The native and mutated A824V xyr1 PCR fragment were amplified from genomic DNA of QM9414 by using primers in Supplementary Table 2 and cloned between the pdc promoter and trpC terminator into the BamHI site of Npxbthg using the ClonExpress MultiS kit (Vazyme Biotech) resulting in vector Npxbthg-OEXyr1 and Npxbthg-OEmXyr1. These vectors were transformed into the recipient strain using A. tumefaciens-mediated fungal transformation.
Clonexpress multis kit
Manufactured by Vazyme
Sourced in China
The ClonExpress MultiS kit is a DNA cloning tool designed for efficient and flexible gene assembly. It enables the seamless joining of multiple DNA fragments in a single reaction.
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Lab products found in correlation
4 protocols using clonexpress multis kit
1
Overexpression of Xyr1 in Trichoderma
2
Versatile Cloning of DCL3 Constructs
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The ClonExpress II or ClonExpress MultiS kit (Vazyme, China) was used to construct the following plasmids by recombination cloning following the manufacturer’s instructions. All oligonucleotide primers used are listed in Table S1 .
pBI121-6myc-DCL3 was generated by cloning the coding sequence (NM_001161190.2, 4743 bp) of DCL3 from Col-0 plants into the BamHI-linearized vector pBI121-Myc.
To generate pBI121-6myc-DCL3mRIII and pBI121-6myc-DCL3ΔRBD constructs, corresponding sequences were amplified from pBI121-6myc-DCL3, and the PCR products were cloned into the BamHI-linearized vector pBI121-Myc. For the plocex-DCL3-EGFP, plocex-DCL3mRIII-EGFP and plocex-DCL3ΔRBD constructs, the DCL3, DCL3mRIII and DCL3ΔRBD sequences were cloned into the SpeI/BamHI-linear vector plocex-EGFP.
For generating translational fusion with glutathione S-transferase (GST) protein, the fragments (F1, F2, F3, F4 and F5) of DCL3 were amplified from pBI121-6myc-DCL3 and cloned into the BamHI/XhoI-linearized vector pGEX-4T-2.
pBI121-6myc-DCL3 was generated by cloning the coding sequence (NM_001161190.2, 4743 bp) of DCL3 from Col-0 plants into the BamHI-linearized vector pBI121-Myc.
To generate pBI121-6myc-DCL3mRIII and pBI121-6myc-DCL3ΔRBD constructs, corresponding sequences were amplified from pBI121-6myc-DCL3, and the PCR products were cloned into the BamHI-linearized vector pBI121-Myc. For the plocex-DCL3-EGFP, plocex-DCL3mRIII-EGFP and plocex-DCL3ΔRBD constructs, the DCL3, DCL3mRIII and DCL3ΔRBD sequences were cloned into the SpeI/BamHI-linear vector plocex-EGFP.
For generating translational fusion with glutathione S-transferase (GST) protein, the fragments (F1, F2, F3, F4 and F5) of DCL3 were amplified from pBI121-6myc-DCL3 and cloned into the BamHI/XhoI-linearized vector pGEX-4T-2.
3
Simultaneous Silencing of NaJOX-like Genes
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We used virus-induced gene silencing (VIGS) technology to create plants in which NaJOX-like-1, -2, -3, and -4 were independently and simultaneously silenced. cDNA fragments of NaJOX-like-1, -2, -3 and -4 were amplified by PCR and fused into BamHI/HindIII-linearized pTV00 vectors. To simultaneously silence the four NaJOX-like genes, all four cDNA fragments were ligated into pTV00 by the infusion cloning method based on homologous recombination using the ClonExpress MultiS kit (Vazyme, China). The primer pairs used for plasmid construction are listed in Table S1 . The Agrobacterium tumefaciens (strain GV3101)-mediated transformation procedure for VIGS was described previously (Saedler and Baldwin, 2004 (link)). Plants for VIGS experiments were grown in a greenhouse at 23 °C/8 h light. To monitor the progress of VIGS, we simultaneously silenced another set of plants with a construct specific for phytoene desaturase (NaPDS), resulting in visible bleaching of green tissue. When the leaves of NaPDS-silenced plants were sufficiently bleached (~6 weeks after germination), leaves of NaJOX-like-silenced (VIGS-NaJOXs), and empty vector-inoculated (VIGS-EV) plants were used for experiments.
4
Generating Msm RNase J Knockout Strain
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Msm-RNase J knockout plasmid was obtained by the homologous recombinant method. The upstream (2,760,206–2,762,205) and downstream (2,763,883–2,765,882) homologous fragments flanking the RNase J were cloned from the genome of Msm MC2155, then constructed into the plasmid containing the resistant gene and SacB gene by ClonExpress® MultiS kit (Vazyme, China). The correct clones were confirmed by restriction enzyme digestion and DNA sequencing. Electroporation of Msm MC2155 was performed as previously described40 (link). After electroporation and recovery, cells were plated on 7H10 plates containing 50 µg/mL kanamycin to screen the positive clones. Then, the positive monoclones were transferred to a non-resistance 7H19 medium for double exchange and plated on 7H10 plates containing 50 µg/mL kanamycin and 10% sucrose for another three days. The correct RNase J knockout (KO) strain was confirmed by the colony PCR method. The RNase J complemented strain was obtained by transforming the pMV261 plasmid expressing RNase J into the KO strain and obtained the RNase J stable-expressing Msm strain through single-clone screening and identification. The correct RNase J complemented (Com) strain was confirmed by colony PCR and qRT-PCR methods.
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