相对定量和绝对定量—以CsSAMDC基因表达分析为例

Relative and Absolute Quantification-Expression Analysis of CsSAMDC Gene As a Case

以无芒隐子草干旱诱导的cDNA文库中获得的SAMDC序列为基础,应用实时荧光定量RT-PCR技术同时结合相对定量和绝对定量方法,以分析无芒隐子草SAMDC基因在不同组织、不同胁迫梯度下的表达为案例,详细介绍并比较相对定量和绝对定量在基因表达方面的应用。分别采集无芒隐子草幼苗自然干旱0,2,4,6,8,10d以及恢复浇水1,4d的叶片和根系材料,相对定量采用2-△△CT法;以相应基因的质粒扩增产物为标准品制作标准曲线,对定量PCR的引物浓度进行了优化,而后筛选最稳定的内参基因,以内参基因获得的标准化因子对绝对定量数据进行标准化。相对定量2-△△CT法结果显示,CsSAMDC基因在干旱胁迫8d后,叶片中的表达量为胁迫前的0.62±0.13倍,根中的表达量为胁迫前的5.93±0.71倍。绝对定量表明,CsSAMDC基因只在胁迫第8d和第10d的根组织中检测到表达量显著增大,胁迫第4～6d和恢复浇水后1～4d,CsSAMDC基因在叶和根组织中的表达较对照降低。相对定量和绝对定量在CsSAMDC基因表达研究中显示相对一致的趋势。

This experiment was based on a full-length SAMDC sequence from the drought stress induced cDNA library of Cleistogenes songorica. Two methods, absolute quantification and relative quantification, were used to analyze the gene expression under different tissue and drought stress treatment by real-time quantitaive PCR experiments and were compared each other, while CsSAMDC gene was used as a case. Leaves and roots tissue were sampled from the plants at 0, 4, 6, 8, and 10 d of drought stress and 1 and 4 d after rewatering. The 2^ΔΔCT method was used to analyze the relative changes in gene expression from quantitative real-time PCR experiments. A standard curve was generated by the purified plasmid DNA. The primer concentration in the realtime PCR was optimized. All absolute quatification data were normalized by the normalization factor from the most stable house-keeping genes in C. songorica. Relative quantification results using 2^ΔΔCT method showed that CsSAMDC transcripts increased 5.93± 0.71 fold in roots, down regulated 0.62 ± 0. 13 fold in leaves, under drought stress. Absolute quantification results showed that CsSAMDC transcripts up-regulated in roots significantly under drought stress as long as 8 to 10 days after drought stress, while down-regulated in both roots and leaves after drought stress within 6 days and 1 to 4 days after rewatering, compared with control. CsSAMDC transcipt showed similar tendency of gene expression compared by using relative and absolute quantification methods.