烟草Nt-syr1基因实时荧光定量PCR检测方法  

A Real-time Fluorescent Quantitative PCR for Detecting Transcripts of Nt-syr1 Gene in Tobacco

作　　者：代晓燕[1] 苏以荣[1] 魏文学[1] 陈风雷 邹焱[3] 龙文 贾志红[1] 范业宽[4] 

机构地区：[1]中国科学院亚热带农业生态研究所亚热带农业生态重点实验室,长沙410125 [2]贵州省铜仁地区烟草公司,贵州铜仁554300 [3]贵州省烟草科学研究所,贵阳550003 [4]华中农业大学资源与环境学院,武汉430070

出　　处：《中国烟草科学》2008年第3期20-24,28,共6页Chinese Tobacco Science

基　　金：中国科学院知识创新重要方向项目(KSCX2-YW-N-48-1);贵州省烟草公司科技计划项目共同资助

摘　　要：根据烟草Nt-syr1基因mRNA序列设计特异引物,建立了SYBR Green I实时荧光定量PCR反应体系,对烟株打顶后叶片Nt-syr1基因进行了mRNA转录水平上的定量分析,为从分子生物学水平上研究烤烟钾素营养调控机理提供新的技术手段。该方法简单实用,获得的荧光定量PCR扩增曲线基线平整,指数区扩增明显,斜率大;稳定性和重现性好,变异系数小;循环阈值Ct与PCR起始模板量的对数值之间存在良好的线性关系。对基因表达结果分析表明,烟株打顶后1 h,Nt-syr1基因在叶片中强烈表达,表达量约是同期不打顶处理的480倍,随后逐渐降低。与打顶处理相比,打顶后涂抹生长调节剂可以降低其表达量。The project aimed to establish real-time fluorescent quantitative PCR detecting Nt-syr1 gene in tobacco and provided a new technique for the mechanism study of potassium regulation at the molecular level. By designing two pairs of amplification primers based the mRNA sequence of Nt-syr1 in tobacco and establishing SYBR Green I reaction system of real-time fluorescent quantitative PCR, quantitative analysis was conducted to detect the changes of Nt-syr1 gene in tobacco leaves after decapitation. The technique showed that the amplification curve had flat baseline, distinct exponential area, large and stable slope, and the coefficient of variation was very little. There was a linear relationship between threshold cycle values at which samples crossed threshold and the logarithmic values of template concentration. The result indicated that the Nt-syr1 gene was strongly expressed in tobacco leaves after decapitation about 1 hour, and the expression was enhanced about 480-fold in comparison to no decapitation tobacco, but using plant growth regulators could decrease the expression caused by decapitation.

关 键 词：实时荧光定量PCR 烟草 Nt—syr1 基因表达 

分 类 号：S572[农业科学—烟草工业]