Chinese Journal of Rice Science

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Establishment and Application of a Real-Time Fluorescence Quantitative PCR for Detecting Transcripts of Low Abundance Gene, OsAMT1;3,in Rice

SUN Shu-bin , LI Bao-zhen , HU Jiang , XU Guo-hua   

  • Received:1900-01-01 Revised:1900-01-01 Online:2006-01-10 Published:2006-01-10

水稻低丰度表达基因OsAMT1;3实时荧光定量PCR方法的建立及其应用

孙淑斌;李宝珍;胡江;徐国华*   

  1. 南京农业大学 资源与环境科学学院, 江苏 南京 210095; *通讯联系人, E-mail: ghxu@njau.edu.cn

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