Study On The Regulation Of KLF10 Gene In Goat Intramuscular Preadipocyte Differentiation

KLF10,as a member of the Krüppel-like factors(KLFs),has three highly conserved Cys2/His2 zinc finger structures that regulate many functions of cells.Recently,researchers have found that KLF10 is involved in adipocyte differentiation in the mouse 3T3-L1 cell line.However,little information have been reported on ruminants,especially goat(Capra hircus).Pre-transcriptome sequencing in our laboratory indicated that KLF10 might participate in the differentiation of intramuscular adipocytes in goats.In this study,taking Jianzhou Big-eared goat as experimental Animals,the regulation of KLF10 on the differentiation of goat intramuscular precursor adipocytes was clarified and its possible mechanism of action was elucidated using RTPCR,real-timequantitative PCR,Western Blot,cell culture,transfection,overexpression,interference,Oil red O staining,Triglyceride determination and Dual fluorescein reporter assay and other methods.The obtained main results of this study are as fallows:(1)The sequence of goat KLF10 gene was 1457 bp with the CDS being 1449 bp,encoding a protein of 482 amino acids.TBP was selected and identified as the most stable internal reference gene in goat tissues.The KLF10 was widely expressed in goat various tissues and was the highest in spleen and lung(P<0.01).(2)In the process of differentiation of intramuscular precursor adipocytes in goats,the expression level of KLF10 increased in general.Lipid accumulation increased after KLF10 overexpression.After overexpressing KLF10,the expression level of adipocyte differentiation marker gene C/EBP? was significantly up-regulated(P<0.05).However,LPL and C/EBP? expression levels were significantly down-regulated(P<0.05),and SREBP1 expression level was significantly down-regulated(P<0.01).The expression of KLF3,KLF14 and KLF17 mRNAs was significantly up-regulated after KLF10 expression(P<0.01),and the mRNA expression levels of KLF1 and KLF16 were also significantly up-regulated(P<0.05),while the expression level of KLF11 was significantly down-regulated(P< 0.05).(3)Lipid accumulation decreased after KLF10 RNA interference.After overexpressing KLF10,the expression level of adipocyte differentiation marker gene C/EBP? was significantly up-regulated(P<0.05).(4)The goat C/EBP? promoter sequence was successfully cloned and its luciferase reporter vector was constructed and transfected into goat intramuscular precursor adipocytes.The results showed that the activity of C/EBP? promoter was significantly increased after KLF10 overexpression(P<0.01).In conclusion,we cloned the gene sequence of goat KLF10 and identified its tissue and cell sequence expression characteristics.Overexpression and interference showed that KLF10 promoted the differentiation of goat intramuscular precursor adipocytes,and this regulation may be achieved through C/EBP? in cooperation with some members of the family.