Quantitative Expression Analysis of Terpene Synthesis Genes

RNAprep pure plant kit (Biofit, Chengdu, China) was used to isolate total RNA from fresh Wanhu No.5 and Wanhu No.6 flowers (100 mg). According to the manufacturer’s instructions, RNA (1 μg) was used to synthesize cDNA using PrimeScript^TM RT kit with gDNA eraser (January, Perfect Real Time, Takara, Tokyo, Japan). Using QuantStudio 6 Flex real-time PCR system (Thermo Fisher, Waltham, MA, United States) and SYBR^® PremixExTaq^TMII (2x) (Japan, Takara), the gene expression level was detected by qRT-PCR. We used NCBI-BLAST online software² to design fluorescent quantitative primers for the key genes in the terpene synthesis pathway (Supplementary Table 1). The results are attached in Figure 11. The reaction steps are 50°C 2 min, 95°C 30 s, 95°C 5 s, 60°C 34 s, 40 cycles, and 72°C for 10 min (Jin et al., 2013 (link)). The 2^–ΔΔCT method was used to calculate the relative gene expression, and the experiment was repeated three times (Livak and Schmittgen, 2001 (link)).