Dataset Information

Identification of valid reference genes for quantitative RT-PCR in Caragana microphylla under salt and drought stresses.

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-3-phosphate 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.

SUBMITTER: Kim SJ

PROVIDER: S-EPMC7548303 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of valid reference genes for quantitative RT-PCR in Caragana microphylla under salt and drought stresses.

Kim Su Jung SJ Na Jung Up JU Kim Ji Seong JS Lee Jeong Eun JE Nie Haulin H Lee Keum Ah KA Woo Su Young SY Kim Sun Hyung SH

Physiology and molecular biology of plants : an international journal of functional plant biology 20201006 10

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 ...[more]

PMID: 33088054

Similar Datasets

Project description:BackgroundDrought and soil salinity are major abiotic stresses. The mechanisms of stress tolerance have been studied extensively in model plants. Caragana korshinskii is characterized by high drought and salt tolerance in northwestern China; unique patterns of gene expression allow it to tolerate the stress imposed by dehydration and semi-desert saline soil. There have, however, been no reports on the differences between C. korshinskii and model plants in the mechanisms underlying their drought and salt tolerance and regulation of gene expression.ResultsThree sequencing libraries from drought and salt-treated whole-seedling- plants and the control were sequenced to investigate changes in the C. korshinskii transcriptome in response to drought and salt stresses. Of the 129,451 contigs, 70,662 (54.12 %) were annotated with gene descriptions, gene ontology (GO) terms, and metabolic pathways, with a cut-off E-value of 10(-5). These annotations included 56 GO terms, 148 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 25 Clusters of Orthologous Groups (COG). On comparison of the transcriptomes of the control, drought- and salt-treated plants, 1630 and 1521 contigs showed significant differences in transcript abundance under drought and salt stresses. Compared to the differentially expressed genes (DEGs) in drought- or salt-treated Arabidopsis in the database, 542 DEGs in drought-treated C. korshinskii and 529 DEGs in salt-treated samples were presumably unique to C. korshinskii. The transcription profiles revealed that genes related to transcription factors, protein kinases, and antioxidant enzymes are relevant to the tolerance of drought and salt stress in this species. The expression patterns of 38 randomly selected DEGs were confirmed by quantitative real-time PCR and were essentially consistent with the changes in transcript abundance identified by RNA-seq.ConclusionsThe present study identified potential genes involved in drought and salt tolerance in C. korshinskii, as well as many DEGs uniquely expressed in drought- or salt-treated C. korshinskii samples compared to Arabidopsis. To our knowledge, this study is the first exploration of the C. korshinskii transcriptome under drought and salt conditions, and these results will facilitate the discovery of specific stress-resistance-related genes in C. korshinskii, possibly leading to the development of novel plant cultivars through genetic engineering.

Project description:BackgroundNormalization in real-time qRT-PCR is necessary to compensate for experimental variation. A popular normalization strategy employs reference gene(s), which may introduce additional variability into normalized expression levels due to innate variation (between tissues, individuals, etc). To minimize this innate variability, multiple reference genes are used. Current methods of selecting reference genes make an assumption of independence in their innate variation. This assumption is not always justified, which may lead to selecting a suboptimal set of reference genes.ResultsWe propose a robust approach for selecting optimal subset(s) of reference genes with the smallest variance of the corresponding normalizing factors. The normalizing factor variance estimates are based on the estimated unstructured covariance matrix of all available candidate reference genes, adjusting for all possible correlations. Robustness is achieved through bootstrapping all candidate reference gene data and obtaining the bootstrap upper confidence limits for the variances of the log-transformed normalizing factors. The selection of the reference gene subset is optimized with respect to one of the following criteria: (A) to minimize the variability of the normalizing factor; (B) to minimize the number of reference genes with acceptable upper limit on variability of the normalizing factor, (C) to minimize the average rank of the variance of the normalizing factor. The proposed approach evaluates all gene subsets of various sizes rather than ranking individual reference genes by their stability, as in the previous work. In two publicly available data sets and one new data set, our approach identified subset(s) of reference genes with smaller empirical variance of the normalizing factor than in subsets identified using previously published methods. A small simulation study indicated an advantage of the proposed approach in terms of sensitivity to identify the true optimal reference subset in the presence of even modest, especially negative correlation among the candidate reference genes.ConclusionsThe proposed approach performs comprehensive and robust evaluation of the variability of normalizing factors based on all possible subsets of candidate reference genes. The results of this evaluation provide flexibility to choose from important criteria for selecting the optimal subset(s) of reference genes, unless one subset meets all the criteria. This approach identifies gene subset(s) with smaller variability of normalizing factors than current standard approaches, particularly if there is some nontrivial innate correlation among the candidate genes.

Dataset Information

Identification of valid reference genes for quantitative RT-PCR in Caragana microphylla under salt and drought stresses.

Publications

Identification of valid reference genes for quantitative RT-PCR in <i>Caragana microphylla</i> under salt and drought stresses.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets