*2.4. Extraction of Total RNA and qRT-PCR*

In the experiment, 0.2 g of tea leaf samples was ground into powder in liquid nitrogen, and the total RNA was extracted using the Plant Total RNA Purification Kit (GeneMark, Taichung, Taiwan). The Moloney Murine Leukemia Virus (MMLV) first-strand synthesis kit (Gene DireX, Las Vegas, NV, USA) was used for reaction of 2 µg of total RNA. In the reaction solution, 1 µL of Oligo dT (1 µg/µL) was mixed with 1 µL of 10 mM dNTP to react for 10 min at 70 ◦C and for 5 min at 4 ◦C. After the reaction, 4 µL of 5× reaction buffer, 2 µL of 0.1 M Dithiothreitol (DTT), 1 µL of diethylpyrocarbonate (DEPC) H2O, and 1 µL of MMLV reverse transcriptase were sequentially added into the solution for 1 h of reaction at 37 ◦C and 10 min of reaction at 65 ◦C, after which the reaction was terminated. Subsequently, 80 µL of DEPC H2O was added into a 0.2-mL microcentrifuge tube to perform qRT-PCR on the obtained cDNA.

For qRT-PCR, the cDNA was amplified using the CFX ConnectTM Real-Time System (Bio-Rad, Hercules, CA, USA), and data were analyzed using Bio-Rad CFX Manager 3.1. The *18S rRNA* of the tea leaf samples were used as the internal control to normalize cDNA levels. The reaction conditions were as follows: 5 min at 94 ◦C; 15 s of 45 circulations at 94 ◦C, 60 ◦C, and 72 ◦C each; and finally, 10 min at 72 ◦C. Nonspecific products or primer dimers were identified based on their lower melting temperature than that of the specific amplicon. The primers used for qRT-PCR analyses were as follows: *CsHCT* forward sequence 50 -caaattaaccaaggaccaactcaac-30 and reverse sequence 5 0 -tgtaattgaccatgttcccatcttc-30 ; and *18S rRNA* forward sequence 50 -ccgctggcaccttatgagaa-30 and reverse

sequence 50 -tttcagccttgcgaccatact-30 . The qRT-PCR experiments were repeated at least 3 times each biologically independently, and the data shown are average values. Statistical analyses were performed using Statistical Analysis System (SAS) 9.4 software.
