Quantitative Analysis of Defense Gene Expression in Radix pseudostellariae

Plants were ground into powder with liquid nitrogen, and plant RNA was extracted with TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China) in accordance with the instructions. Furthermore, RNA concentration was measured using NanoDrop 2000C Spectrophotometer (Thermo Scientific, United States). According to the kit’s instructions, the first-strand cDNA was synthesized using TransScript^®, miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Each sample used 1 μg of total RNA, and the products were immediately diluted to 80 μl with DEPC water as a template.
Based on the previous transcriptome data of R. pseudostellariae in our laboratory (Qin et al., 2017 (link)), nine primer pairs were used (Supplementary Table 2) to analyze the expression of defense-related genes in R. pseudostellariae as a result of Trichoderma and/or F. oxysporum infection. The actin gene (Supplementary Table 2) was used as an internal reference gene. The 15 μl of the PCR reaction contains 7.5 μl of 2 × SYBR Green qPCR Master Mix (TransGen Biotech, Beijing, China), 1 μl of each primer (10 μM), 0.6 μl of cDNA template, and 5.9 μl H₂O. The PCR program was as follows: 94°C for 30 s, followed by 40 cycles of 94 C for 5 s and 60°C for 30 s. After RT-PCR, the 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)) was used to calculate the relative gene expression levels.