Condori Jose, Nopo-Olazabal Cesar, Medrano Giuliana, Medina-Bolivar Fabricio
Arkansas Biosciences Institute, Arkansas State University, P,O, Box 639, State University, AR 72467, USA.
BMC Res Notes. 2011 Oct 10;4:392. doi: 10.1186/1756-0500-4-392.
Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments.
A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc).
This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2) and a gene encoding a ribosomal protein (RPL8C). A commonly used reference gene GAPDH showed low stability of expression suggesting that its use may lead to inaccurate gene expression profiles when used for data normalization in stress-stimulated hairy roots. Likewise the A. rhizogenes transgene rolC showed less expression stability than GAPDH. This study proposes that a minimum of two reference genes should be used for a normalization procedure in gene expression profiling using elicited hairy roots.
通过发根农杆菌介导的转化产生的毛状根培养物已成为阐明特殊代谢产物生物合成的实用生物学模型。为了有效了解参与这些化合物代谢途径的基因的表达模式，需要根据MIQE（定量实时PCR实验发表的最低信息）指南在特定实验条件下系统地验证参考基因。在本报告中，我们描述了花生毛状根培养物中用于RT-qPCR的参考基因的首次验证，该培养物在诱导剂处理后产生芪类化合物。
共评估了21个候选参考基因。19个基因是根据先前在植物中的qPCR研究选择的，另外两个来自发根农杆菌转移的T-DNA。利用花生候选基因在拟南芥中的同源序列获得了其核苷酸序列。为了鉴定合适的引物，为每个候选参考基因获得了校准曲线。数据分析后，选择了12个符合标准效率标准的候选基因。使用geNorm和NormFinder算法分析了这些基因的表达稳定性，并根据基因的表达稳定性建立了排名。结果表明，与用醋酸钠（NaOAc）处理的根培养物相比，茉莉酸甲酯（MeJA）处理的根培养物中候选参考基因的表达变化较小。
这项工作是首次在毛状根中验证用于RT-qPCR的参考基因。虽然这些基因是在NaOAc和MeJA处理条件下选择的，但我们预计这些基因将为各种胁迫条件下的毛状根参考基因提供良好的靶点。主要参考基因是一个编码TATA盒结合蛋白（TBP2）的基因和一个编码核糖体蛋白（RPL8C）的基因。常用的参考基因GAPDH显示出较低的表达稳定性，这表明在胁迫刺激的毛状根中用于数据归一化时，其使用可能导致不准确的基因表达谱。同样，发根农杆菌转基因rolC的表达稳定性也低于GAPDH。本研究提出，在使用诱导毛状根进行基因表达谱分析的归一化过程中，应至少使用两个参考基因。
