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当前位置: 首页 > 文章 > 水稻GA20ox-2基因mRNA的TaqMan荧光定量RT-PCR检测 中国水稻科学 2009,23 (3) 309-314
Position: Home > Articles > Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of mRNA of GA20ox-2 in Rice Chinese Journal of Rice Science 2009,23 (3) 309-314

水稻GA20ox-2基因mRNA的TaqMan荧光定量RT-PCR检测

作  者：
高东;何霞红;王云月;李成云;朱有勇
单  位：
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
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
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。
译  名：
Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of mRNA of GA20ox-2 in Rice
作  者：
GAO Dong,HE Xia-hong,WANG Yun-yue,LI Cheng-yun,ZHU You-yong(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)
关键词：
rice;semi-dwarf gene;gene expression;real-time quantification;reverse transcription-polymerase chain reaction;probe
摘  要：
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.

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