Pyes2

The vector pREP3X was used for the overexpression of Schizosaccharomyces pombe56 (link). In this system, thiamine was used as a repressor of the pREP3X vector. For the assays of sensitivity to the H₂O₂ stimuli, the transformed cells were cultured in yeast medium with thiamine at 30 °C with an initial starting optical density at 600 nm (OD600) of 0.2. During the logarithmic phase, the cells were collected by centrifugation, washed thrice with sterile water and finally diluted to densities of 10⁶, 10⁵ and 10⁴ cell/ml using a blood-counting chamber and then assayed on yeast solid media plates with or without 1.5 mM H₂O₂ or thiamine. (B) The pYES2-TaMCA1 vector and empty vector were introduced into yca1Δ (KFY729) strain according to the user manual of pYES2 (Invitrogen). The transformed cells were assayed on yeast solid media plates with or without 1.2 mM H₂O₂.

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).

The 1.1-kb fragment of MoPEX19 cDNA was amplified using the primer pair 19pYES2cds4/19pYES2cds5 and inserted into the SmaI/BstXI sites of the yeast expression vector pYES2 (Invitrogen) to generate pYES2-PEX19. pYES2-PEX19 was transformed respectively into S. cerevisiae wild-type strain BY4741 (MATa: his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and its derivative ScPEX19 (YDL065C) deleted mutant (Thermo Scientific, San Jose, CA, USA) using the lithium acetate method. To assess the ability of the transformants to utilize oleic acid, the yeast cells were precultured in liquid YPD medium (1% yeast extract, 2% peptone and 2% dextrose) to mid-log phase, washed three times with sterilized double distilled water, adjusted to OD₆₀₀ = 1, and then grown on YNO medium [0.67% yeast nitrogen base with amino acids (Sigma), 0.1% oleic acid and 0.05% Tween 40, adjusted to pH 6.0] and SD medium (0.67% yeast nitrogen base with amino acids and 2% dextrose, adjusted to pH 6.0) in 5-μl aliquots of 10-fold serial dilutions at 30°C for 13–15 h.

Yeast growth assay was conducted according to the method described by Bienert et al. [34 (link)] with modification. The Saccharomyces cerevisiae strain INVSc1 was transformed with either an empty pYES2 (Invitrogen) as control or derivate of pYES2 carrying MfPIP2-7 coding sequence. Yeast cells were grown on SD/-Ura synthetic medium containing 2 % glucose until an A_600nm of 0.6 to 0.8, followed by two times washing with liquid SG/-Ura synthetic medium containing 2 % galactose to an A_600nm of 0.6. After a series of dilution, 10 μl were spotted on solid SG/-Ura medium containing various concentrations of H₂O₂ as indicated. Differences in growth and survival were recorded after 4 days of incubation at 30 °C.

The ORFs of HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E were PCR-amplified using cDNA as a template and cloned into the yeast expression vector pYES2.0 (Invitrogen). After confirmation by restriction enzyme digestion and sequencing, the recombinant pYES2.0-HpDGAT2s plasmids were transformed into the S. cerevisiae TAG-producing strain INVSc1 or TAG-deficient quadruple mutant strain H1246 with the S.c. EasyComp Transformation Kit (Invitrogen) [20 (link)]. The expression of HpDGAT2 genes in the yeast strain was verified at the transcript level by qRT-PCR. For the feeding experiments, yeast cultures were induced as described above but in the presence of 1% (w/v) Tergitol NP-40 (Sigma Aldrich, St. Louis, MO, USA) in the medium. At the beginning of induction, the appropriate FAs (C18:2n6, C18:3n3, C18:3n6, and C18:4n3) were added to the culture to a final concentration of 100 μM. Samples at an OD600 of 2.5 were harvested for lipid extraction, separation by TLC and analysis by GC.