Identification of valid reference genes for quantitative RT-PCR in Caragana microphylla under salt and drought stresses
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SHORT COMMUNICATION
Identification of valid reference genes for quantitative RT-PCR in Caragana microphylla under salt and drought stresses Su Jung Kim1 • Jung Up Na1 • Ji Seong Kim1 • Jeong Eun Lee1 • Haulin Nie1 Keum Ah Lee1 • Su Young Woo1 • Sun Hyung Kim1
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Received: 3 June 2020 / Revised: 3 August 2020 / Accepted: 24 August 2020 / Published online: 6 October 2020 Ó Prof. H.S. Srivastava Foundation for Science and Society 2020
Abstract Caragana microphylla is a leguminosae plant and grows mainly in semi-arid areas of northwest China and Mongolia. However, the lack of studies on C. microphylla reference genes limits the accurate understanding of the molecular biology mechanisms in this crop under abiotic stresses. In this study, we selected nine candidate genes from salt-treated C. microphylla transcriptome data and evaluated their stability by using geNorm, NormFinder, BestKeeper, and RefFinder in salt and drought conditions. In addition, the relative expressions of Delta 1-pyrroline-5-carboxylate synthase 2 (P5CS2) and Catalase 2 (CAT2) were examined to confirm the stability of the candidate reference genes. As a results, glyceraldehyde-3phosphate dehydrogenase C2 (GAPC2) and 26S proteasome regulatory subunit (RPN5) were the most stable in both salt and drought treatments. The relative expression of P5CS2 and CAT2 also showed more stable levels in normalization by GAPC2 and RPN5 than the most unstable gene, Ubiquitin 4 (UBQ4). Therefore, it is believed that these candidate reference genes selected and validated in our study could be used to study the molecular biological study of response to salt and drought stress in C. microphylla.
Su Jung Kim and Jung Up Na have contributed equally to this work.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12298-020-00874-3) contains supplementary material, which is available to authorized users. & Sun Hyung Kim [email protected] 1
Department of Environmental Horticulture, University of Seoul, Seoul 02504, Korea
Keywords Reference genes Gene expression Caragana microphylla qRT-PCR Salt stress Drought stress
Introduction The genus Caragana has more than 100 species and belongs to the family of Leguminosae. The genus Caragana mainly grows in arid and semi-arid areas and is known to comprise ecologically important plants because they reduce wind erosion and enhance water conservation (Wang et al. 2006; Zhang et al. 2006). Some studies investigated the mechanism of response to abiotic stress using biochemical and molecular biological experiments in Caragana because the genus Caragana have been known to resist salt and drought (Zhang et al. 2006; Wang et al. 2011). Moreover, in order to explore the extensive response genes depending on abiotic stresses, Yang et al. (2014) selected the reference genes for reverse transcription polymerase chain reaction (RT-PCR) and Li et al. (2016) performed transcriptome analysis by using salt—and drought—treated Caragana korshinskii. Quantitative real-time PCR (qRT-PCR), which
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