摘要
实时荧光定量PCR(qRT-PCR)技术具有高灵敏性、高保真性和高特异性,被广泛应用于基因表达的分析。在数据处理过程中,选用稳定表达的基因作为内参基因对准确分析实验结果非常关键。以毛果杨(Populus trichocarpa)的不同组织以及锌胁迫下的组培苗为材料,使用荧光定量PCR方法分析了TUA8、TUB6、ubiquitin、GAPDH、actin、18S rRNA和EF1α7个看家基因的表达情况。通过geNorm、NormFinder和BestKeeper 3个程序的综合分析,发现actin、ubiquitin、EF1α和18S rRNA的稳定性较好,可用作毛果杨基因表达研究的内参基因;而TUB6在不同组织中稳定性最差;GAPDH在锌胁迫下的组织中稳定性最差,因此不适宜作为内参基因。毛果杨NAC基因的表达分析,进一步验证了上述结果。该研究对采用qRT-PCR方法分析毛果杨基因表达过程中内参基因的选择具有指导作用,同时对揭示NAC基因的功能也有一定的意义。
Quantitative RT-PCR （qRT-PCR） has been widely used in gene expression analysis because of its sensitivity, specificity, and reproducibility. Application of suitable reference genes to normalize qRT-PCR data is critical in analyzing PCR results. We analyzed the expression patterns of 7 housekeeping genes, including TUA8, TUB6, ubiquitin, GAPDH, actin, 18S rRNA and EFla, in various tissues of greenhouse-grown Populus trichocarpa and Zn-treated in vitro plantlets. The stability of housekeeping gene expression was analyzed with use of 3 software packages, including geNorm, Norm- Finder, and BestKeeper. The genes actin, ubiquitin, EFla and 18S rRNA were suitable reference genes for efficient normalization of qRT-PCR data, whereas TUB6 and GAPDH were not suitable for analysis of greenhouse-grown plants and Zn-treated plantlets, respectively. These findings were confirmed by comparative profiling of 4 P. trichocarpa NAC genes. This study provides useful information for reference gene selection in qRT-PCR analysis of gene expression in P. trichocarpa. It is also helpful to elucidate the function of P. trichocarpa NAC genes.
出处
《植物学报》
CAS
CSCD
北大核心
2013年第5期507-518,共12页
Chinese Bulletin of Botany
基金
国家重点基础研究发展规划(No.2012CB114502)
国家自然科学基金(No.31070534)
