RealTime TaqManFluorescence Quantitative RT-PCR Assay for Detection and Quantification of mRNA of   GA20ox-2  in  Rice

Abstract

Abstract: A technique for realtime quantification of GA20ox-2 expression in rice using TaqMan fluorescence quantitative PCR with specific primers and probe was established. The preparation of standard plasmid DNA for realtime quantification of transcripts of GA20ox-2 had a good practicability in the established system. The technique was exact, authentic and convenient. Standard curve showed the established system had a strict specificity and sensitivity, and had a 102 to 107 copies respondent capability of initiative templates, and had a good stability and repeatability, with the coefficients of variation in intra and interbatch were 0.12% to 0.31% and 0.21% to 0.34%, respectively. There were a high PCR efficiency (E=1003%) and a good linear relationship between threshold cycle value at which sample crosses threshold and the logarithmic value of template concentration (correlation coefficient = 0.999). A series of standards for realtime PCR analysis have been constructed successfully, and realtime TaqManfluorescence quantitative RTPCR is reliable to quantitatively evaluate mRNA of GA20ox-2 in rice.

Key words: rice, semidwarf gene, gene expression, realtime quantification, reverse transcriptionpolymerase chain reaction, probe

摘要： 成功建立了一项基于TaqMan 实时荧光定量的RT-PCR技术，定量分析水稻半矮化关键基因之一GA20ox-2转录水平。该技术体系中重组质粒标准品的制备方法具有很好的实用性；质粒标准品对基因GA20ox-2表达的实时定量准确、可靠、便捷。标准曲线表明，所建立的GA20ox-2基因mRNA表达实时荧光定量PCR检测方法，特异性好，灵敏度高，可达102拷贝；线性范围广，可达102～107拷贝；扩增效率高（E=100.3%）；稳定性、重复性好，可靠性高，批内和批间变异系数仅分别为0.12%～0.31%和0.21%～0.34%；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（^r＝0.999），可对GA20ox2基因表达进行准确实时定量。

关键词: 水稻, 矮秆基因, 基因表达, 实时定量, 逆转录聚合酶链式反应, 探针

GAO Dong,HE Xiahong,WANG Yunyue,LI Chengyun,ZHU Youyong*. RealTime TaqManFluorescence Quantitative RT-PCR Assay for Detection and Quantification of mRNA of GA20ox-2 in Rice
[J]. Chinese Journal of Rice Science.

高东, 何霞红, 王云月, 李成云, 朱有勇. 水稻GA20ox-2基因mRNA的TaqMan荧光定量RT-PCR检测 [J]. 中国水稻科学.

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RealTime TaqManFluorescence Quantitative RT-PCR Assay for Detection and Quantification of mRNA of GA20ox-2 in Rice

水稻GA20ox-2基因mRNA的TaqMan荧光定量RT-PCR检测

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