| Buffalo somatic cell cloning is an effective technical means for expansion of buffalo with good traits rapidly,but it is not widely applied because of its low efficiency.Studies have shown that there are significant differences in cloning efficiency among individual buffalo cells,while the mechanism leading to this difference is still unclear.The cloning efficiency of somatic cells depends mainly on the reprogramming,which may be closely related to the chromatin openness and remodeling of cells.In order to explore the mechanism of the difference in cloning efficiency of different individual buffalo somatic cells,this study was designed to compare the expression of chromatin remodeling-related genes,chromatin openness and transcriptional levels in cell lines with different clonal efficiency,and explore the effect of ATP content on chromatin remodeling-related genes expression.The results were looking forward to lay a foundation for improving the efficiency of buffalo somatic cell cloning.The study obtained the following results:1.Analysis of buffalo somatic cell characteristics with different cloning efficiencyTwo buffalo fetal somatic cell lines with significant difference cloning efficiency established in our laboratory(BFF3 with higher cloning efficiency and BFF1 with lower cloning efficiency)were selected to analyze the expression of the ATP-dependent core enzyme gene SMARCA4,which belonged to the chromatin remodeling SWI/SNF complex.The results showed that the expression level of SMARTAC4 in BFF1 was significantly lower than that in BFF3(P<0.05).The growth characteristics of the two cells showed that there was no significant difference in the proliferation rate in logarithmic phase.The proliferation rate of BFF1 cells after plateau was lower than that of BFF3.After contact inhibited,the proportion of G0/G1 phase of the two cells was 90.75%,91.18%.In G0/G1 phase,the expression levels of chromatin remodeling SWI/SNF family SMARCC1,SMARCC2 and SMARCE1 genes in BFF3 were significantly higher than those of BFF1(P<0.05),while the expression levels of SMARCA4,SMARCA2,SMARCAL1 and SMARCB1 genes were not significantly different.2.Analysis of chromatin opening and transcription levels of buffalo donor cells with different cloning efficiencyTo understand the relationship between cloning efficiency and chromatin openness,transcription level,ATAC-seq and RNA-seq were carried out for BFF1 and BFF3 cells.The ATAC-seq results showed that in BFF3 cells,at the genome-wide level,chromatin,SMARCA4,SMARCA2 and RNA-binding protein-mRNA processing factors were more open than BFF1 cells.KEGG functional enrichment showed that BFF3 cells were specific in the oxytocin signaling pathway and regulating the pluripotency pathway of stem cells.RNA-seq results showed that the transcription levels of ATP1B 1 and ATP11A in BFF3 cells had highly significant difference with those of BFF1 cells,which was lower(P<0.01).3.Effect of ATP content on the expression of genes related to SWI/SNF family BFF1 and BFF3 cells were treated with 0.5 ?M rotenone for 24 h,which is a functional inhibitor of mitochondrial complex I,cells apoptotic rate was 10.16% and 20.85% respectively,cell proliferation was blocked partially,mitochondrial membrane potential decreased,and ATP content decreased.Compared with untreated group,the expressions of SMARCA4,SMARCA2,SMARCAL1,SMARCC1,SMARCC2,SMARCB1 and SMARCE1 were all decreased,and the decrease degree of BFF1 cells was larger.After treated cells with 5 mM glycolysis inhibitor 2-deoxyglucose for 12 h,the ATP levels of the two cells increased.Compared with untreated group,the expressions of SMARCA4,SMARCA2,SMARCAL1,SMARCC1,SMARCC2,SMARCB1 and SMARCE1 in BFF1 cells was decreased,and except SMARCE1,the expression of other genes was significantly decreased(P<0.05).The expression of other genes was increased in BFF3 cells except the expression of SMARCA2 decreased,and the difference between SMARCA4 and SMARCAL1 was extremely significant(P<0.001).In conclusions,buffalo somatic cells with different cloning efficiency differed in chromatin openness,transcriptional levels of chromatin remodeling-related and ATP functional genes,and ATP content on chromatin remodeling genes expression.Cells with higher cloning efficiency were more dependent on ATP,and the expression of chromatin remodeling genes was higher. |
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