| Adipose tissue of beef cattle is mainly distributed under the skin,around the internal organs and intramuscular.The adipose tissue located in the muscle,namely intramuscular fat(IMF),is the main factor affecting the quality of beef.The content of IMF not only affects the sensory and color of beef,but also contains a large number of fatty acids and proteins that cannot be synthesized by ourselves.Evidences suggested that serum-and glucocorticoid induced kinase 1(SGK1)is a key kinase widely involved in regulating a variety of metabolic processes.Previous studies showed that SGK1 was mainly involved in regulating the kidney diseases.The results of transcriptome sequencing in our laboratory showed that SGK1 was differentially expressed genes in the adipose tissue of six-month old and adult cattle.However,whether SGK1 regulates the process of bovine adipogenesis and its regulatory mechanism is still unclear.This study explored the role and regulation mechanism of SGK1 during the proliferation and differentiation of bovine preadipocytes to provide a theoretical basis for genetic improvement and molecular breeding of beef cattle.The main results are as follows:(1)The coding region amplification and expression pattern detection of bovine SGK1Collect bovine 7 tissues including fat,heart,liver et al.Detecting the expression level of SGK1 in bovine tissues by qPCR.The result showed that compared with heart,SGK1 highly expressed in kidney(P<0.01),followed by liver,spleen,fat and other tissues.The expression of SGK1 on day 6 was significantly higher than that on day 0 during adipocytes differentiation(P<0.05).(2)Packaging overexpression adenovirus and short hairpin RNA lentivirus of SGK1Then,CDs region of SGK1 was connected with adenovirus shuttle vector pAd-track-CMV and skeleton vector pAd-easy to obtain adenovirus recombinant plasmid.The recombinant plasmid was transfected into HEK293A.The titer of bovine SGK1 overexpressed adenovirus and negative control virus were 2.51×1010 pfu/mL and 3.16×1010 pfu/mL,respectively.Three shRNA sequences for SGK1 were designed and the shRNA1 was selected the best interference efficiency to SGK1 by using the double luciferase reporter gene system.Using shRNA1 to perform single target interference lentivirus packaging The titer of interfering lentivirus and negative control virus were 1 ×108 TU/mL and 3 × 108 TU/mL,respectively.(3)Overexpression of SGK1 promoted bovine adipogenesis and loss-of-function SGK1 inhibited bovine adipogenesisOverexpression of SGK1 in bovine preadipocytes significantly increased the content of lipid droplets compared to NC.The results of qPCR and western blot showed that the mRNA(PPARy and C/EBPα)expression of adipocyte differentiation marker genes increased significantly(P<0.05)and proliferation related genes(CCND2,MCM6 and PCNA)decreased significantly(P<0.05).The protein expression of differentiation related genes was not significantly increased,while cell cycle related proteins(CDK1 and CCND2)were extremely significantly inhibited(P<0.01).When SGK1 was interfered,it was detected that the content of lipid droplets in adipocytes decreased significantly,the mRNA(C/EBPα,C/EBPβ,SREBP-1c and FABP4)and protein expression of adipocyte differentiation related proteins(PPARγ and C/EBPα)were significantly inhibited(P<0.05),while the mRNA of cell cycle related genes(CCND2 and MCM6)increased significantly,but the protein expression didn’t change significantly.(4)Overexpression of SGK1 in adipocytes significantly inhibited the mRNA expression of FOXO1 and FOXO3(P<0.01),and the results were opposite when SGK1 deficiency(P<0.05).Overexpression of SGK1 significantly increased the phosphorylation level of FOXO1(P<0.05),but its total protein expression did not change significantly,while the phosphorylation level of FOXO3 did not change significantly,and it’s total protein significantly decreased(P<0.05).SGK1 interfered only increased the total protein level of FOXO3(P<0.05).The results of transcriptome sequencing showed that a total of 1,704 differentially expressed genes were obtained in SGK1 expressed bovine adipocytes,including 1,126 up-regulated genes and 578 down-regulated genes.The results of GO function annotation showed that the DEGs were enriched in the items of metabolism,development and activation of biological processes.The results of KEGG enrichment analysis showed that SGK1 significantly changed the genes expression of enriched on PI3K/Akt signaling pathway and lipid metabolism related signaling pathway.In conclusion,SGK1 is a key positive regulator during bovine adipogenesis,which changed the genes expression of related-proliferation and related-differentiation in adipocytes regulating bovine adipogenesis.This study explored the molecular mechanism of SGK1 regulating bovine adipogenesis and provide an important theoretical basis for constructing the network regulating of bovine fat deposition,and provided reference information for beef cattle genetic improvement and molecular breeding. |
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