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实时定量PCR技术(qRT-PCR)能够快速地对多个样品进行基因表达分析,因此已成为芯片和微点阵数据验证的常用手段.为了得到更精确的数据,通常需要一个和多个内对照基因来平衡化qRT-PCR数据.目前一般选择持家基因(housekeeping gene)作为内对照,但很多持家基因的表达水平随着环境的改变而改变.在棉花纤维发育和体细胞胚发生机制研究过程中已经产生大量芯片和微点阵数据,但目前在采用实时定量PCR验证这些数据时都只用了一个持家基因来平衡化数据.为了验证其可靠性,本研究根据常用的持家基因(18S rRNA,Histone3,UBQ7,Actin,Cyclophilin,Gbpolyubiquitin-1和Gbpolyubi-quitin-2)设计了7对引物,用相对的绝对定量法验证了在棉花不同组织和不同发育阶段(21个样品)表达水平的稳定性,发现在纤维发育的后期(17 DPA(day post anthesis)以后),这些基因的表达都下调,而纤维发育后期特异表达基因AGP的表达从15DPA开始到27DPA一直都呈上升趋势.因此对于纤维系列特别是纤维发育后期基因的qRT-PCR更好的选择是相对的绝对定量.而对于非纤维系列,这些持家基因的表达水平相对纤维系列变化幅度较小,但为了得到更精确的表达数据,应该同时用Histone3,UBQ7和Gbpolyubiquitin-1一起来平衡化qRT-PCR数据.
Real-time quantitative PCR (qRT-PCR) enables rapid gene expression analysis of multiple samples and has therefore become a popular means of chip and microarray data validation. In order to obtain more accurate data, one and more internal controls are usually required Genes to balance qRT-PCR data.Generally housekeeping genes are commonly used as internal controls, but many housekeeping genes change with environmental changes.In the process of cotton fiber development and somatic embryogenesis A large number of chips and microarray data have been generated but only one housekeeping gene has been used to validate these data by real-time quantitative PCR to balance the data.To verify the reliability, this study based on the commonly used housekeeping genes (18S rRNA, Histone3, UBQ7, Actin, Cyclophilin, Gbpolyubiquitin-1 and Gbpolyubi-quitin-2) were used to design seven pairs of primers. The relative absolute quantitative method was used to verify the stability of expression levels in different tissues and different developmental stages of cotton (21 samples) , And found that the expression of these genes was down-regulated in the late stage of fiber development (after 17 DPA day-post anthesis), whereas the late-stage fiber-specific gene AG P expression has been increasing from 15 DPA to 27 DPA, so a better choice for qRT-PCR in fiber series, especially in late-developmental genes, is relative absolute quantification, whereas for non-fiber series these housekeeping gene expression levels The relative changes in fiber series are small, but qRT-PCR data should be equilibrated with Histone 3, UBQ7 and Gbpolyubiquitin-1 simultaneously in order to obtain more accurate expression data.