The aim of this study was to clone the goat RPL29 gene and analyze its effect on lipogenesis in intramuscular adipocytes. Using Jianzhou big-eared goats as the object, the goat RPL29 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR), the gene structure and expressed protein sequence were analyzed by bioinformatics, and the mRNA expression levels of RPL29 in various tissues and different differentiation stages of intramuscular adipocytes of goats were detected by quantitative real-time PCR (qRT-PCR). The RPL29 overexpression vector pEGFP-N1-RPL29 constructed by gene recombination was used to transfect into goat intramuscular preadipocytes and induce differentiation. Subsequently, the effect of overexpression of RPL29 on fat droplet accumulation was revealed morphologically by oil red O and Bodipy staining, and changes in the expression levels of genes related to lipid metabolism were detected by qRT-PCR. The results showed that the length of the goat RPL29 was 507 bp, including a coding sequence (CDS) region of 471 bp which encodes 156 amino acid residues. It is a positively charged and stable hydrophilic protein mainly distributed in the nucleus of cells. Tissue expression profiling showed that the expression level of this gene was much higher in subcutaneous adipose tissue and inter-abdominal adipose tissue of goats than in other tissues (P < 0.05). The temporal expression profile showed that the gene was expressed at the highest level at 84 h of differentiation in goat intramuscular adipocytes, which was highly significantly higher than that in the undifferentiated period (P < 0.01). Overexpression of RPL29 promoted lipid accumulation in intramuscular adipocytes, and the optical density values of oil red O staining were significantly increased (P < 0.05). In addition, overexpression of RPL29 was followed by a highly significant increase in ATGL and ACC gene expression (P < 0.01) and a significant increase in FASN gene expression (P < 0.05). In conclusion, the goat RPL29 may promote intra-muscular adipocyte deposition in goats by up-regulating FASN, ACC and ATGL.
Animals ; Lipogenesis/genetics* ; Adipogenesis/genetics* ; Goats/genetics* ; Adipocytes ; Cell Differentiation/genetics* ; Sequence Analysis ; Cloning, Molecular

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPβ lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPβ promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPβ gene expression.
Animals ; Goats/genetics* ; Cell Differentiation/genetics* ; Adipogenesis/genetics* ; Gene Expression Regulation ; Proteins/genetics* ; Cloning, Molecular

Objective: To explore the effect of adenoassociated virus sense transfection up-regulating the expressionlevel of the growth and differential factor 11 ( GDF11) in vivo on aortic injury in type 2 diabetic mellitus rats(T2DM). Methods: Nine-week-old male SD rats were randomly selected to establish a T2DM model by usinghigh -sugar and high-fat chow plus small-dose streptozotocin (STZ) combined induction. Both normal rats and diabetic model rats were randomly divided into five groups: blank control group ( Control group) , negative virus control group ( NC group), GDF11 adeno-associated virus group ( GDFl1 group), diabetic group ( DM group), anddiabetic + GDF11 adeno-associated virus group ( DM + GDFl1 group). After 8 weeks of feeding, the serum con-centrations of insulin ( INS ), advanced glyeosylation end products ( AGES), recombinant growth transforming fac.tor 11 ( GDF11 ), total cholesterol (T-CH0 ), triglycerides (TG), low-density lipoproteins (LDL-C), high-density lipoproteins ( HDL-C), asymmetric dimethylarginine ( ADMA), and malondialdehyde ( MDA) were assayed inthe rats ; periodic acid-schiff stain( PAS stain) was used to observe the sites of glycogen deposition, and hematoxylin-eosin staining ( HE) was used to observe vascular damage. Scanning electron microscopy was used to observethe damage of vascular endothelial cells and vascular elastic fibers, and protein blotting and immunohistochemistrwere used to detect the expression levels of vascular injury-related proteins. Protein blotting and immunohistochemistry were used to detect the expression levels of vascular injury-related proteins. Results: The biochemical indexes showed that the serum concentrations of AGES, T-CHO, TG, LDL-C and MDA were higher in the DM groupthan those in the Control group (P <0. 05), the concentrations of INS, GDF11, HDL-C and ADMA were signifi.cantly lower than those in the Control group (P <0. 05 ), and the concentrations of AGE'S and HDL-C were not sig.nificantly lower in the DM + GDF1l group compared with the DM group ( P <0. 05). HDL-C was not significantlydilerent from the DM group, and several other data were improved ( P<0. 05 ). Pathological staining suggestedthat PAS staining in the DM group suggested that glycogen particles deposited in the endothelium and subendotheli.um of the aorta, HE staining observed thickening of the aortic mesentery, endothelial cells and elastic fibers brokeolf in an irregular alignment , and electron microscopy observed endothelial damage in the vasculature and elastic fibers broke off in the DM group, and these changes attenuated in the DM + GDFl1 group. Protein blotting and im.munohistochemistry indicated that the expression of endothelial cell-associated proteins decreased in the DM groug( P <0. 05), and mesenchymal markers elevated in the DM group ( P <0. 05 ), these proteins were regressed inthe DM + GDFl1 group, and the difference was statistically significant (P<0.05). Conclusion Inereasing theexpression level of GDFl1 in vivo can improve aortic vascular injury caused by diabetes mellitus, which is inferredthat it may be related to the inhibition of endothelial mesenchymal transition to protect the function of vascular endo.thelial cells and thus improve vascular injury.