Easygeno assembly cloning kit

The full length of the GmPK21 CDS was amplified by PCR using the primer GmPK21-F2 and GmPK21-R2 (Table S5) and then was introduced into the linearized plasmid pC3300s (digested by SacI and BamHI) using the EasyGeno Assembly Cloning kit (Tiangen, VI201), generating the pC3300s-35S::GmPK21. The construct was then transformed into Arabidopsis (Col-0) using Agrobacterium GV3101. The seeds of transformants were selected by 1/2 MS medium containing glufosinate solution. The RT-PCR and qRT-PCR was then performed to confirm the presence of the GmPK gene in the glufosinate resistant lines (The RT-PCR primers RT-GmPK21-F/ RT-GmPK21-R and qRT-PCR primers qGmPK21-F (Ara)/qGmPK21-R(Ara) were shown in Table S5). The PK activity of 20-day-old transgenic Arabidopsis and WT was determined by using the Pyruvate Kinase (PK) Activity Assay Kit (BC0540, Solarbio life sciences). 3 biological replicates were performed and 10 seedlings (aboveground parts) were pooled per biological replicate. Data were presented as means ± SD, and Student’s t-tests were used by SPSS statistics 19 to assess the significance of differences.

The primers of proteolytic system genes used in the study which were designed by primer premier 5 (Premier, Canada) are listed in Supplementary Table S2. These genes were directly amplified from L. lactis F44 or B. subtilis 168 via polymerase chain reaction (PCR). The restriction enzyme cutting sites were simultaneously inserted into the amplified gene. The resulting fragments were digested with BamHI and HindIII (or SmaI and NcoI), and then ligated into plasmid pLEB124, cut with BamHI and HindIII (or SmaI and NcoI) to generate the resulting plasmids. The resulting plasmids were transformed into E.coli TG1 by heat shock transformation for enrichment. After antibiotics selection, the plasmids were extracted with TIANprep Mini Plasmid Kit (TIANGEN, China), and then transformed into the L. lactis F44 by electroporation transformation. To construct strain BAFM, nprB, oppA, pepF and pepM were fused with the linear plasmid using the EasyGeno Assembly Cloning kit (TIANGEN, China). All the constructed plasmids were confirmed by restriction enzyme digestion and DNA sequencing.

The M13KO7 helper phage was used to infect a culture of TG1 cells. After growth overnight at 37°C, the TIANprep Mini Plasmid Kit (TIANGEN) was used to extract the double-stranded DNA (dsDNA) phage chromosome. The insert genes and the M13KO7 phage chromosome were amplified by PCR with primers containing 20 base pair overlap. The PCR products were assembled by an EasyGeno Assembly Cloning Kit (TIANGEN) at 50°C for 30 min. The assembly mix was transformed into competent DH5α cells. The point mutations in the RBD region were generated by QuikChange Lightning (Agilent). The recombinant phage chromosome was sequenced (GENEWIZ), and the sequencing results were analyzed by SnapGene 4.2 software. To produce the recombinant phage, the DH5α strain transformed with the recombinant phage chromosome was grown overnight at 37°C and centrifuged for 15 min at 8,000 × rpm at 4°C. The supernatant was collected, and the recombinant phage particles were precipitated by adding 20% (w/v) polyethylene glycol (PEG) 8,000 solution to 2.5 M NaCl (LABLEAD) in distilled water. Following incubation on ice for 1 h, the mixture was centrifuged for 30 min at 10,000 × rpm at 4°C, and the pellet was resuspended in 1 ml of PBS and stored at 4°C. After incubation at 95°C for 15 min, real-time fluorescent quantitative PCR was used to detect the titer of the phage.

The CDS without the termination codon of GmPK21 was amplified by PCR using the primer GmPK21-F1 and GmPK21-R1 (Table S5) and then was introduced into the plasmid linearized pAN580 vector (digested by XbaI and BamHI) using the EasyGeno Assembly Cloning kit (Tiangen, VI201), generating the GmPK21-GFP fusion plasmid. The empty vector pAN580 was used as the control. The GmPK21-GFP and empty vector pAN580 were transformed into the Arabidopsis or tobacco protoplasts [50 (link)] respectively. The GFP signal was then analyzed using the LSM710 confocal microscope (Zeiss, Oberkochen, Germany).

The full-length human MDR1 3′-UTR containing the predicted miR-298 binding site (445–451 bp) was amplified from the genomic DNA using specific primers (Table 1). The Duo-Luciferase reporter vector pEZX-FR2 (GeneCopoeia Inc., Rockville, MD, United States) was linearized with EcoRI and XhoI restriction enzymes, and the PCR product was cloned into the linearized vector using an EasyGeno Assembly Cloning kit (Tiangen Biotech Co., Ltd., Beijing, China) to create the vector pEZX-MDR1-3′-UTR. The consensus miR-298 binding site was mutated using a QuikChange II site-directed mutagenesis kit (Stratagene, Agilent Technologies, Santa Clara, CA, United States) to construct a clone of the mutant MDR1 3′-UTR, which was referred to as pEZX-MDR1-3′-UTR-mut. All clones were verified by DNA sequencing. For the luciferase reporter assay, HEK293T cells seeded in 24-well plates were transiently transfected with pEZX-MDR1-3′-UTR or pEZX-MDR1-3′-UTR-mut reporter plasmids together with a miR-298 mimic or miR-NC using Lipofectamine iMax (Invitrogen, Carlsbad, CA, United States). Twenty-four hours after transfection, cells were lysed, and their luciferase activities were determined using a Luc-Pair^TM Duo-Luciferase Assay Kit (GeneCopoeia), according to the manufacturer’s instructions.

The promoter region (~300 bp) of genes in the cluster (BAB_RS27025-RS27055) was amplified by PCR. The PCR products were purified by gel extraction and ligated into the KpnI- and BamHI-digested pMCR-LacZ plasmid using the EasyGeno Assembly Cloning kit (TIANGEN) to construct the LacZ reporter plasmids. The successfully constructed recombinant plasmids were electroporated into 2308, 2308ΔvdtR, and 2308ΔvdtR-Rev, while the empty pMCR-LacZ plasmid was also transformed into Brucella strains as a negative control. LacZ activation was determined using o-nitrophenyl-β-d-galactoside as the substrate described in our previous study (46 (link)).

For minigene construction, mouse genomic DNAs were extracted from TA muscle using a TIANamp Genomic DNA kit (TIANGEN Biotech). The corresponding mouse genomic DNA fragments were amplified from mouse genomic DNA using high-fidelity polymerase (TransGen Biotech) with KpnI and XhoI restriction enzyme sites flanking at the end. Subsequently, the fragments were purified, recycled and cloned into pcDNA3.1 (+) plasmid using EasyGeno Assembly Cloning kit (TIANGEN Biotech). Similarly, the human Rbm24 coding sequence was amplified with the PCR primers containing XhoI and KpnI restriction enzyme sequences and were cloned into the pXJ40 vector 32 (link). Plasmids expressing Rbm24 and the minigene (1:1) were transfected into 293T cells using Lipofectamine 3000 (Invitrogen). After 48 h of transfection, total RNAs were extracted and reverse transcribed. Splicing assay was performed by RT-PCR. The sequences of the primers used are provided in Table S6.