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.
Highlights
Gene expression research has become an important means of revealing gene function and molecular mechanisms
In order to further verify the reliability of the reference gene selection results, we analyzed the expression pattern of NtCER7, a gene related to the regulation of the cuticular wax biosynthesis of N. tangutorum under salt stress and in different organs
These results provide a theoretical basis for subsequent research on the regulation of functional gene expression in N. tangutorum
Summary
Gene expression research has become an important means of revealing gene function and molecular mechanisms. With our increasing understanding of the agricultural ecological value of non-model plants, it has become important to study their molecular genetic mechanisms and to determine their appropriate reference genes under different experimental conditions. In this study, based on our previous transcriptome analysis, 10 commonly used reference genes (ACT,UBC, TUA,TUB, GAPDH, CYP, PP2A, His, HSP, and EF1-α) in N. tangutorum were cloned Their expression levels under a series of experimental conditions, including in different organs (root, stem, and leaf), under various abiotic stresses (salt, drought, heat, and cold), and under hormone stimuli [abscisic acid (ABA)] were detected by qPCR. In order to further verify the reliability of the reference gene selection results, we analyzed the expression pattern of NtCER7, a gene related to the regulation of the cuticular wax biosynthesis of N. tangutorum under salt stress and in different organs. These results provide a theoretical basis for subsequent research on the regulation of functional gene expression in N. tangutorum
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