Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jan 21;15(1):131.
doi: 10.3390/genes15010131.

Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing

Wei Guo  1 , Yihui Yang  1 , Bo Ma  1 , Wenbo Wang  1 , Zenghui Hu  2 , Pingsheng Leng  1
Affiliations

Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing

Wei Guo et al. Genes (Basel). .

Abstract

Euonymus japonicus is one of the most low-temperature-tolerant evergreen broad-leaved tree species in the world and is widely used in urban greening. However, there are very few molecular biology studies on its low-temperature tolerance mechanism. So far, no researcher has selected and reported on its reference genes. In this study, 21 candidate reference genes (12 traditional housekeeping genes and 9 other genes) were initially selected based on gene expression and coefficient of variation (CV) through RNA-Seq (unpublished data), and qRT-PCR was used to detect the expression levels of candidate reference genes in three different groups of samples (leaves under different temperature stresses, leaves of plants at different growth stages, and different organs). After further evaluating the expression stability of these genes using geNorm, NormFinder, Bestkeeper, and RefFind, the results show that the traditional housekeeping gene eIF5A and the new reference gene RTNLB1 have good stability in the three different groups of samples, so they are reference genes with universality. In addition, we used eIF5A and RTNLB1 as reference genes to calibrate the expression pattern of the target gene EjMAH1, which confirmed this view. This article is the first to select and report on the reference gene of E. japonicus, laying the foundation for its low-temperature tolerance mechanism and other molecular biology research.

Keywords: E. japonicus; RNA-seq; new reference genes; qPT-PCR; traditional housekeeping genes.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Agarose gel electrophoresis of RNA. (a) Leaves under different temperature stresses; (b) leaves of plants at different growth stages; (c) different organs.
Figure 2
Figure 2
Agarose gel electrophoresis of PCR products of 21 candidate RGs. (a) The 12 candidate traditional housekeeping genes; (b) the 9 candidate new RGs.
Figure 3
Figure 3
Specificity of primer pairs of qRT-PCR amplification. Melting curves of 21 candidate RGs exhibiting only single peaks.
Figure 4
Figure 4
The expression profiles of candidate RGs across three different groups of samples. (a) The 12 candidate traditional housekeeping genes; (b) the 9 candidate new RGs. Each point in the box is the Ct value size, and the line across the box represents the median. The box points out the 25th and 75th percentiles. Whiskers represent the maximum and minimum values.
Figure 5
Figure 5
Stability values of candidate RGs analyzed by GeNorm. (A–C) Candidate traditional housekeeping genes in leaves under different temperature stresses, leaves of plants at different growth stages, and different organs, respectively; (D–F) Candidate new RGs in leaves under different temperature stresses, leaves of plants at different growth stages, and different organs, respectively.
Figure 6
Figure 6
Expression patterns of EjMAH1 in three groups of samples using eIF5A, RTNLB1, and a combination of these genes as reference genes. (a) Leaves under different temperature stresses; (b) leaves of plants at different growth stages; (c) different organs. NA indicates that no expression was detected. Note: Different capital letters indicate significant differences at 0.05 levels between treatments using the same RG, and different lowercase letters indicate significant differences at 0.05 levels between the same treatments using different RGs.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Xue D. Master’s Thesis. Minzu University of China; Beijing, China: 2023. Multi-Omics Analysis of the Adaptation of Euonymus japonicus to Winter Freezing Stress. - DOI
    1. Shiyuan M., Guiyun L., Mingzhong Z., Zhihao Z., Ye W., Huatian W., Xiumei L. Physiolocical Response to Cold Stress and Evaluation of Cold Resistance for Five Species of Euonymus Linn. Acta Bot. Boreali Occident. Sin. 2020;40:624–634.
    1. Yankai L., Lijun W., Xiaohe S. Survey and ldentification of Foliar Diseases on Euonymus japonicus. J. Yangtze Univ. Nat. Sci. Ed. 2017;14:5–8+3. doi: 10.16772/j.cnki.1673-1409.2017.10.002. - DOI
    1. Huiwen X. Master’s Thesis. Central South University of Forestry and Technology; Changsha, China: 2011. The Research on the Propagation Techniques of Euonymus japonicus cv. Microphyllus Butterscotch.
    1. Ma L., Wu J., Qi W., Coulter J.A., Fang Y., Li X., Liu L., Jin J., Niu Z., Yue J., et al. Screening and verification of reference genes for analysis of gene expression in winter rapeseed (Brassica rapa L.) under abiotic stress. PLoS ONE. 2020;15:e0236577. doi: 10.1371/journal.pone.0236577. - DOI - PMC - PubMed

Publication types

LinkOut - more resources