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Reference genes selection for Calotropis procera under different salt stress conditions.

ABSTRACT: Calotropis procera is a perennial Asian shrub with significant adaptation to adverse climate conditions and poor soils. Given its increased salt and drought stress tolerance, C. procera stands out as a powerful candidate to provide alternative genetic resources for biotechnological approaches. The qPCR (real-time quantitative polymerase chain reaction), widely recognized among the most accurate methods for quantifying gene expression, demands suitable reference genes (RGs) to avoid over- or underestimations of the relative expression and incorrect interpretation. This study aimed at evaluating the stability of ten RGs for normalization of gene expression of root and leaf of C. procera under different salt stress conditions and different collection times. The selected RGs were used on expression analysis of three target genes. Three independent experiments were carried out in greenhouse with young plants: i) Leaf100 = leaf samples collected 30 min, 2 h, 8 h and 45 days after NaCl-stress (100 mM NaCl); ii) Root50 and iii) Root200 = root samples collected 30 min, 2 h, 8 h and 1day after NaCl-stress (50 and 200 mM NaCl, respectively). Stability rank among the three algorithms used showed high agreement for the four most stable RGs. The four most stable RGs showed high congruence among all combination of collection time, for each software studied, with minor disagreements. CYP23 was the best RG (rank of top four) for all experimental conditions (Leaf100, Root50, and Root200). Using appropriated RGs, we validated the relative expression level of three differentially expressed target genes (NAC78, CNBL4, and ND1) in Leaf100 and Root200 samples. This study provides the first selection of stable reference genes for C. procera under salinity. Our results emphasize the need for caution when evaluating the stability RGs under different amplitude of variable factors.

SUBMITTER: Coelho MRV

PROVIDER: S-EPMC6472812 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Reference genes selection for Calotropis procera under different salt stress conditions.

Coêlho Maria R V MRV Rivas Rebeca R Ferreira-Neto José Ribamar C JRC Pandolfi Valesca V Bezerra-Neto João P JP Benko-Iseppon Ana Maria AM Santos Mauro G MG

PloS one 20190418 4

Calotropis procera is a perennial Asian shrub with significant adaptation to adverse climate conditions and poor soils. Given its increased salt and drought stress tolerance, C. procera stands out as a powerful candidate to provide alternative genetic resources for biotechnological approaches. The qPCR (real-time quantitative polymerase chain reaction), widely recognized among the most accurate methods for quantifying gene expression, demands suitable reference genes (RGs) to avoid over- or unde ...[more]

PMID: 30998798

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Project description:Reverse transcription-quantitative real-time PCR (RT-qPCR) is a widely used technique for gene expression analysis. The reliability of this method depends largely on the suitable selection of stable reference genes for accurate data normalization. Hypericum perforatum L. (St. John's wort) is a field growing plant that is frequently exposed to a variety of adverse environmental stresses that can negatively affect its productivity. This widely known medicinal plant with broad pharmacological properties (anti-depressant, anti-tumor, anti-inflammatory, antiviral, antioxidant, anti-cancer, and antibacterial) has been overlooked with respect to the identification of reference genes suitable for RT-qPCR data normalization. In this study, 11 candidate reference genes were analyzed in H. perforatum plants subjected to cold and heat stresses. The expression stability of these genes was assessed using GeNorm, NormFinder and BestKeeper algorithms. The results revealed that the ranking of stability among the three algorithms showed only minor differences within each treatment. The best-ranked reference genes differed between cold- and heat-treated samples; nevertheless, TUB was the most stable gene in both experimental conditions. GSA and GAPDH were found to be reliable reference genes in cold-treated samples, while GAPDH showed low expression stability in heat-treated samples. 26SrRNA and H2A had the highest stabilities in the heat assay, whereas H2A was less stable in the cold assay. Finally, AOX1, AOX2, CAT1 and CHS genes, associated with plant stress responses and oxidative stress, were used as target genes to validate the reliability of identified reference genes. These target genes showed differential expression profiles over time in treated samples. This study not only is the first systematic analysis for the selection of suitable reference genes for RT-qPCR studies in H. perforatum subjected to temperature stress conditions, but may also provide valuable information about the roles of genes associated with temperature stress responses.

Dataset Information

Reference genes selection for Calotropis procera under different salt stress conditions.

Publications

Reference genes selection for Calotropis procera under different salt stress conditions.

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