2.4 Expression Analysis and Cloning of CsHSP24.6 andCspTAC5
Total RNA was extracted from young leaves of C. sinensis using the RNAiso-Mate kit for plant tissue (Takara, Dalian, China) according to the supplier protocols. The quality and quantity of RNA were verified by 1.2% w/v agarose gel and spectrophotometric analysis. The resulting RNA sample was used as the template for reverse transcription to synthesize the first strand of cDNAs using a PrimeScript RT Reagent Kit (Takara, Dalian, China) by following the manufacturer’s protocol.
According to the cDNA sequences of CsHSP24.6 (TEA033542) andCspTAC5 (TEA000936), specific primers (Table S5 ) were designed to obtain the open reading frames (ORFs) of CsHSP24.6using Phusion High-Fidelity Polymerase (Thermo Scientific, Vilnius, Lithuania). An end-to-end PCR program was performed at 98 °C for 30 s, which was followed by 30 cycles at 98 °C for 10 s and 60 °C for 20 s, 72 °C for 45 s, and finally 10 min at 72 °C. The PCR products were purified from and cloned into a pEASY-Blunt Simple cloning vector following the manufacturer’s protocol (TransGen Biotech, Beijing, China) and then transformed into Escherichia coli DH5α competent cells for DNA sequencing.
The primers (Table S6) for qRT-PCR were designed using Primer5 software. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is stably expressed in tea tissues40, was used as an internal standard. The PCR cycling parameters and relative expression level were set and calculated by a published method41.