牛转FAD3B基因胎儿成纤维细胞中内参基因的稳定性评估

Evaluations of Reference Genes in Bovine Transfected FAD3B Gene Fetal Fibroblasts

采用RT-PCR扩增亚麻种子(Linum usitatissimumLinn)FAD3B基因,构建不同启动子的两种重组真核表达载体pIRES-AcGFP-CMV-FAD3B和pIRES-AcGFP-CAG-FAD3B,通过qRT-PCR检测转基因细胞中FAD3B基因的表达水平。以不同过表达水平的转基因细胞为试验对象,评价9种候选内参基因ACTB、GAPDH、18S rRNA、UXT、PPP1R11、RPS15A、SF3A1、EEF1A2和HMBS的稳定性。根据GeNorm、NormFinder和BestKeeper 3种统计学算法得到的稳定性值对基因进行排序。结果显示,内参基因稳定性的综合排序为PPP1R11>EEF1A2>18S rRNA>RPS15A>GAPDH>HMBS>UXT>ACTB>SF3A1,其中PPP1R11和EEF1A2是最稳定的内参基因。稳定内参的选择可以更加准确地校正基因的表达水平,从而为阐述基因的功能奠定了坚实的基础。

The FAD3B was cloned from the seed of flax（Linum usitatissimum Linn）using RT-PCR.The gene was constructed as the recombinant eukaryotic expression vectors pIRES-AcGFP-CMV-FAD3B and pIRES-AcGFP-CAG-FAD3B with different promoters.The expression level of FAD3B was detected by qRT-PCR in transfected cells.Different over-expression levels of transfected cells were used as the experimental subjects.Nine candidate reference genes were evaluated,including ACTB,GAPDH,18S rRNA,UXT,PPP1R11,RPS15A,SF3A1,EEF1A2 and HMBS.Three statistical algorithms（geNorm,NormFinder and BestKeeper）were used to rank genes by their stability values.The overall rank ordering of candidate internal control genes was PPP1R11EEF1A218S rRNARPS15AGAPDHHMBSUXTACTBSF3A1,PPP1R11 and EEF1A2 can be considered as the most stable reference genes.Selection of stable control genes can normalize the expression level of gene,laid a solid foundation so as to elaborate the function of genes.