Transcriptome Analysis of DcCHIs in D. cambodiana

The response of DcCHIs to injection of the inducer was analyzed based on transcriptome data of D. cambodiana in previous reports (Supplementary Table 1; Zhu et al., 2016 (link)). Tissue expression, stress response expression, and overexpression analysis of DcCHIs were determined by quantitative real-time PCR (qRT-PCR) analysis. Total RNA from different samples were extracted using plant RNA Isolation Kit (FOREGENE, Chengdu, China) according to the manufacturer’s instructions. cDNA was synthesized from the total RNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara, Dalian, China). The qRT-PCR analysis was carried out in triplicate using the Mx3005P Real-Time PCR System (Stratagene, La Jolla, CA, United States). qRT-PCR conditions were set as follows: 5 min at 95°C for initial denaturation, followed by 40 cycles of denaturation for 10 s at 94°C, annealing for 30 s at 60°C, and extension for 30 s at 72°C. The actin genes in D. cambodiana (Zhu et al., 2016 (link)) and tobacco (Liu et al., 2012 (link)) were used as the internal controls to normalize qRT-PCR data. Primers used in this study were listed in Supplementary Table 2.