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Artículo

Selection and optimization of reference genes for RT-qPCR normalization: a case study in Solanum lycopersicum exposed to UV-B

Fernández, María BelénIcon ; Lukaszewicz, GermánIcon ; Lamattina, LorenzoIcon ; Cassia, Raul OscarIcon
Fecha de publicación: 22/03/2021
Editorial: Elsevier France-Editions Scientifiques Medicales Elsevier
Revista: Plant Physiology and Biochemistry
ISSN: 0981-9428
Idioma: Inglés
Tipo de recurso: Artículo publicado
Clasificación temática:
Bioquímica y Biología Molecular

Resumen

Quantitative RT- PCR is one of the most common methods to study gene expression in response to stress. Therefore, it is crucial to have suitable reference genes (RGs) for result normalization. Although several reports describe UV-B-modulated gene expression in Solanum lycopersicum, there are no suitable RGs identified until now. The aim of this work was to evaluate the suitability of seven traditional genes: actin (ACT), tubulin (TUB), ubiquitin (UBI), glyceraldehyde- 3 phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), phosphatase 2A catalytic subunit (PP2A) and GAGA binding transcriptional activator (GAGA); and two non-traditional genes: thioredoxin h1 (TRX h1) and UV-B RESISTANCE LOCUS 8 (UVR8), as candidate RGs for their potential use as reliable internal controls in leaves, stems and roots of tomato seedlings exposed to acute and chronic UV-B. The stability of these genes expression was evaluated using five statistical algorithms: geNorm, NormFinder, BestKeeper, Delta Ct and ANOVA. Considering the comprehensive stability ranking, we recommend ACT+TUB as the best pair of RGs for leaves, PP2A+GAPDH+TRX h1 for stems and TUB+UVR8 for roots. The reliability of the selected RGs for each tissue was verified amplifying tomato chalcone synthase 1 (CHS1) and cyclobutane pyrimidine dimer (CPD) photolyase (PHR1-LIKE). Under UV-B treatment, CHS1 was upregulated in leaves, stems and roots whereas PHR1-LIKE was only upregulated in leaves and stems. This interpretation differs when the most and least stable RGs are chosen. This is the first report regarding suitable RGs selection for accurate normalization of gene expression in tomato seedlings exposed to UV-B irradiation.
Palabras clave: CHS1 , PHR1-LIKE , QUANTITATIVE RT-PCR , REFERENCE GENE , SOLANUM LYCOPERSICUM , UV-B , UVR8
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info:eu-repo/semantics/restrictedAccess Excepto donde se diga explícitamente, este item se publica bajo la siguiente descripción: Creative Commons Attribution-NonCommercial-ShareAlike 2.5 Unported (CC BY-NC-SA 2.5)
Identificadores
URI: http://hdl.handle.net/11336/148920
URL: https://linkinghub.elsevier.com/retrieve/pii/S0981942821000371
DOI: http://dx.doi.org/10.1016/j.plaphy.2021.01.026
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Articulos(IIB)
Articulos de INSTITUTO DE INVESTIGACIONES BIOLOGICAS
Citación
Fernández, María Belén; Lukaszewicz, Germán; Lamattina, Lorenzo; Cassia, Raul Oscar; Selection and optimization of reference genes for RT-qPCR normalization: a case study in Solanum lycopersicum exposed to UV-B; Elsevier France-Editions Scientifiques Medicales Elsevier; Plant Physiology and Biochemistry; 160; 22-3-2021; 269-280
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