Abstract: One of the ammonium transporter genes in rice,OsAMT1;3,normally expressed in low abundance,which is difficult to quantify its expression by using traditional methods,such as Northern blot,PCR technique. A technique for real-time quantification of OsAMT1;3 relative to a housekeeping gene for encoding an actin in rice using real-time SYBR Green quantitative PCR(RQ-PCR) with specific primers was established,which showed:the amplification curve had flat baseline,distinct exponential area,large and stable slope; The coefficient of variation of the technique was 0.47%; There was a linear relationship between threshold cycle value at which sample crosses threshold and the logarithmic value of template concentration; The expression of OsAMT1;3 was enhanced 4-fold by nitrogen starvation in comparison to supply of ammonium as sole source of nitrogen.

Key words: real-time fluorescence quantitative PCR, amplification curve, standard curve, ammonium transporter gene, gene expression, methodology

摘要： 采用实时荧光定量PCR技术，通过使用特异引物，对水稻中的低丰度表达基因OsAMT1;3进行了转录水平上的定量分析，成功建立了可检测低丰度表达基因的SYBR Green实时荧光定量PCR技术平台。该方法具有很好的准确性和实用性。获得的荧光定量PCR扩增曲线，基线平整，指数区明显，斜率大且固定；线性范围广，17～36个循环都能测出；稳定性、重复性好，变异系数仅为0.47%；标准曲线表明，循环阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系，可对基因表达进行相对定量；缺氮条件下OsAMT1；3 与纯NH4+处理相比表达量增加4倍以上。

关键词: 实时荧光定量PCR , 扩增曲线, 标准曲线, 铵转运蛋白基因, 基因表达, 研究方法