Quantitative Real-Time PCR and Metabolomics of Tea Petals

The quantitative real-time PCR (qRT-PCR) was performed as described before (Zhou et al., 2020 (link)). Briefly, total RNA was extracted from petals by using RNAqueous™ Total RNA Isolation Kit (Thermo, MA, United States). Primers of selected gene members were designed with the Primer Premier 5.0 software and listed in Supplementary Table 4. β-Actin was used as the reference gene. The fluorescence PCR reagent was the Hieff™ qPCR SYBR Green Master Mix (No Rox) (Yaesen Biotech Co., Ltd., Shanghai, China). The experiment and analysis were carried out on the LightCycler^® 480 II Real-Time System (Roche, CA, United States). Metabolome was carried out by using liquid chromatography–mass spectrometry.
The determination of the non-volatile metabolome of tea petals was described as earlier (Zhou et al., 2020 (link)). Briefly, 100 mg powder samples were extracted in 1.0 ml methanol (70%) at 4°C for 24 h, and 5 μl supernatant was injected into ultra-performance liquid chromatography (UPLC, Shimadzu Co., Kyoto, Japan) with a mass system (MS, Applied Biosystems 6500 Q TRAP, MA, United States). Metabolites were identified using MWDB (Metware Database, Metware Biotechnology Co., Ltd., Wuhan, China) and subject to the partial least squares (PLS) discriminant analysis. The significant dissimilarities of metabolites were set as the variable importance (VIP) ≥1 and the fold change ≥2 or ≤0.5.