Chinese Journal of Rice Science

• 实验技术 • Previous Articles     Next Articles

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

GAO Dong, HE Xiahong, WANG Yunyue, LI Chengyun, ZHU Youyong*   

  1. The National Center for Agricultural Biodiversity/Key Laboratory of Agricultural Biodiversity for Plant Disease Management, Ministry of Education/Key Laboratory of Plant Pathology, Yunnan Agricultural University, Kunming 650201, China; *Corresponding author, E-mail: yppl@public.km.yn.cn
  • Received:1900-01-01 Revised:1900-01-01 Online:2009-05-10 Published:2009-05-10

水稻GA20ox-2基因mRNA的TaqMan荧光定量RT-PCR检测

高 东, 何霞红, 王云月, 李成云, 朱有勇*   

  1. 云南农业大学 农业生物多样性应用技术国家工程研究中心/农业生物多样性和控制病虫害教育部重点实验室/云南省植物病理重点实验室, 云南 昆明 650201; *通讯联系人,E-mail: yppl@public.km.yn.cn

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