Ecodrytm premix kit

Total RNA was isolated from the cultured cells utilizing TRIzol reagent (Thermo Fisher Scientific). The conversion of RNA into cDNA was carried out with the RNA to cDNA EcoDry^TM Premix Kit (TaKaRa, Tokyo, Japan), following the manufacturer’s instructions. The qPCR analysis was executed using the Thermal Cycler Dice Real Time System III (TaKaRa) and the Power SYBR Green PCR Master Mix (TaKaRa), following the manufacturer’s protocols. The primers used in this research are detailed in Table 1. Relative mRNA transcription levels were quantified using the 2^−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) being used as the internal control for normalization.

Total RNA with high integrity 28S:12S ratio of 2:1 was extracted from flash-frozen leaf and root tissues from the control and drought samples using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s recommendations. Using the (1 μg) of total RNA cDNA was synthesized by RNA-to-cDNA EcoDryTM Premix Kit, (Takara, Kusatsu, Shiga Prefecture, Japan). By a PowerUp SYBR Green Master Mix Kit from Thermo Fisher Scientific Inc., VaAQP genes were processed to a quantitative real-time polymerase chain reaction (qRT-PCR) on an ABI StepOne PCR System (Applied Biosystems, Foster City, CA, USA). Briefly, 100 ng template cDNA and 10 nM each primer in a final volume of 20 μL according to manufacturer’s instructions used the following protocol: UDG activation at 50 °C for 2 min, followed by polymerase activation at 95 °C for 2 min, denaturation 95 °C 15 s (Hold) and annealing and extension at 60 °C for 1 min (40 cycles), followed by melt curve analysis 95 °C 15 s, 60 °C 1 min, and dissociation 95 °C 15 s. Four biological replicates were used during the analysis, and adzuki bean actin gene was used as the internal control for normalization. The primer set used for qRT-PCR were designed using Primer QuestTM Tool (https://sg.idtdna.com/pages/tools/primerquest (22 April 2022)). The details of primer pairs used in this study are provided in Supplementary Table S1.