Total RNA isolation, reverse transcription, and gene expression
analysis
Total RNA was isolated from frozen plant samples using the TaKaRa
MiniBEST Plant RNA Extraction Kit (Takara, Dalian, China) according to
the manufacturer’s instructions. RNA purity and concentration were
determined using an Epoch Microplate spectrophotometer (BioTek,
Winooski, VT, USA), whereas the integrity of the RNA was evaluated by
1% agarose gel electrophoresis. Following the manufacturer’s
recommendations, TaKaRa PrimerScript ™ RT Master Mix (Takara, Dalian,
China) was used for reverse transcription and total RNA (1 μg) was used
for cDNA synthesis. qRT-PCR was performed using a Quantstudio 6 Flex
real-time PCR system (Thermo Fisher, Carlsbad, California, California,
USA) and 2x FAST qPCR Master Mixture (with OOX II) (DiNing, Dalian,
China). Every qRT-PCR sample contained 1 μl of cDNA, including those for
antioxidant related genes, osmoprotectants related genes and
symbiosis-related genes (Table S1), 10 μl of EvaGreen 2× qPCR MasterMix
(DiNing, Dalian, China), 2 μl of primer and 7 μl of sterilized water.
The qRT-PCR cycle parameters were as follows: 10 min at 95°C, and then
40 cycles of 30 s at 95°C and 1 min at 60°C. Three independent
replicates were performed for each sample. The Gmactin andGm60s were used as endogenous Control genes were expressed as
2−ΔΔCt using the comparative threshold cycle (Ct)
method (Livak & Schmittgen, 2001).