TaqMan荧光定量RT-PCR检测水稻RAc1基因mRNA方法的建立  

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

作　　者：高东[1] 王云月[1] 何霞红[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：《云南植物研究》2009年第1期75-81,共7页Acta Botanica Yunnanica

基　　金：国家973计划项目(2006CB100200)

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-TSimple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260nm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。The cDNA was cloned as the standard for real-time quantifying mRNA of RAcl of rice and the TaqMan fluorescence quantitative PCR assay for detecting RAcl 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. RAcl was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The method of RAcl 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 ken constructed successfully, and real-time TaqMan-Fluorescence Quantitative RT-PCR was reliable to quantitatively evaluate mRNA of RAcl of rice. Furthermore, RAcl gene as the internal control gene for detectingother gene expression of rice.

关 键 词：水稻 肌动蛋白 基因表达 实时定量RT-PCR TAQMAN探针 

分 类 号：Q943[生物学—植物学] S511[农业科学—作物学]