Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon.
Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon.
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DOI：
10.1371/journal.pone.0087197
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Bie Z
中科院分区：
文献类型：
--
作者：
Kong Q;Yuan J;Niu P;Xie J;Jiang W;Huang Y;Bie Z
甜瓜（Cucumis梅洛. L）不仅是一种重要的经济作物，而且是研究许多生物学特性的理想模式作物。筛选合适的参考基因是进行实时荧光定量PCR（RT-qPCR）的关键。在这项研究中，14个候选的参考基因，并在植物的根和叶片中进行生物胁迫，非生物胁迫，和植物生长调节剂处理的表达变化进行了RT-qPCR评估。使用geNorm和Normalt确定所选基因表达的稳定性并进行排序。geNorm为每组条件鉴定了两个最稳定的基因：所有样品中的CmADP和CmUBIep，根中的CmUBIep和CmRPL，叶中的CmRAN和CmACT，非生物胁迫条件下的CmADP和CmRPL，生物胁迫条件下的CmTUA和CmACT，以及植物生长调节剂处理下的CmRAN和CmACT。Normandy确定CmRPL是根和生物胁迫条件下的最佳参考基因，而CmADP在其他实验条件下是最佳参考基因。发现CmUBC 2和CmPP 2A在许多实验条件下不适合。在甜瓜基因组中鉴定了过氧化氢酶家族基因CmCAT 1、CmCAT 2和CmCAT 3，并将其作为靶基因来验证所鉴定的参考基因的可靠性。过氧化氢酶家族基因在接种枯萎病后3天表现出最大的上调，之后它们被下调。当使用不合适的参考基因进行标准化时，它们的表达水平被显著高估。这些结果不仅为甜瓜基因表达分析中参考基因的选择提供了指导，而且为研究过氧化氢酶家族基因在逆境响应中的功能提供了有价值的信息。
Melon (Cucumis melo. L) is not only an economically important cucurbitaceous crop but also an attractive model for studying many biological characteristics. Screening appropriate reference genes is essential to reverse transcription quantitative real-time PCR (RT-qPCR), which is key to many studies involving gene expression analysis. In this study, 14 candidate reference genes were selected, and the variations in their expression in roots and leaves of plants subjected to biotic stress, abiotic stress, and plant growth regulator treatment were assessed by RT-qPCR. The stability of the expression of the selected genes was determined and ranked using geNorm and NormFinder. geNorm identified the two most stable genes for each set of conditions: CmADP and CmUBIep across all samples, CmUBIep and CmRPL in roots, CmRAN and CmACT in leaves, CmADP and CmRPL under abiotic stress conditions, CmTUA and CmACT under biotic stress conditions, and CmRAN and CmACT under plant growth regulator treatments. NormFinder determined CmRPL to be the best reference gene in roots and under biotic stress conditions and CmADP under the other experimental conditions. CmUBC2 and CmPP2A were not found to be suitable under many experimental conditions. The catalase family genes CmCAT1, CmCAT2, and CmCAT3 were identified in melon genome and used as target genes to validate the reliability of identified reference genes. The catalase family genes showed the most upregulation 3 days after inoculation with Fusarium wilt in roots, after which they were downregulated. Their levels of expression were significantly overestimated when the unsuitable reference gene was used for normalization. These results not only provide guidelines for the selection of reference genes for gene expression analyses in melons but may also provide valuable information for studying the functions of catalase family genes in stress responses.
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