Sinha Pallavi, Singh Vikas K, Suryanarayana V, Krishnamurthy L, Saxena Rachit K, Varshney Rajeev K
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, 502324, India.
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, 502324, India; School of Plant Biology and Institute of Agriculture, The University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia.
PLoS One. 2015 Apr 7;10(4):e0122847. doi: 10.1371/journal.pone.0122847. eCollection 2015.
Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.
使用定量实时聚合酶链反应（qRT-PCR）进行基因表达分析是一项非常灵敏的技术，其灵敏度取决于研究中所使用的参照基因的稳定性能。许多持家基因已被用于多种作物的各类表达研究中，然而，人们发现它们在不同胁迫条件下的表达并不一致。因此，针对几种作物的不同表达研究，已推荐使用物种特异性持家基因。然而，尽管木豆（Cajanus cajan）已有可用的基因组序列，但尚未见有关木豆特异性持家基因的报道。为了鉴定木豆在干旱胁迫条件下用于表达分析的稳定持家基因，研究了阿莎（ICPL 87119）根、茎和叶组织中10个常用持家基因（EF1α、UBQ10、GAPDH、18SrRNA、25SrRNA、TUB6、ACT1、IF4α、UBC和HSP90）的相对表达变化。使用geNorm、NormFinder和BestKeeper三种统计算法来确定候选基因的稳定性。geNorm分析确定IF4α和TUB6为最稳定的持家基因，然而，NormFinder分析确定IF4α和HSP90为干旱胁迫条件下最稳定的持家基因。随后，在基于qRT-PCR的uspA基因表达分析中对鉴定出的候选基因进行了验证，uspA基因在木豆干旱胁迫条件下起重要作用。uspA基因的相对定量根据内部对照（稳定和最不稳定基因）而变化，从而突出了在此类实验中选择以及验证内部对照的重要性。鉴定出的稳定且经过验证的持家基因将有助于木豆的基因表达研究，尤其是在干旱胁迫条件下。
