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EnglishThe aim of this study was to analyze the expression of putative reference genes in sugarcane under drought stress. The varieties RB72454 and RB72910 were cultivated and the treatments control and drought stress applied to 135-day-old plants grown under field conditions. The stress level of the plants was measured by rate of photosynthesis, transpiration, and stomatal conductance. For each biological replicate, expression analyses were conducted using quantitative polymerase chain reaction for the genes α-tubulin, β-tubulin, β-actin, cyclophilin, eukaryotic elongation factor 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H3 and ubiquitin. Stability was evaluated using the geNorm, NormFinder, and BestKeeper software packages. Among the candidate genes, GAPDH was identified as the most stable in all software, indicating its suitability for gene expression studies in sugarcane undergoing drought stress; the gene β-actin was the second most stable. These findings suggest using GAPDH and β-actin for normalization in relative gene expression in sugarcane. Key words: gene expression; drought stress; reference gen; Saccharum portuguesO objetivo do estudo foi analisar a expressao de genes de referencia em cana-de-acucar sob estresse hidrico. As cultivares RB72454 e RB72910 foram cultivadas em campo e os tratamentos controle e estresse foram aplicados apos 135 dias. O estresse das plantas foi mensurado utilizando as variaveis fisiologicas: taxa fotossintetica, transpiracao e condutância estomatica. Para cada repeticao biologica, a expressao genica foi conduzida usando PCR tempo real para os genes α-tubulina, β-tubulina, β-actina, ciclofilina, fator 1 de alongamento eucariotico, gliceraldeido 3-fosfato desidrogenase (GAPDH), histona H3 e ubiquitina. A estabilidade dos genes foi avaliada usando os softwares geNorm,NormFinder e BestKeeper. Entre os genes candidatos, GAPDH foi identificado como o mais estavel nos tres metodos de analises, indicando ser estavel para estudos de expressao genica em cana-de-acucar sob estresse hidrico; seguindo o gene β-actina como o segundo mais estavel. Os resultados concluem que os genes GAPDH e β-actina sao indicados para normalizacao em expressao genica relativa, associada com estresse hidrico em cana-de-acucar. Palavras-chave: expressao genica; estresse hidrico; genes de referencia; Saccharum.

ciência rural

Validation of reference genes for accurate normalization by quantitative polymerase chain reaction in sugarcane drought stress studies using two cultivars