Clonexpress one step cloning kit

We used the wheat GS cDNAs from wheat variety Yumai49 as the template and amplified the CDS (coding sequence) region for TaGS1;1, TaGS1;2, TaGS1;3, and TaGS2 cDNA by PCR with the specific primers (Supplementary Table 3). The PCR products were cloned into the pET21a vector (Novagen) and fully sequenced. The recombinant vectors were constructed using ClonExpress One Step Cloning Kit (Vazyme). The CDSs of wheat GS were cloned into the Nde I and Hind III sites of pET21a vector (Novagen). The recombinant vectors and empty pET21a vector were transformed into Rosetta (DE3) pLysS cell. Protein production was induced by the addition of IPTG (isopropyl-b-D-thiogalactoside) to a final concentration of 1 mM and incubation in a shaker at 180 rpm. TaGS1;1, TaGS1;2, TaGS1;3, and TaGS2 were induced at 30°C for 5 h, 12°C for 17 h, 37°C for 5 h, and 25°C for 7 h, to obtain soluble protein. After induction, cells were harvested by centrifugation at 5,000g for 10 min at 4°C. The pellet was suspended in breaking buffer [10 mM Tris, 10 mM MgCl₂, 0.05% Triton X-100, 100 μg ml⁻¹ phenylmethanesulfonyl fluoride (PMSF), pH 7.5] and sonicated using an ultrasonic homogenizer JY92-2D (Ningbo Scientz Biotechnology Co. Ltd., Ningbo, China). The lysate was centrifuged at 12,000g for 15 min at 4°C and the supernatants were collected and used for identification of TaGS specific antibodies by western blot.

All PCR reactions were performed using KOD One PCR Master Mix -Blue- (TOYOBO). Recombinational cloning was performed with the ClonExpress One Step Cloning Kit (Vazyme).

Our previously reported T. foenum-graecum transcriptome (Zhou et al., 2019 (link)) was used for gene isolation. Five sterol C3-glucosyltransferase (S3GT) candidates (Cluster-2140.105632, Cluster-2140.95550, Cluster-2140.71031, Cluster-2140.131704, and Cluster-2140.319) were identified by a BLAST search against the known Dioscorea zingiberensis S3GTs (namely Dz3GT1 or Dz3GT2) that we previously have reported (Li et al., 2018 (link)). Except for the cluster-2140.95550, the other four gene candidates could be amplified by standard RT-PCR from the methyl jasmonate (MeJA)-treated T. foenum-graecum seedlings, which are the same set of plant materials that we previously used for establishing the T. foenum-graecum transcriptome (Zhou et al., 2019 (link)). The successfully amplified four candidates were designated as TfS3GT1 (cluster-2140.105632), TfS3GT2 (cluster-2140.71031), TfS3GT3 (cluster-2140.319), and TfS3GT4 (cluster-2140.131704), respectively, and they were subsequently cloned into an Escherichia coli expression vector pGEX-2T via BamHI/EcoRI sites using a ClonExpress^® One Step Cloning Kit (Vazyme, China). The N-terminus of TfS3GT1-4 was designed to be fused in a frame with a glutathione-S-transferase (GST) tag present in the pGEX-2T vector.

The ORFs of six candidate PR genes and SfruORco gene were amplified using primers with a cutting site of EcoRI or XbaI (Table S1), and were then cloned into pGH19 vector that was double-digested with EcoRI and XbaI, using the ClonExpress^® One Step Cloning Kit (Vazyme, Nanjing, China). The plasmid was extracted by the Miniprep method and purified with phenol-chloroform-isoamyl alcohol. The purified plasmid was linearized with a restriction enzyme (NotI/NdeI) and used as templates to synthesize cRNAs by using T7 polymerase of mMESSAGE mMACHINE^® T7 Kit (Thermo Fisher Scientific, Waltham, MA, USA). The purified cRNAs were diluted with nuclease-free water at a concentration of 2 µg/µL and stored at −80 °C until use.

The vector used for promoter screening and validation was pUC19, and the reporter gene gfp was used to characterize the promoter strength. The construction procedure of gfp expression plasmid pUC19-P_{BBa_23118}-gfp served as an example. In brief, promoter P_{BBa_23118} and gene gfp were amplified by corresponding primers (Table S2) and fused by overlap extension PCR. Then, the fused fragment was inserted into the linearized vector pUC19 using ClonExpress One Step Cloning Kit (Vazyme Biotech Co., Nanjing, China), resulting in plasmid pUC19-P_{BBa_23118}-gfp. The recombinant plasmid was then transformed into E. coli JM109 and selected on an LB agar plate containing ampicillin (100 µg/mL). Similarly, the other GFP expression vectors under the control of different promoters were constructed using this approach. As for the gene expression plasmids constructed based on expression vectors pTrc99a and pUCP18 for the overexpression of genes ilvCDE and pigFN, respectively, the genes ilvCDE and pigFN were amplified and purified, and then the purified PCR products were ligated with the linearized plasmids pTrc99a and pUCP18 through Gibson assembly (ClonExpress^® II One Step Cloning Kit, Vazyme Bio Inc., Nanjing, China). The recombinant plasmids were transformed into strains E. coli W3110 and S. marcescens JNB5-1 by electroporation, and selected on LB agar plate containing ampicillin (100 µg/mL).

To generate FgVps23-GFP fusion vector, the FgVPS23 gene with its 1515-bp upstream promoter region was amplified with 29F and 29R using genomic DNA extracted from wild type (PH-1) as template. The resulting fragment was cloned into the Nde I and EcoR I sites of the PKNTG vector harboring the GFP allele and the neomycin gene as a selection marker via using the ClonExpress One Step Cloning Kit (Vazyme, Nanjing, China). Other ESCRT protein GFP fusion vectors were generated by using the same strategy. The resulting vector was verified by sequencing and then transformed into the protoplasts of corresponding ESCRT gene deletion mutant strains. The neomycin-resistant transformants were picked and then screened by PCR and GFP signal.