Characteristics Of Intramuscular Fat Deposition And Mechanism Of Adipocyte Differentiation In Cashmere Goat

Intramuscular fat content is a key indicator that influences the meat quality of cashmere goat, it is directly related to the economic value and meat value of the cashmere goat. Inner Mongolia cashmere goat has low cholesterol, juicy and delicious, which shows excellent traits of meat quality. Therefore, this study takes Inner Mongolia cashmere goat as the experimental animal model, discuss the different parts of the muscle intramuscular fat deposition, and development change features of fat deposition metabolism-related gene expression, construct intramuscular preadipocytes of cashmere goat in vitro, to induce and differentiate, research the relevant lipid gene expression during the cell differentiating process.Use RNAi technology to identification mechanism that regulate the cashmere goat fat deposition of PPARy gene at he transcriptional level. Basing on this study to construct four RNA library of intramuscular preadipocyte, mature adipocytes, interference group of fat cells, and control group of fat cells.The high-throughput sequencing for transcriptome was performed by Illumina MiSeq. Apply bioinformatics software to analyze and screen the key indicators that regulate the fat deposition from sequencing assessment, gene function annotation, identification of gene alternative splicing, and other bioinformatics, Adequate sources of genes were provided for meat quality traits of livestock in transgenic breeding. Main study results are shown as follows:1.The expression of key genes for fatty deposition in the different muscle tissue and analysis of the correlation with IMFPPARy gene expression is significantly different in different parts of the muscle, for instance, such expression is highest in the diaphragm muscle, next moment chest muscle, longissimus muscle, and quadriceps muscle, which are significantly higher than that in semitendinosus muscle, biceps femoris muscle, psoas major muscle and obliquus externus abdominis muscle (P<0.01). Secondly, such expression level is much higher in the intercostal muscle and trapezius muscle. FTO gene expression level in different tissues is higher than that of other genes, the expression levels in quadriceps muscle, and trapezius muscle are higher than in other parts, and the difference between the above two muscles are significant, while the gene expression levels in longissimus muscle and intercostal muscle are much lower. Lipin gene expression level in different muscles is much lower, while such expression level is much higher in diaphragm muscle, and next moment chest muscle; secondly, such expression level is extremely low in intercostal muscle, quadriceps muscle, psoas major muscle, obliquus externus abdominis muscle, longissimus muscle, semitendinosus muscle, trapezius muscle, and biceps femoris muscle. LPL and HSL are key enzymes in the fat synthesis and splitting, their expression trends are basically the same, HSL gene expression level in psoas major muscle is higher than that in trapezius muscle. LPL expression pattern is the opposite. A-FABP and Perilipin gene expression patterns are more special, the overall expression level is lower, there is no significant difference among tissues, the former’s expression level is higher in next moment chest muscle, while the latter’s expression level is higher in biceps femoris muscle. Generally speaking, the PPARy gene mRNA of cashmere goat forms negative correlation with intramuscular fat; while other adipogenic genes HSL, LPL, FTO, lipin, perilipin, A-FABPmRNA form positive correlation with intramuscular fat content.2.Construction of intramuscular preadipocyte of cashmere goat, and expression of key gene of fat deposition in the differentiation processSample the longissimus muscle of one-day-old cashmere goat, and then use Type-II collagenase digestion method to obtain intramuscular preadipocyte of cashmere goat with sound homogeneity, and in shuttle-shape. Add IBMX+DEX+INS in vitro to induce adipogenic differentiation, oil red O staining can be seen for the red intracellular lipid droplets. Expression of key gene for RT-PCR detection fat deposition against intramuscular preadipocyte in three periods of early (D3), middle (D7), and end (D11), as well as in key points of induced differentiation time (24h、48h、72h、96h), the expression level of FTO gene in each time point is higher than that of other genes; the expression patterns of LPL, lipin, PPARy, A-FABP and perilipin are similar, as the day number of cultivating the preadipocytes, and the triglyceride accumulation, the corresponding expression level increases gradually; the expression level of LPL gene in early differentiation of adipocytes is highest; while the expression level of PPARy in the cell differentiation process increases gradually, after48h induction, the expression reaches the top, and then the expression abundance gradually reduces; thereof the expression abundance of lipin gene in each time point is extremely low; the expression level of HSL reduces gradually as the differentiation degree increases; A-FABP gene has the same expression pattern with PPARy; the expression level of perilipin gene is higher only in the latter period of induction and differentiation.3.The effect of PPARy gene silence against the fat cell proliferation and differentiationIn order to prove the important role of PPARy gene in the process of adipocyte differentiation, this study preliminarily succeeds in constructing the PPARy lentiviral vector, which has the infection efficiency of80%against the intramuscular fat cells, while fluorescence quantitative and Western blot detection of lentiviral vector significantly inhibit PPARy expression. At the same time, the proliferation of fat cells is enhanced significantly, while the differentiation is reduced significantly. The expression levels of other adipocyte differentiation related genes like LPL, FTO, A-FABP, and perilipin reduce for a varying degree due to the decrease of PPARy gene expression level. There are no significant changes being detected against the HSL and Lipin. Therefore, PPARy gene is located in the central regulation position in the process of adipocyte differentiation, and it plays a key role in regulating the expression of other genes.4.Comparative transcriptome analysis before and after the mature of intramuscular fat cells, as well as before and after interferenceBased on the RNA-seq high-throughput sequencing technology, this study totally obtains16G transcriptome data, conduct two two comparison against the four samples of intramuscular preadipocyte, mature adipocytes, interference group of fat cells, and control group of fat cells through the TopHat software, and the differential genes screened with152,450,456,412,39and445different expressions. Thereof compare and analyze the interference group and control group to screen to pref-1,FGF,TGF, TNF,RA,IL-21and EGF,which has higher expression in shRNA-PPARγ intramuscular fat cells. Conduct GO classification and KGEE pathway preliminary analysis against these differential genes, so obtains10pathways related adipocyte differentiation, thereof the most significant pathways indicate PPARy signal pathway, WNT signal pathway, mTOR signal pathway, and MAPK signal pathway. Thereof6genes, namely, LPL, FABP, FATP, AP2, RXRG and CD36enriched in PPARy signal pathway, which shall be served as key candidate gene of cashmere goat intramuscular fat deposition.