Quantitative Gene Expression Analysis

Total RNA was extracted using an RNA-solve reagent (OMEGABio-Tek, Norcross, GA, USA) as described previously in Zhu et al. (2020 (link)) and treated with RNase-free DNase I (Invitrogen, United States) to eliminate genomic DNA contamination. The amount and purity of RNA in each sample were evaluated by the Nano-drop spectrophotometer (Thermo, United States). Approximately 1 μg of RNA was used to synthesize the first-strand cDNA using MMLV-reverse transcriptase (Promega, USA) following the instructions. The qRT-PCR was performed and analyzed using SYBR Green PCR master mix (Promega, USA) on an ABI7500 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). For target gene expression analysis, the specific primers used are listed in Supplementary Table S1. Soybean housekeeping gene GmEF1-α (Glyma.17G186600) and Arabidopsis housekeeping gene AtEF1-α (At5G60390) were used as an endogenous control to normalize the expression of corresponding genes in soybean and Arabidopsis (Zhu et al., 2020 (link); Lin et al., 2021 (link)). The relative expression level was calculated by the ratio of the expression level of the target genes to that of the housekeeping gene (Qin et al., 2012 (link)).