【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。更多还原

【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.更多还原