Selection of Reference Genes for Gene Expression Normalization in Peucedanum praeruptorum Dunn under Abiotic Stresses, Hormone Treatments and Different Tissues

Abstract

Peucedanum praeruptorum Dunn is one of the main traditional Chinese medicines producing coumarins and plenty of literatures are focused on the biosynthesis of coumarins. Quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used method in studying the biosynthesis pathway and the selection of reference genes plays a crucial role in accurate normalization. To facilitate biosynthesis study of coumarins, twelve candidate reference genes were selected from the transcriptome database of P. praeruptorum according to previous studies. Then, BestKeeper, geNoFrm and NormFinder were used for selecting stably expressed reference genes in different tissues and under various stress treatments. The results indicated that, among the twelve candidate reference genes, the SAND family protein (SAND), actin 2 (ACT2), ubiquitin-conjugating enzyme 9 (UBC9), protein phosphatase 2A gene (PP2A) and polypyrimidine tract-binding protein (PTBP1) were the most stable reference genes under different experimental treatments, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin beta-6 (TUB6) were the least stable genes. In addition, the suitability of SAND, TIP41-like protein (TIP41), UBC9, ACT2, TUB6 and their combination as reference genes were confirmed by normalizing the expression of 1-aminocyclopropane-1-carboxylate oxidase (ACO) in different treatments. This work is the first survey of the stability of reference genes in P. praeruptorum and provides guidelines to obtain more accurate qRT-PCR results in P. praeruptorum and other plant species.

Fig 1. The comparison of transcript abundances of the twelve candidate reference genes.

Boxes indicate the 25th/75th percentiles, the lines represent the median, squares represent the means and whiskers represent the maximum and minimum values.

Fig 2. Expression stability of twelve candidate genes in P. praeruptorum as predicted by geNorm analysis.

Average expression stability (M) in each treatment is calculated. The least stable gene which has a high M value is on the left, and the most stable gene on the right. The methods of treatment or group classification are listed in the picture correspondingly.

Fig 3. Determination of the optimal numbers of reference genes for normalization by pairwise variation using geNorm.

Pairwise variation (Vn/n+1) analysis between the normalization factors (NFn and NFn+1) was performed in all treated samples. Different treatments are included and markerd as square frame with different colors. The total group refers to all samples. The V (variation) value with which below 0.15 was labeled with asterisk, representing that addition of a further reference gene does not result in any improvement of normalization.

Fig 4. Validation of the reference genes. Relative expression level of ACO was normalized using candidate reference genes under different treatments.

(A) Expression level was normalized using most and least stable reference genes under cold treatment. (B) Expression level was normalized using different reference genes under NaCl treatment. TIP41, SAND and ACT2 represent the three most stable reference genes and UBC9 is a least stable gene. (C) Expression level was normalized using different combination. Data are displayed as means ± SEM, and the statistical analyses were performed using the Student’s t-test to compare those between two reference genes or combination for normalization. *P< 0.05; ***P< 0.001; N.S.: No significant difference.