Trizol reagent

RNA samples were isolated from the leaves of 5-day-old seedlings using plant Trizol reagent (Vigorous Biotechnology, China). 2 µg aliquot of total RNA was used for cDNA synthesis by M-MLV Reverse Transcriptase (TAKARA, Japan). The ORF primers of GmACSL2 are: GmACSL2-1F: 5′-ATGGCGACAATTCCTATCACCTAC-3′ and GmACSL2-1R: 5′ -TTACATGTATAGATTGTCTATTTGCTCCC-3′. The primers were designed by Primer Premier 5.0 [29] . The PCR conditions were as follows: 95°C for 5 min; 35 cycles of 95°C for 40 s, 58°C for 40 s, and 72°C for 2 min; and an additional step of 72°C for 10 min. The amplified products were cloned into pMD18-T vector (TAKARA, Japan) and then sequenced.
The PCR product of GmACSL2 from pMD18-T vector was digested with BamHI/Xbal, and then subcloned into vector pYES2 (Invitrogen, America) to generate the pYES2-GmACSL2 plasmid for yeast vector construction. GmACSL2 was first subcloned into the pENTR vector for subcellular localization, and then LR recombined with pK7FWG2.0 to obtain pK7FWG-GmACSL2-eGFP through gateway system according to the protocol of Invitrogen [30] (link). The Plasmids of pCXDR-SSE1-dsRed were constructed following the method described by Chen et al [31] (link). GmACSL2 was subcloned into the vector pGFPGUSPlus (pCAMBIAl305.1113) for soybean hairy root induction, with the HPTII gene as the selective marker and GUS/GFP as the reporter gene [32] (link).

Genomic DNA was isolated from tail biopsies following the HotSHOT method (Truett et al. 2000 ) and genotyping was performed using standard PCR methods with sequence-specific primers (listed in Supplemental Materials, Table S3). Total RNA was extracted from the liver and various tissues using Trizol reagent (Vigorous Biotechnology) according to the manufacturer’s protocols. RNA was converted to cDNA using M-MLV reverse transcriptase (M170A; Promega) according to the manufacturer’s protocols. qRT-PCR was performed with SYBR Green master mix (DRR420A; Takara) using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Relative RNA quantifications were normalized to the endogenous control (Gapdh). Data were analyzed using the 2^–△△Ct method. All of the experiments were repeated independently at least three times. The primers used are listed in the Supplemental Materials (Table S4).