Chan Pek-Lan, Rose Ray J, Abdul Murad Abdul Munir, Zainal Zamri, Low Eng-Ti Leslie, Ooi Leslie Cheng-Li, Ooi Siew-Eng, Yahya Suzaini, Singh Rajinder
Advanced Biotechnology and Breeding Centre, Malaysian Palm Oil Board (MPOB), No. 6, Persiaran Institusi, Bandar Baru Bangi, Kajang, Selangor, Malaysia.
Australian Research Council Centre of Excellence for Integrative Legume Research, School of Environmental and Life Sciences, The University of Newcastle, New South Wales, Australia.
PLoS One. 2014 Jun 13;9(6):e99774. doi: 10.1371/journal.pone.0099774. eCollection 2014.
The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels.
In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization.
Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.
体细胞胚胎发生组织培养过程已被用于繁殖高产油棕。由于愈伤组织形成率和胚胎发生率较低，因此开展了分子研究以鉴定调控该过程的基因，其表达水平通常使用逆转录定量实时PCR（RT-qPCR）进行定量分析。随着油棕基因组序列的最新发布，采用RT-qPCR建立合适的基因分析策略至关重要。为准确量化基因表达水平，应选择最合适的内参基因。
在本研究中，使用RT-qPCR对从cDNA微阵列研究和文献综述中挑选出的8个候选内参基因，在26个组织培养样本中进行了全面评估。这些样本取自两个组织培养系和培养基处理组，包括叶片外植体培养物、连续发育阶段的愈伤组织和胚状体。三种统计算法（geNorm、NormFinder和BestKeeper）证实，新型内参基因（pOP-EA01332、PD00380和PD00569）的表达稳定性优于传统管家基因（GAPDH、NAD5、微管蛋白、泛素和肌动蛋白）。PD00380和PD00569被确定为在所有样本、MA2和MA8组织培养系中表达最稳定的基因。它们在验证一个假定的乙烯响应转录因子3样基因表达谱方面的适用性，证明了使用两个基因的几何平均值进行标准化的重要性。
在油棕组织培养样本中建立了用于RT-qPCR的最稳定表达内参基因的系统选择方法。选择了PD00380和PD00569用于RT-qPCR基因表达数据的准确可靠标准化。这些数据对于与组织培养过程相关的研究将具有重要价值。此外，本文所述方法将有助于在其他油棕组织以及与产量、生物和非生物胁迫相关基因的表达谱分析中选择合适的内参基因。
