2.4 Total RNA extraction and RT-qPCR
Total RNA from leaves was extracted using Trizol (Invitrogen, USA) in accordance with the manufacturer’s instructions. First-strand cDNA was synthesized from total RNA (1 μg) using the HiScript II Q RT for qPCR (+gDNA viper) kit (Vazyme, China). RT-qPCR was performed on a QuantStudioTM 6 Flex Real-Time PCR System (Applied Biosystems, Singapore) using a CHamQ SYBR RT-qPCR Master Mix kit (Vazyme, China), following the supplier’s protocol. Expression levels were normalized against expression of the housekeeping gene OsUBQ5 and analyzed by the comparative Ct method (2-∆∆Ct method) (Livak & Schmittgen, 2001). At least three biological replicate samples were used. Differences were considered significant at P < 0.05. The primers used in this study are listed in Table S1.