Systematic selection and validation of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Nitraria tangutorum

Abstract

Suitable reference genes can be used to calibrate the error in quantitative real-time PCR (qPCR) experiments, making the results more credible. However, there are no reference genes suitable for multiple species and under different experimental conditions. Nitraria tangutorum Bobr. is a typical plant native to desert areas. It is drought-resistant, saline-alkali resistant, extreme temperatures-resistant, and has strong adaptability. To date, the importance of this germplasm has not been sufficiently understood; therefore, it is still unclear which genes can be used as reference genes to calibrate qPCR data of N. tangutorum. In this study we analyzed the expression levels of 10 candidate reference genes (ACT, GAPDH, TUA, TUB, CYP, UBC, His, PP2A, HSP, and EF1-α) in N. tangutorum seedlings under a series of experimental conditions, including in different organs (root, stem, and leaf) and under abiotic stresses (salt, drought, heat, and cold) and hormone stimuli (abscisic acid) by qPCR. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of the ten genes. Comprehensive analysis showed that EF1-α and His had the best expression stability, whereas HSP was the least suitable as a reference gene. The expression profile of NtCER7, a gene related to the regulation of cuticular wax biosynthesis in N. tangutorum, verified the accuracy of the experimental results. Based on this study, we recommend EF1-α and His as suitable reference genes for N. tangutorum. This paper provides the first data on stable reference genes in N. tangutorum, which will be beneficial to studying the gene expression of N. tangutorum and other Nitraria species in the future.

Figure 1

Cq values of 10 candidate reference genes in all N. tangutorum samples. The block diagram shows the quartile range. The outer box indicates the 25th to 75th percentile, and the inner box indicates the average. The horizontal lines inside the box are the median lines. Whiskers indicate the minimum and maximum values. The figures were generated by using OriginPro software (version 2018; https://www.originlab.com/).

Figure 2

Average expression stability values (M) of the 10 candidate reference genes based on the geNorm algorithm. The lower the M value, the more stable the expression. The most stable genes are to the right and the most unstable genes are on the left. The figures were generated by using OriginPro software (version 2018; https://www.originlab.com/).

Figure 3

Pairwise variation (V) of the 10 candidate reference genes calculated by geNorm to determine the optimal number of reference genes for accurate normalization. The threshold used was 0.15. The figures were generated by using OriginPro software (version 2018; https://www.originlab.com/).

Figure 4

Relative expression of NtCER7 under salt stress and in different organs of N. tangutorum. EF1-α and His and EF1-α + His were used as one or two of the most stable reference genes; HSP was used as the least stable reference gene. Different letters indicate significant differences in the expression of the target gene based on three biological replications (P < 0.05). The figures were generated by using OriginPro software (version 2018; https://www.originlab.com/).