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。
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.
出处
《中国水稻科学》
CAS
CSCD
北大核心
2006年第1期8-12,共5页
Chinese Journal of Rice Science
基金
国家自然科学基金资助项目(30471037)
国家973计划资助项目(2005cb120903)。
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
real-time fluorescence quantitative PCR
amplification curve
standard curve
ammonium transporter gene
gene expression
methodology
