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Plant Diversity ›› 2009, Vol. 31 ›› Issue (1): 75-81.DOI: 10.3724 SP.J.1143.2009.08142

• 研究论文 • 上一篇    下一篇

TaqMan 荧光定量RT-PCR 检测水稻RAc1基因mRNA 方法的建立

高 东, 王云月, 何霞红, 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
  • 收稿日期:2008-07-19 出版日期:2009-02-25 发布日期:2009-02-25
  • 通讯作者: 朱有勇

Establishment of Real-time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of mRNA Expression of RAc1 of Rice

GAO Dong, WANG Yun-Yue, HE Xia-Hong, LI Cheng-Yun , ZHU You-Yong   

  1. The National Center for Agricultural Biodiversity, Ministry of Education Key Laboratory of Agricultural Biodiversity for Plant Disease Management , Key laboratory of Plant Pathology , Yunnan Agricultural University , Kunming 650201 , China
  • Received:2008-07-19 Online:2009-02-25 Published:2009-02-25
  • Contact: ZHU You-Yong

摘要: 构建包含RAc1 基因cDNA 片段的质粒, 作为水稻肌动蛋白基因RAc1 之mRNA 定量检测的标准品,建立检测方法, 为水稻其他基因的定量建立内参。从水稻叶总RNA 中逆转录扩增总cDNA, PCR 扩增RAc1基因中设计的目的片段, 将纯化的目的片段与pMD19-T Simple 载体进行连接, 转化宿主菌JM-109 , 提取重组质粒DNA, PCR 鉴定并测序分析。纯化质粒并检测260 nm 吸光值, 确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR 梯度浓度标准品, 进行实时荧光定量PCR 实验。建立了RAc1 基因mRNA 表达实时荧光定量PCR 检测方法, 特异性好, 检测灵敏度达102 拷贝, 线性范围为102 ~107 拷贝, 阈值循环数(Ct )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= 1.000 ), 扩增效率高(E= 98. 2% )。建立了基因RAc1 实时定量PCR 的质粒标准品。

关键词: 水稻, 肌动蛋白, 基因表达, 实时定量RT-PCR, TaqMan 探针

Abstract: The cDNA was cloned as the standard for real-time quantifying mRNA of RAc1 of rice and the TaqMan fluorescence quantitative PCR assay for detecting RAc1 gene expression was established as internal control gene for detecting other gene expression of rice . Total RNA extracted from leaf of rice was reverse transcribed to cDNA . The prospective amplicon was amplified and purified , then was ligated with pMD19-T Simple vector and transformed into bacterium JM-109. Plasmid DNA extracted from positive clones were verified by PCR amplification and sequenced . The concentration of purified DNA
template was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for FQ-PCR . RAc1 was amplified by real- time fluorescence quantitative PCR from the plasmid DNA . The method of RAc1 mRNA real-time PCR was well established , which detected as low as 102 copies with the linear range from 102 to 107 copies
. The standard curves showed high correlations (r = 1.000) and PCR efficiency ( E = 98. 2%) . A series of standards for real-time PCR analysis had been constructed successfully, and real-time TaqMan-Fluorescence Quantitative RT-PCR was reliable to quantitatively evaluate mRNA of RAc1 of rice. Furthermore, RAc1 gene as the internal control gene for detecting other gene expression of rice .

Key words: Rice