Abstract
Cucumber green mottle mosaic virus (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (Cucumis sativus). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression. Most commonly used reference genes involved in CGMMV-infected cucumber are not universally expressed depending on tissue types and stages of plant development. It is therefore crucial to identify suitable reference genes in investigating the role of miRNA expression. In this study, seven reference genes, including Actin, Tubulin, EF-1α, 18S rRNA, Ubiquitin, GAPDH and Cyclophilin, were evaluated for the most accurate results in analyses using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene expression was assayed on cucumber leaves, stems and roots that were collected at different days post inoculation with CGMMV. The expression data were analyzed using algorithms including delta-Ct, geNorm, NormFinder, and BestKeeper as well as the comparative tool RefFinder. The reference genes were subsequently validated using miR159. The results showed that EF-1α and GAPDH were the most reliable reference genes for normalizing miRNA expression in leaf, root and stem samples, while Ubiquitin and EF-1α were the most suitable combination overall.
Highlights
Cucumber (Cucumis sativus) is an economically important member of the gourd family, Cucurbitaceae, that is cultivated throughout the world [1, 2]
Melting curve and agarose gel electrophoresis analysis showed that a specific fragment of the expected size and a single peak were observed, respectively, in reverse transcriptase-PCR (RT-PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) amplifications (S1 and S3 Figs)
We have evaluated and validated the expression stability of seven selected reference genes, including Actin, Tubulin, EF-1α, 18S rRNA, Ubiquitin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Cyclophilin, to determine the most suitable reference genes used in RT-qPCR analysis of miRNA in leaf, stem and root tissues of cucumber infected by Cucumber green mottle mosaic virus (CGMMV)
Summary
Cucumber (Cucumis sativus) is an economically important member of the gourd family, Cucurbitaceae, that is cultivated throughout the world [1, 2]. Selection of reference genes for the normalization of microRNA expression affected by a range of biotic and abiotic stresses, and viral diseases constitute a major constraint to both cucumber quality and yield [3]. Cucumber green mottle mosaic virus (CGMMV) is one of the important viral pathogens of cucumber. CGMMV seriously affects the host’s physiology, leading to sterile flowers and abnormal fruit [4], which often results in reduced yields and a lowered market value [5]. Strict quarantine regulations have been imposed to prevent the spread of CGMMV between different regions of cultivation, and limit its negative effect on global cucumber production [6, 7]
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