Rna solve reagent

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)).

Total RNA was extracted from various plant tissues using an RNA-solve reagent (OMEGA Bio-Tek, Norcross, GA, USA) following the manufacturer’s instructions. The RNA samples were added with RNase-free DNase I (Invitrogen, Carlsbad, CA, USA) to remove genomic DNA. The first complementary DNA was synthesized from 1 µg RNA by using GoScript (Promega, Madison, WI, USA), according to the manufacturer’s instructions. The qRT-PCR analysis was conducted using an ABI7500 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Each reaction contained 2 μL of 1:20 diluted reverse transcription product, 0.4 μL of forward and reverse primers (0.2 μM final concentration), 7.2 of μL DNase-Free ddH₂O and 10 μL of 2 × SYBR™ Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA). The PCR cycling system consisted of incubation at 95 °C for 1 min and followed by 40 cycles of 95 °C for 15 s, 60 °C for 60 s and 72 °C for 30 s. The dissociation curve was obtained after the last cycle. For target genes expression analysis, the specific primer pairs were designed as listed in Supplementary Table S2. Expression levels of the soybean housekeeping gene GmEF1-α (Glyma.17G186600) and the Arabidopsis housekeeping gene AtEF-α (At5G60390) was used to normalize the samples, as described previously [67 (link)].

Total RNA was extracted from soybean roots using the RNA-solve reagent (OMEGA Bio-tek, Norcross, GA, USA). After being treated with RNase-free DNase I (Invitrogen, Carlsbad, CA, USA), total RNA was used to synthesize the first-strand cDNA using MMLV-reverse transcriptase (Promega, Madison, WI, USA) following instructions in manuals. The qRT-PCR analysis was conducted using SYBR Green-monitored on a ABI StepOne Plus real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Primer pairs (Table S1) for qRT-PCR were designed. Housekeeping gene GmEF-1a (accession no. X56856) was used as an internal control to normalize the expression of corresponding genes in soybean.