Clonexpresstm 2 one step cloning kit

The full length CDS of GmMT-II was cloned into the binary vectors pA7-GFP using the ClonExpress TM II One Step Cloning Kit (Vazyme, Nanjing, China) and inserted into pA7-GFP vector to construct a GmMT-II-pA7-GFP fusion expression vector. The fusion construct and the empty control vector (pA7-GFP) were transformed into the A.tumefaciens and transient transformation of onion epidermal was performed through the particle bombardment method. The plants were kept at 28 °C and 80% relative humidity (RH) with a 16/8 h photoperiod for 48 h until examination. Fluorescent signals were observed using a confocal laser scanning microscopy (CLSM) (Leica TCS SP2 confocal microscope, Jena, Germany). All CLSM images were obtained using Leica confocal software and the HCX PL APO 63×/1.2 W CORR water immersion objective. The GFP channel was acquired by excitation at 488 nm with detection at 500–530 nm. At least three replicates were used to essay all transient expression.

For PCR experiments, standard protocols were applied with a PCR amplification kit (TaKaRa, Cat. # R011). Fungal RNA was extracted by means of the RNAsimple Total RNA Kit (TIANGEN Cat. # DP419). Plasmid DNA was isolated from E. coli using the TIANprep Rapid Mini Plasmid Kit (TIANGEN Cat. # DP105–03). DNA fragments separated in an agarose gel were extracted with the Universal DNA Purification Kit (TIANGEN Cat. # DP214–03). Multiple fragments were assembled via the ClonExpress^TM II One Step Cloning Kit (Vazyme Biotech Co., Ltd., China). Strains P. pastoris GS115 and E. coli TOP10 and yeast vectors pPICZ B and pPIC3.5 K were purchased from Invitrogen. Transformation of yeast cells and screening of transformants were executed according to Pichia protocols³⁹ . Yeast two-hybrid (Y2H) assay were described in detail in supplementary data file (Supplementary Fig. S7).

The mutant 6C4 was obtained from our previous T-DNA insertion mutagenesis experiments [25 (link)]. In the mutant, the exon of VdNUC-2 (VdLs.17 homolog VDAG_00896) was truncated by T-DNA (S1 Fig). The deletion construct for VdNUC-2 knockout was prepared in accordance with the protocol reported by Paz et al. [51 (link)] and then used in the ATMT of V07DF2 to obtain independent mutant strains. A 7.0 kb genomic DNA fragment, which included the putative promoter and coding sequence of VdNUC-2, was amplified and cloned into the complementation plasmid named pCambia1300-ble (S6 Fig) by ClonExpress^TM II One Step Cloning Kit (Vazyme, Nanjing, China). The plasmid was used in the ATMT of 6C4 to generate VdNUC-2 complementation. All primers used for plasmid construction are shown in S1 Table. Additionally, the VdNUC-1 and VdPHO-2 (VdLs.17 homolog VDAG_03154 and VDAG_01304) targeted deletion mutants were also obtained using the same protocol with the specific primers shown in S1 Table.

A mutant library of the ido gene was constructed by error-prone PCR from pET-ido using an instant error-prone PCR kit(Tiandz, Inc.; 101005) and the primers ido-1 and ido-2. The mutated gene products were consequently ligated into the vector pWSK29, which had been digested by Xba I via the ClonExpressTM II One Step Cloning Kit(Vazyme Biotech Co., Ltd; C11201), using a homologous recombinase. The recombinant plasmids were transformed into E. coli ΔsucAΔaceA, and the cells were grown on plates containing M9 medium(glycerol as a carbon source) supplemented with L-Ile and α-KGA(1 g/L) as well as ampicillin(100 mg/L) and IPTG(0.05 mM).
The ido gene was amplified from pWSK-ido or pWSK-ido ^M3 by PCR using primers ido-3 and ido-4. The obtained PCR products were digested by BamH I and Hind III and were cloned into the expression vector pET-His. The recombinant plasmids(pET-IDO and pET-IDO^M3) were further transformed into competent E. coli BL21(DE3) cells, and the cells were selected on LB plates containing 100 mg/L ampicillin, resulting in BL-ido and BL-ido^M3.

We used pSPL3 vector, a generous gift from Dr. Irene Bottillo (Sapienza University of Rome, Italy) and Dr. Leping Shao (Qingdao University, China). The exons and flanking introns of the ATP7B gene were amplified by Phanta^® Super-Fidelity DNA Polymerase (Vazyme Biotech Co., Ltd., China) with the primers indicated in Supplementary Table 2. The primers used in this step were edited by Primer 5 software, and the corresponding viscous terminal sequence was added to the end. pSPL3 vector was cut with XhoI and BamHI (Invitrogen, United States). All of the indicated fragments were cloned into a pSPL3 vector with the XhoI and BamHI using ClonExpressTM II One Step Cloning Kit (Vazyme Biotech Co., Ltd., China). All constructs were sequenced to confirm by Sanger sequencing (TsingKe Co., Ltd., China), and the corresponding exon wild-type plasmids (Minigene ATP7B_ex) were screened. All clones were functionally checked in 293T cells (Cell Bank of the Chinese Academy of Sciences, China., Cat# GNHu17), and 14 kinds of wild-type plasmids were constructed by inserting exons into the splicing vector pSPL3, covering 16 exons (except exons 1, 2, 8, 9, and 21). The pattern for constructing the plasmids can be found in Supplementary Figure 1. The sequencing data of the RT-PCR products of the 14 kinds of wild-type plasmids which covered 16 exons of ATP7B are shown in Supplementary Figure 2.

To create hybrid minigene constructs, we used the pSPL3 minigene reporter vector, which includes a conventional expression system with two exons (SD6 and SA2) to analyze the resultant mRNA transcripts. The minigene vector mainly produces two transcripts, one composed of exon SD6, an inserted exon, and exon SA2 (upper), and the other composed only of exon SD6 and SA2 (lower) (Figure 2A). To perform a minigene assay, we generated fragments containing the target exons (3, 12, and 13) where the variants were located, and 150-200 bp of flanking intronic regions with XhoI and BamHI restriction sites. These inserts were amplified by PCR from the patients' genomic DNA using primers described in Supplementary Table 2. Both edges of the shortened introns were properly designed by the Human Splicing Finder to avoid the activation of cryptic splicing. The pSPL3 vector was digested by restriction enzymes XhoI and BamHI, and then ligated with the purified PCR products to construct the wild-type and mutant minigene vectors using the ClonExpressTM II One Step Cloning Kit (Vazyme Biotech Co., Ltd). All constructs were confirmed by bidirectional sequencing.

To generate an effector construct, the full-length ORF of PtHB13 was fused into the pGreenII 62-SK vector using the ClonExpress^TM II One Step Cloning Kit (Vazyme Biotech Co.,Ltd., Nanjing, China), while the original and mutated PtFLC promoter fragments were cloned into pGreenII 0800-LUC to generate reporters. For transient gene expression analysis, the effector and reporter constructs were co-transformed into tobacco leaf cells. Transformation and detection of LUC activity were performed as previously described [44 (link)]. The transformed tobacco leaf cells were detected by using the Dual-Luciferase^® Reporter Assay System (Promega (Beijing) Biotech Co., Ltd., Beijing, China).