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. 2022 Dec 17;13(12):2396.
doi: 10.3390/genes13122396.

Identification of Reliable Reference Genes under Different Stresses and in Different Tissues of Toxicodendron succedaneum

Dongxiao Ma  1   2 , Qin Zhang  1   2 , Jintao Zhou  1   2 , Yu Lu  1   2 , Xiaomeng Duan  1   3 , Chengzhong He  1   2   3 , Jinde Yu  1
Affiliations

Identification of Reliable Reference Genes under Different Stresses and in Different Tissues of Toxicodendron succedaneum

Dongxiao Ma et al. Genes (Basel). .

Abstract

Toxicodendron succedaneum (L.) Kuntze (T. succedaneum) is an economic tree species that produces urushiol and urushi wax, and it is of great value in industry and medicine. However, the stability of reference genes (RGs) has not been systematically reported in T. succedaneum to date. In this study, the expression of 10 candidate RGs was analyzed by RT-qPCR in different tissues (roots, stems, leaves), stress treatments (high/low temperature, drought), and hormone stimulation (jasmonic acid, JA). Then, the stability ranking of 10 candidate genes was evaluated by ∆Ct analysis and three software programs: geNorm, NormFinder, and BestKeeper. Finally, RefFinder was used to comprehensively analyze the expression stability of 10 candidate genes. The comprehensive analysis showed that TsRG05/06, TsRG01/06, and TsRG03/ACT were stable under high/low-temperature stress, drought stress, and JA treatment, respectively. TsRG03 and ACT had stable expression in different tissues. While the TsRG03 and ACT were recommended as the suitable RGs for T. succedaneum in all samples. Meanwhile, UBQ was the least suitable as a reference gene for T. succedaneum. In addition, the results of geNorm showed that the combination of two stable RGs could make the results of gene expression more accurate. These results provide alternative RGs for the study of gene function, correction, and normalization of target gene expression and directed molecular breeding in T. succedaneum.

Keywords: RT-qPCR; Toxicodendron succedaneum; gene expression; reference gene.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The expression levels of 10 candidate RGs across all experimental samples. The box plot represents the distribution interval of Ct values of 10 RGs in all samples: the upper side represents the upper quartile (75%), the middle line represents the median (50%), the lower side represents the lower quartile (25%), the upper edge represents the maximum value of sample data, and the lower edge represents the minimum value of sample data.
Figure 2
Figure 2
Average expression stability values (M) of 10 candidate RGs under different conditions by geNorm analysis. The direction of the arrows indicates the most and least stable RGs. (a) temperature treatments, (b) drought treatments, (c) JA treatments, (d) different tissues, (e) all samples.
Figure 3
Figure 3
Determination of the optimal number of RGs for normalization by pairwise variation (V) using geNorm. The average pairwise variations (Vn/Vn+1) were analyzed to measure the effect of adding RGs during the RT-qPCR.
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