Gene Expression Analysis via qRT-PCR

RNAs were isolated from 100 mg sample using Plant RNA Kit (Omega BioTek, United States) by following the instruction provided in the kit. After treatment with DNase I, the RNAs were reversely transcribed into cDNA using SuperScript^TM II reverse transcriptase (Takara, Japan) and a poly(dT)₁₈ primer. An ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems, United States) was employed to detect the changes in gene expression after stress treatments. A Power SYBR^® Green polymerase chain reaction (PCR) Master Mix (Applied Biosystems, United States) was used in the reaction. HcUBC10 and Atactin2 were used as the reference genes in these experiments (Shi et al., 2012 (link); Zeng et al., 2015 (link)). The relative gene expression level was measured by using the 2^–ΔΔCT comparative method (Livak and Schmittgen, 2001 (link)). The information of all genes in the quantitative reverse transcription (qRT)–PCR experiments was listed in the Supplementary Table 2.