Trizol reagent

Trizol Reagent (CW0580S, CWBIO, Beijing, China) was used to extract the total RNA, and first-strand complementary DNA (cDNA) was synthesized using HiScript^® III-RT SuperMix for qPCR (+gDNA wiper) (R223-01, Vazyme, Nanjing, China). The quantitative real-time PCR mixture contained 2 μL of cDNA, 0.8 μL of forward primer and 0.8 μL of reverse primer, 10 μL of 2× ChamQ Universal SYBR qPCR Master Mix (Q311-01, Vazyme, Nanjing, China), 6.4 μL of dd H₂O, totally in a 20 μL final volume. All the reactions were done in three replications in 96-well plates using the Roche Real-Time PCR system (LightCycler480 II, Roche) following thermal conditions: 95 °C for 30 s, 95 °C for 5 s, and 57 °C for 45 s for 40 cycles. To normalize the sample variance, the soybean β-Tubulin gene (NM_001252709.2) was used as the internal control. The relative expression was measured through comparison with that of the normal condition plants (set as 1.0) and analyzed using the 2^−△△Ct method [64 (link)]. The gene-specific primers used for quantitative real-time PCR analysis are listed in Table S1.

Total mRNA was extracted using TRIzol reagent (CWBIO, CW0580S, Beijing, China) and RNA concentrations were determined using an ultraviolet spectrophotometer. cDNA was performed using a Prime Script™ RT Master Mix Kit (TransGen, AE301-02, Beijing, China) according to the manufacturer’s instructions. qPCR was performed using an Ultra SYBR Mixture Kit (CWBIO, CW0957C, Beijing, China) and a Bio-Rad CFX Maestro system. The reaction parameters were as follows: 95 °C for 15 min, 45 cycles of amplification with three steps: denaturation at 95 °C for 10 s, annealing at 55 °C for 20 s and extension at 72 °C for 30 s.
The primers used are listed below:
GAPDH forward 5′ AACGGATTTGGTCGTATTGG and
GAPDH reverse 5′ GGCTGCTGTCACCCATGAA
SPHK1 forward 5′ AACGGATTTGGTCGTATTGG and
SPHK1 reverse 5′ TCACTCTCTAGGTCCACATCAG
PBX1 forward 5′ CAGTGAGGAAGCCAAAGAGG and
PBX1 reverse 5′ CAGCTGTTTTGGCAGCATAA
S1PR1 forward 5′ GCCTACACAGCTAACCTGCTCTTG and
S1PR1 reverse 5′ TGGCGATGGCGAGGAGACTG
S1PR2 forward 5′ CCACCACCTCCTGCCACTCC and
S1PR2 reverse 5′ CACCGTGTTGCCCTCCAGAAAC
S1PR3 forward 5′ GATCCTCTACGCACGCATCTACTTC and
S1PR3 reverse 5′ ACACGCTCACCACAATCACCAC
S1PR4 forward 5′ GAAGCCGTAGACGCGGCTGG and
S1PR4 reverse 5′ GAAGCCGTAGACGCGGCTGG
S1PR5 forward 5′ GTGAGGTGGGAGCCATAGAA and
S1PR5 reverse 5′ TTGGCTGAGTCTCCCAGAGT

Total RNA was isolated from maize seeds with TRIzol reagent (CWBIO, Beijing, China), and cDNA was generated with a reverse transcriptase kit (TaKaRa, Tokyo, Japan) for quantitative real-time RT-PCR. Approximately 20 μL of mixed solution was prepared for qRT-PCR reaction using SYBR Green PCR Master Mix (TaKaRa, Tokyo, Japan), and the reactions were performed on an Mx3005P sequence detection system according to the manufacturer’s instructions. The maize endogenous gene zSSIIb was used as an internal control to normalize the amount of template cDNA. qRT-PCR primer pairs were designed with Primer 5.0 software (Table S1). Data were analyzed with MxPro software (version 4.10).

Trizol reagent (CWBIO) was used to extract total RNA according to the manufacturer's instructions, and RNA was converted to complementary DNA (cDNA) using the qPCR RT kit (TOYOBO) and oligo (dT) primers. The method of miRNA extraction was similar to total RNA extraction. MiRNA was reversely transcribed into cDNA using the miRNA qRT‐PCR Starter kit (Riobobio). PCR primers for U6 and miR‐6882 were purchased from Riobobio. Quantitative real‐time PCR was performed using the SYBR Green PCR Mix kit (Takara). The detailed primer sequences are shown in Table S1. Results were normalized according to β‐actin mRNA or U6 miRNA expression levels. The results were expressed using the ∆∆CT (cycle threshold) method for quantification.