Kong Qiusheng, Yuan Jingxian, Gao Lingyun, Zhao Shuang, Jiang Wei, Huang Yuan, Bie Zhilong
Key Laboratory of Horticultural Plant Biology, Ministry of Education/College of Horticulture and Forestry, Huazhong Agricultural University, Wuhan, China.
PLoS One. 2014 Feb 28;9(2):e90612. doi: 10.1371/journal.pone.0090612. eCollection 2014.
Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT-PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT-PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT-PCR analyses involving watermelon.
西瓜是主要的葫芦科作物之一，其基因组序列的公布极大地促进了对它的基础研究。定量实时逆转录聚合酶链反应（qRT-PCR）是基因表达分析的首选方法，使用经过验证的内参基因进行标准化对于确保该方法的准确性至关重要。然而，尚未对西瓜的内参基因进行系统验证。在本研究中，使用qRT-PCR对西瓜中15个候选内参基因的转录本进行了定量，并使用geNorm和NormFinder比较了这些基因的稳定性。geNorm分别确定ClTUA和ClACT、ClEF1α和ClACT、ClCAC和ClTUA为正常生长条件、非生物胁迫和生物胁迫下西瓜器官和组织中最佳的内参基因对。NormFinder分别确定ClYLS8、ClUBCP和ClCAC为上述实验条件下最佳的单个内参基因。ClYLS8和ClPP2A被确定为所有样本中最佳的内参基因。根据实验条件，需要2至9个内参基因进行更可靠的标准化。在实验条件下，广泛使用的西瓜内参基因18SrRNA比其他内参基因稳定性差。在西瓜基因组中鉴定出过氧化氢酶家族基因，并用于验证所鉴定内参基因的可靠性。在低温胁迫下，ClCAT1和ClCAT2在前24小时被诱导并上调，而ClCAT3在叶片中被下调。然而，当使用18SrRNA作为内参基因时，这些基因的表达水平被显著高估和错误解读。这些结果为涉及西瓜的qRT-PCR分析中的内参基因选择提供了一个良好的起点。
