells from Qinchuan Cattle(1)Qinchuan cattle muscle satellite cells were isolated by combined enzyme digestion and purified by differential attachment method.The morphological changes of the satellite cells in the process of proliferation were observed.The results showed that the muscle satellite cells were spindle shaped and in good growth condition.(2)The marker genes of myosatellite cells were detected by immunofluorescence,Western blotting and qPCR,and myogenic differentiation fluid induced myosatellite cell differentiation.The purity of Pax7 was 94.27%,Western blotting and qPCR showed that the myosatellite cells were in static or proliferative phase,and the induced differentiation experiment showed that the myosatellite cells had high purity and good myogenic differentiation performance,which could be used in the next experiment3.The effect of overexpression of DGAT1,FABP4 and PPAR?2 on cell differentiation(1)The total RNA was extracted and detected by qPCR.The results of qPCR showed that the overexpression vector pegfp-n1-dgat1,pegfp-n1-fabp4,pegfp-n1-ppar?2 could be expressed in skeletal muscle.(2)QPCR showed that the expression of ACC in PPAR?2 overexpression group was significantly higher than that in DGAT1 and FABP group,and the expression of FASN in PPAR?2 overexpression group was significantly higher than that in DGAT1 and FABP 4;the expression of LPL in PPAR?2 overexpression group was significantly lower than that in DGAT1 and FABP 4.The results of ELISA showed that the content of ACC in PPAR?2 overexpression group was significantly higher than that in DGAT1 and FABP4,while the content of LPL was significantly lower than that in DGAT1 and FABP4.The results showed that PPAR?2 was better than DGAT1 and FABP4 in regulating the expression of fat metabolism related genes.(3)After transfection of myosatellite cells with over expression vector,lipogenic differentiation was induced,and the formation of lipid droplets was detected by oil red O staining.The results showed that the OD value of oil red O staining in PPAR?2 overexpression group was significantly higher than that in DGAT1 group and FABP4 group.It shows that PPAR?2 is better than DGAT1 and FABP4 in promoting fat deposition(4)The content of triglyceride and free fatty acid in PPAR?2 overexpression group was significantly higher than that in DGAT1 and FABP4,and the concentration of free fatty acid in PPAR?2 overexpression group was significantly lower than that in DGAT1 and FABP4,that is to say,PPAR?2 was better than DGAT1 and FABP4 in promoting fat depositionTo sum up,three different genes related to fat deposition,DGAT1,FABP4 and PPAR?2 overexpression vectors,were successfully constructed in this experiment.The transfection of bovine muscle satellite cells showed that PPAR?2 had the strongest ability of fat deposition in muscle. |
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