摘要
从水稻花序分生组织总RNA中逆转录扩增总cDNA,PCR扩增OsCKX2基因中设计的目的片段,将纯化的目的片段克隆到pMD19-TSimple载体中,PCR鉴定并测序分析,制备成荧光定量PCR梯度浓度质粒标准品。对重组质粒进行实时荧光定量PCR实验。标准曲线结果表明,建立的OsCKX2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1.0×102~1.0×107拷贝;扩增效率高（E=95.9%）;稳定性、重复性好,可靠性高,批内和批间变异系数（CV）仅为：0.14%~0.29%和0.16%~0.35%;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=1.000）,可对OsCKX2基因表达进行准确实时定量。一个基于TaqMan实时荧光定量RT-PCR技术,定量分析控制水稻谷粒数关键基因之一OsCKX2转录水平的技术体系被成功建立。该技术体系重组质粒标准品的制备方法具有很强的实用性;对基因OsCKX2的表达实时定量准确、可靠、便捷。
Total RNA extracted from inflorescence meristem of rice was reverse transcribed to cDNA.The prospective amplicon was amplified and purified,then was ligated with pMD19-T Simple vector.Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced.The combined plasmid was diluted to series as standard for FQ-PCR.OsCKX2 was amplified by real-time fluorescence quantitative PCR from the plasmid DNA.Standard curve showed： the established system had strict specificity and sensitivity,and had 1.0×102~1.0×107 copies respondent capability of initiative templates,and had good stability and repeatability,the coefficient of variation intra-and inter-batch were 0.14%~0.29% and 0.16%~0.35%.There was a well linear relationship between threshold cycle value at which sample crosses threshold and the logarithmic value of template concentration（correlation coefficient = 1.000）.A technique assay for real-time quantification of OsCKX2 expression in rice using TaqMan fluorescence quantitative PCR with specific primers and probe was established,which showed： the preparation of standard plasmid DNA for real-time quantifying transcripts of OsCKX2 had corking practicability in the established system,and had exact,authentic and convenient strongpoint.
出处
《云南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2010年第3期351-357,共7页
Journal of Yunnan Agricultural University:Natural Science
基金
国家“973”计划资助项目(2006CB100200)
