Abstract
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.
Highlights
Melon (Cucumis melo L.), a member of the Cucurbitaceae family, ranks as the 9th most cultivated horticultural crop in terms of total world production [1]
A total of 14 genes that have been used in the studies of cucurbit and other crops were selected for use as candidate reference genes for melons
To further confirm the specificity of the primer pairs, the PCR amplification products were analyzed by agarose gel electrophoresis using genomic DNA and mixed cDNA as templates
Summary
Melon (Cucumis melo L.), a member of the Cucurbitaceae family, ranks as the 9th most cultivated horticultural crop in terms of total world production [1]. The full sequence of melon genome is readily available, which greatly facilitates the identification of genes underlying certain traits and the elucidation of mechanisms that regulate relevant characteristics [2]. Gene expression analysis is a major experimental approach in functional genomics studies. Reverse transcription quantitative real-time PCR (RT-qPCR) is a standard tool for the quantification of levels of gene expression. It is rapid, sensitive, and specific [4]. The reliability and accuracy of RT-qPCR is lost if inappropriate reference gene is selected [7]. The selection of suitable reference gene is vital to RT-qPCR analysis
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