Agrobacterium vacuum infiltration was performed as described in Matoba et al. (Matoba et al.,
Pri101 an vector
Manufactured by Takara Bio
Sourced in China
The PRI101-AN vector is a plasmid DNA vector designed for laboratory use. It contains the necessary genetic elements for cloning and expressing recombinant proteins in various cell lines. The core function of this vector is to serve as a tool for molecular biology research and experimentation.
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10 protocols using pri101 an vector
1
Transient Protein Expression via Agrobacterium Infiltration
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Agrobacterium vacuum infiltration was performed as described in Matoba et al. (Matoba et al.,2010 ). pICH11599‐GRFT, pICH14011 integrase promodule, pICH17388 apoplast module (Marillonnet et al., 2004 ), pICH38077‐NahG (PVX one component expression vector) and pRI101‐AN vector (for AtXYL1 overexpression, Clontech) were transformed into Agrobacterium tumefaciens GV3101 by electroporation. Each Agrobacterium culture was suspended in infiltration buffer (10 mm MES, 10 mm MgSO4, pH 5.5) and mixed at OD600 0.1. The Agrobacterium suspension was infiltrated into leaves using vacuum pump. At 5 days postinfiltration (dpi), infected leaves were harvested and analysed.
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Agrobacterium vacuum infiltration was performed as described in Matoba et al. (Matoba et al.,
2
Cloning and Characterization of Wheat HSFA2h
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The full-length wheat HSFA2h sequence was amplified using the Fp_HSFA2h-pRI101 and Rp_HSFA2h-pRI101 primers (Table 1 ).
The amplified cDNA and pRI101-An vector (from Clontech) were restricted with NdeI and KpnI restriction enzymes, gel purified, and ligated with T4 ligase enzyme (NEB) in order to clone the HSFA2h into the plant expression vector pRI 101-AN under the regulation of the cauliflower mosaic virus (CaMV)-35S constitutive promoter. The recombinant plasmids were mobilized into Agrobacterium tumefaciens EHA105 and further used to transform A. thaliana ecotype Columbia by the floral dip method [39 ]. Antibiotic-resistant transformed lines were further validated through qRT-PCR and Northern blot analyses. Homozygous T3 lines were further characterized for their thermotolerance using different biochemical and molecular parameters under HS.
The amplified cDNA and pRI101-An vector (from Clontech) were restricted with NdeI and KpnI restriction enzymes, gel purified, and ligated with T4 ligase enzyme (NEB) in order to clone the HSFA2h into the plant expression vector pRI 101-AN under the regulation of the cauliflower mosaic virus (CaMV)-35S constitutive promoter. The recombinant plasmids were mobilized into Agrobacterium tumefaciens EHA105 and further used to transform A. thaliana ecotype Columbia by the floral dip method [39 ]. Antibiotic-resistant transformed lines were further validated through qRT-PCR and Northern blot analyses. Homozygous T3 lines were further characterized for their thermotolerance using different biochemical and molecular parameters under HS.
3
Transient Expression of VvMYB14 in Tobacco
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The VvMYB14 ORF was cloned and ligated into a pRI101-AN vector (Takara, Dalian, China) downstream of the 35 S promoter, yielding a 35 S::MYB14 plasmid. The VvACS1 promoter, the region up to 1500 bp upstream of ATG, and its mutated form with the mutant MBS element were used to replace the 35 S promoter within pRI101-GUS, yielding a Pacs1::Gus plasmid and a mutant Pacs1::Gus plasmid. The plasmids were subsequently introduced into Agrobacterium strain GV3101. The Agrobacterium-mediated transient transformation of tobacco leaves was performed according to the methods of Yang et al55 (link). GUS histochemical staining and activity detection were then performed according to the methods of Jefferson et al.56 (link).
4
Overexpression of MdPIF4 in Apple
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The full-length cDNA of MdPIF4 was cloned into the pRI 101-AN vector (Takara, Dalian, China) to obtain the MdPIF4 overexpression construct. Then, the pRI 101-AN empty vector and recombinant construct were transformed into Agrobacterium tumefaciens strain LBA4404. These agrobacteria were grown in lysogeny broth (LB) medium supplemented with 50 mg/mL kanamycin and 50 mg/mL rifampicin [68 (link)].
Transgenic apple calluses were obtained by agrobacterium-mediated transformation [69 (link)]. Briefly, the 14-day-old apple calluses were co-cultured with agrobacterium strains carrying the empty vector or recombinant constructs in the dark for 20 min. Then the transformed apple calluses were plated on selective medium supplemented with 250 mg/L carbenicillin and 30 mg/L kanamycin [70 (link)]. The successfully transgenic apple calluses grew in about four weeks.
Transgenic apple calluses were obtained by agrobacterium-mediated transformation [69 (link)]. Briefly, the 14-day-old apple calluses were co-cultured with agrobacterium strains carrying the empty vector or recombinant constructs in the dark for 20 min. Then the transformed apple calluses were plated on selective medium supplemented with 250 mg/L carbenicillin and 30 mg/L kanamycin [70 (link)]. The successfully transgenic apple calluses grew in about four weeks.
5
Regulation of TaPHO2 Degradation by tae-miR399 and TaIPS1
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To investigate the regulation of tae-miR399 and TaIPS1 on TaPHO2 degradation, a 1.6 Kb cDNA fragment containing the five putative miR399 binding sites of the three TaPHO2 genes were cloned into pRI101-AN vector (Takara). The full length of tae-miR399 and TaIPS1.1 were also cloned into pRI101-AN vector. All the resulting binary vectors were introduced into the GV3101 strain of Agrobacterium tumefaciens. The Agrobacterium mediated transient expression assays in tobacco leaves were conducted as described by Liu et al.17 (link). The Primers used for vector construction were listed in Supplementary Table S1 .
6
Subcellular Localization of MdGSTF6
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MdGSTF6 was cloned into the pRI101-AN vector (Takara, Dalian, China) to construct the 35S::MdGSTF6-GFP recombinant vector. The vector was then transformed into Agrobacterium tumefaciens LBA4404 competent cells. Transgenic calli of 35S::MdGSTF6-GFP were obtained as described by Wang et al.57 (link), while 35S::GFP was used as a negative control. Protoplasts were isolated from the two transgenic calli as described previously58 (link). Fluorescence was observed under a fluorescence microscope (Nikon Ni-E, Tokyo, Japan).
The membrane protein and cytoplasmic protein were separated using the Membrane Protein and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The extracted proteins were tested by protein gel blotting using the anti-GFP antibody. A Coomassie blue-stained gel served as the loading control.
The membrane protein and cytoplasmic protein were separated using the Membrane Protein and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The extracted proteins were tested by protein gel blotting using the anti-GFP antibody. A Coomassie blue-stained gel served as the loading control.
7
Subcellular Localization of AIM24 Proteins
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To investigate the subcellular localization of AIM24-A and AIM24-B proteins, the GFP sequence was fused to the 5′ terminus of PpAIM24, MpAIM24-A1, MpAIM24-A2, MpAIM24-B, AtAIM24-B1, and AtAIM24-B2 and cloned into the pRI101-AN vector (Takara cat. #3262). The RFP-HDEL peptide was used as the marker for ER localization. The coupled plasmids were co-transformed into tobacco leaf epidermal cells using the Agrobacterium-mediated method (Sparkes et al., 2006 (link)). GFP and RFP fluorescence signals were observed using an Olympus FV1000 MPE confocal microscope. GFP fluorescence was excited with a 488-nm laser, and emitted fluorescence was recorded at 505–525 nm. RFP fluorescence was excited with a 543-nm laser, and emitted fluorescence was recorded at 560–660 nm. In addition, ER-tracker red (Beyotime, cat. #C1041) was used to label the ER. ER staining was performed following the kit manual. In brief, concentrated ER-tracker red was diluted (1:1000) to obtain a working solution. One-week-old plants were then incubated in ER-tracker red working solution for 30 min at room temperature. The fluorescence was detected using a confocal microscope (Olympus FV1000) with excitation at 587 nm and emission at 615 nm.
8
Agrobacterium-Mediated Gene Transformation
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For gene transformation, MdAGO4‐1/2 and MdDRM2‐1/2 were each cloned into the pRI101‐AN vector (TaKaRa) containing a 35S promoter and a GFP tag sequence to construct the 35S::AGO4‐1/2‐GFP and 35S::DRM2‐1/2‐GFP recombinant vectors, respectively. For RNAi assays, the partial CDS of MdNRPE1 was cloned into the pFGC1008 vector (http://www.chromdb.org ) using the restriction sites AscI/SwaI and BamHI/SpeI for forward and reverse cloning, respectively. These vectors were then transformed into Agrobacterium tumefaciens LBA4404 competent cells. The transformed Agrobacterium cells were incubated with 2‐week‐old apple callus on MS medium without antibiotics for 48 h in the dark at 24 °C. Then, the apple callus was transferred to medium containing kanamycin and carbenicillin to select cells harbouring the genes.
9
Overexpressing DEK33 and COG3236 in N. benthamiana
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For protein overexpression in N. benthamiana, full-length and truncated DEK33 were cloned into a pHB vector under the CaMV 35S promoter. DEK33, DEK33*, COG3236, and COG3236* were cloned into a modified pRI101-AN vector (Takara). For modification of pRI101-AN, a 6×FLAG fragment was cloned into the EcoRI and SacI sites. A GFP fragment was cloned into the NdeI and SalI sites, and EcoRI and SacI sites. A GST fragment was cloned into the NdeI and SalI sites. DEK33 and DEK33* were cloned into pRI101-AN with a FLAG tag. COG and COG* were cloned into pRI101-AN with a GFP tag. The positions at which the various truncations were made are shown in Supplementary Fig. S11 . All primers are listed in Supplementary Table S4 . Vectors were transformed into Agrobacterium tumefaciens strain GV3101. The agro-infiltration procedure was performed as previously described (Liu et al., 2010 (link)).
10
Subcellular Localization of SaWRKY1-GFP
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Coding sequence (CDS) of SaWRKY1 and GFP was amplified (Table S7 ). Both SaWRKY1 and GFP were cloned in binary plant expression pRI101‐ANvector (Takara Bio Inc., Kusatsu, Shiga, Japan). pRI101‐AN:SaWRKY:GFP and pRI101‐AN:GFP clones were transformed into Agrobacterium tumefaciens strain GV3101 using a standard transformation protocol. Empty pRI101‐AN vector was used as a negative control. A. tumefaciens cultures with respective clones were grown at 28 °C in Luria‐Bertani medium containing selection markers (25 mg/L rifampicin and 50 mg/L kanamycin) at 120 rpm for 24 h. Cells were harvested by centrifugation at 1370 g for 5 min and suspended in infiltration buffer (half strength MS medium, pH 5.6 and 200 μM acetosyringone). These were pelleted and re‐suspended in infiltration buffer by adjusting an OD 1.0 at 600 nm. Cultures were incubated at 24 °C for 3–4 h and were infiltrated into leaves of 2‐week‐old Nicotiana benthamiana plants that were maintained at 18 to 24 °C in a growth chamber. Leaf sections were visualized for subcellular localization of GFP at 6 dpi after agro‐infiltration using confocal laser scanning microscope (Zeiss LSM 710, Oberkochen, Germany).
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