Rice (Oryza sativa) Protoplast Isolation and Its Application for Transient Expression Analysis

Feng He1, Songbiao Chen2, Yuese Ning1, Guo‐Liang Wang3

1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 2 Biotechnology Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, 3 Department of Plant Pathology, The Ohio State University, Columbus, Ohio
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20026
Online Posting Date:  June, 2016
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Rice (Oryza sativa) is not only the staple food for half of the world's population but also a model monocot plant for molecular biology studies. Although rice genes have been extensively investigated in the last two decades, the functions of many genes in the rice genome are still not known. One of the rapid and efficient approaches for determining gene function in vivo is protoplast‐based transient expression analysis. We established a rice protoplast system about 10 years ago, which has been recently used in many laboratories. This protocol is useful for protein expression, subcellular localization, bimolecular fluorescence complementation, and co‐immunoprecipitation assays. © 2016 by John Wiley & Sons, Inc.

Keywords: rice (Oryza sativa); protoplast isolation; transient expression; BiFC and Co‐IP

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Table of Contents

  • Introduction
  • Basic Protocol 1: Protoplast Isolation and Transfection
  • Support Protocol 1: Fluorescence Detection
  • Support Protocol 2: Protein Extraction for Western Blot and IP/CO‐IP Assays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Protoplast Isolation and Transfection

  • Rice seeds
  • 1/2× MS medium (see recipe)
  • 75% ethanol
  • Sodium hypochlorite solution (Sigma‐Aldrich, cat. no. 239305)
  • Enzyme solution (see recipe)
  • W5 medium (see recipe)
  • Suspension medium (see recipe)
  • Plasmid DNA in sterilized double‐distilled water (ddH 2O; ∼1 μg/µl)
  • 40% PEG solution (see recipe)
  • Miracloth (Millipore, cat. no. 475855‐1R)
  • Razor blade
  • 50‐ml conical flask
  • 50‐ml conical tubes and compatible centrifuge
  • 1.5‐ and 2.0‐ml microcentrifuge or EP tubes and compatible centrifuge
  • 20‐, 200‐, and 1000‐µl pipet tips and pipets

Support Protocol 1: Fluorescence Detection

  Additional Materials (also see Basic*Protocol)
  • Transfected protoplasts
  • Microscope slide and cover slide
  • Fluorescence or confocal microscope

Support Protocol 2: Protein Extraction for Western Blot and IP/CO‐IP Assays

  Additional Materials (also see Basic*Protocol)
  • Transfected protoplasts
  • Native protein extraction buffer (see recipe)
  • 4× protein loading buffer (see recipe)
  • Phosphate‐buffered saline/Tween 20 (PBST) buffer (see recipe)
  • Agarose beads for immunoprecipitation (IP) assay (optional)
  • Antibodies (optional)
  • Agarose beads and antibodies for Co‐IP assay (example: Protein G Agarose, Fast Flow [Millipore, cat. no. 16‐266]; Anti‐HA High Affinity antibody [Roche, cat. no. 11867423001]; Anti‐cMyc Tag Antibody [Genscript, cat. no. A00704])
  • Platform rocker
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Literature Cited

Literature Cited
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