Pyes2.1 v5 his topo vector

Total RNA from the leaves of lettuce (L. sativa var. crispa cv. Cheongchima) was extracted using an RNeasy plant mini kit (Qiagen, Germany) following the manufacturer’s instructions. cDNA was obtained from the extracted mRNA using an ImProm-2 Reverse Transcription System (Promega Co., Madison, WI, United States). To construct an expression plasmid vector for yeast, the ORF from LsTAT1 amplified from cDNA was cloned into the pYES2.1/V5-His-TOPO vector (Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) and transformed into Escherichia coli. The primer pairs used to isolate the LsTAT1 cDNAs are shown in Supplementary Table 1. The ORF was then ligated to the GAL1 promoter in the sense orientation. After confirmation of the nucleotide sequence of the inserted DNA, LsTAT1 and an empty vector were expressed in the Saccharomyces cerevisiae strain INVSc1 (Invitrogen). INVSc1 yeast cells were transformed using a modified lithium acetate procedure, as described previously (Gietz et al., 1992 (link)). Transformed cells were selected by SC-T (SC minimal medium lacking tryptophan) and subcultured on YPG medium (Kribii et al., 1997 (link)).

To assess Cd tolerance in yeast cells, purified full-length SaREFl products without stop codon were cloned into the pYES2.1/V5-His-TOPO vector (Invitrogen, Carlsbad, CA, USA) and positive clones were sequenced by general primers GAL1 Forward and V5 C-term Reverse sequencing primers provided by the kit (Table 2).
For the gene overexpressing and subcellular location in Arabidopsis, the cDNA of SaREFl was also amplified by PCR using High Fidelity KOD-Plus DNA Polymerase (Toyobo, Japan) with primers pENTR-F and pENTR-R listed in Table 2. The purified PCR products were then cloned into the Gateway entry vector pENTR and positive clones were further sequenced to verify the direction and sequence accuracy. The verified plasmid was then recombined in pH2GW7 and pK7WGF2.0 to generate pH2GW7-SaREFl for overexpression and pK7WGF2.0-SaREFl for determination of their subcellular locations, respectively.
All the constructs above were verified by sequencing prior to use to confirm error- free amplification and cloning. Primers used during the construction and their uses for preparation of specific constructs were listed in Table 2.

RNAs were extracted from the leaves of C. lanceolata using an RNeasy plant mini kit (Qiagen, Hilden, Germany) and reverse transcribed to generate cDNA. The primers for cloning ClOSC1-4 genes are shown in Table S3. The PCR products of the four genes were ligated into a pYES2.1/V5-HIS-TOPO vector (Invitrogen, Waltham, MA, USA) and transformed into Escherichia coli. After gene sequences of plasmids, plasmids were transformed into the erg7 yeast mutant (MATa erg7 ura3-1 trp1-1) via electroporation. The culture of transformed yeasts and extraction of triterpenes followed the protocols described previously [14 (link)].