Visualizing OsRZF1 expression in rice protoplasts

The ORF of the OsRZF1 gene without the stop codon was amplified (Supplemental Table S1) and cloned to the entry vector with a pENTR™/D-TOPO® Cloning Kit (Invitrogen, USA). Then the 2 × 35 s promoter (Marquès-Bueno et al., 2016 (link)) and amplified OsRZF1 ORF fused with EGFP marker were inserted into the pUC19 plasmid (Takara Bio Inc. Japan) using a Gibson assembly system (New England BioLabs, USA). Rice protoplasts were isolated and transformed according to the method previously reported (Saito et al. 2013 (link)). After 18 h, the EGFP fluorescence was detected using Zeiss LSM880 AxioObserver, equipped with an Argon laser as the excitation source, through a 20x/0.8 plan-Apochromat objective lens. EGFP was excited with an Argon laser at 561 nm and an intensity of 0.5% and detected between 597–624 nm with a detector gain value of 900. Images were analyzed and merged by ImageJ (NIH).

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Kobayashi N.I., Takagi H., Yang X., Nishizawa-Yokoi A., Segawa T., Hoshina T., Oonishi T., Suzuki H., Iwata R., Toki S., Nakanishi T.M, & Tanoi K. (2023). Mutations in RZF1, a zinc-finger protein, reduce magnesium uptake in roots and translocation to shoots in rice. Plant Physiology, 192(1), 342-355.

Publication 2023

pENTR™/D-TOPO® Cloning Kit pUC19 plasmid Gibson assembly system LSM880 AxioObserver

Argon laser Fluorescence Gene Lens Plasmid Protoplasts Rice Stop codon Topo Vector

Corresponding Organization : The University of Tokyo

Other organizations : Ishikawa Prefectural University, National Agriculture and Food Research Organization, Institute of Agrobiological Sciences, Utsunomiya University, National Institutes for Quantum and Radiological Science and Technology, Tohoku University, Yokohama City University, Ryukoku University, Hoshi University, Ebara (Japan)

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Variable analysis

independent variables

2 × 35 s promoter
Amplified OsRZF1 ORF fused with EGFP marker

dependent variables

EGFP fluorescence

control variables

Rice protoplasts

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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