定量检测牛雌性发育候选基因FOXL2表达方法的建立

Construction of Bovine Sex Related Genes FOXL2 Standard Plasmid and Standard Curve by Real-time RT-PCR

本研究根据GenBank中登录的剂量敏感的翼状螺旋/叉头转录因子2(forkhead transcription factor 2,FOXL2)设计引物,构建包含FOXL2基因cDNA片段的质粒,作为中国荷斯坦牛FOXL2基因mRNA定量检测的标准品,建立了FOXL2基因mRNA表达实时荧光定量PCR检测方法。结果表明,该方法特异性好,检测灵敏度达101拷贝,线性范围为101～106拷贝,阈值循环数(Ct)与PCR体系中起始模板量的对数值之间有着良好的线性关系(r=0.998689),扩增效率高(E=99.62%),可以作为检测牛FOXL2基因mRNA定量检测方法。

The primer was designed based on forkhead transcription factor 2（FOXL2）,according to the sequence which was submitted on GenBank.A recombinant plasmid contained cDNA fragment of FOXL2 gene was constructed and used for the standard substance for quantitative detection of mRNA of the FOXL2 gene of Holstein,a Real-time PCR was developed for detection of FOXL2 gene.The results revealed this method had a good specificity,and the sensitivity was up to 101,range from 101 to 106.The cutoff value Ct had a good linear correlation with PCR samples.This method can detecte FOXL2 gene in bovine quantitatively.