水稻GA20ox-2基因mRNA的TaqMan荧光定量RT-PCR检测  

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

作　　者：高东[1] 何霞红[1] 王云月[1] 李成云[1] 朱有勇[1] 

机构地区：[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

出　　处：《中国水稻科学》2009年第3期309-314,共6页Chinese Journal of Rice Science

基　　金：国家973计划资助项目(2006CB100200)

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。A technique for real-time quantification of GA2Oox-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 GA2Oox-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 intraand inter-batch were 0.12% to 0.31% and 0. 2100 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 GA2Oox-2 in rice.

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 

分 类 号：S511[农业科学—作物学]