Ling Hui, Wu Qibin, Guo Jinlong, Xu Liping, Que Youxiong
Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China.
PLoS One. 2014 May 13;9(5):e97469. doi: 10.1371/journal.pone.0097469. eCollection 2014.
The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species.
越来越多地用于基因表达分析的实时定量逆转录聚合酶链反应（qRT-PCR）方法需要一个或几个作为标准化因子的参考基因。为了促进对甘蔗（一种基因组信息有限的非模式植物）基因表达的研究，对13个候选参考基因的稳定性进行了评估。采用geNorm、NormFinder和deltaCt方法来选择在不同组织和各种实验处理条件下稳定表达的内参基因。这些结果表明，在这13个候选参考基因中，甘油醛-3-磷酸脱氢酶（GAPDH）、真核生物延伸因子-1α（eEF-1a）和真核生物翻译起始因子-4α（eIF-4α）是最稳定的，适合用作所有各种实验样品的标准化因子。此外，腺嘌呤磷酸核糖转移酶（APRT）可能是用于研究甘蔗组织样品中基因拷贝数与转录水平之间关系的候选基因。根据geNorm评估的结果，在激素处理实验中将CUL和eEF-1α组合；在非生物胁迫试验中将CAC和CUL组合；在所有处理样品中将GAPDH、eEF-1a和CUL组合，在品种组织中将CAC、CUL、APRT和TIPS-41组合作为标准化组，将导致甘蔗中更准确可靠的表达定量。这是首次对甘蔗转录本表达谱定量的参考基因进行系统验证。本研究应为选择参考基因以更准确地定量甘蔗和其他植物物种中的基因表达提供有用信息。
