基因枪法介导玉米胚的瞬时转化

Matilde  Jose-Estanyol

doi:10.21769/BioProtoc.865

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Peer-reviewed

Maize Embryo Transient Transformation by Particle Bombardment

基因枪法介导玉米胚的瞬时转化

MJ Matilde Jose-Estanyol email

发布: 2013年08月20日第3卷第16期 DOI: 10.21769/BioProtoc.865 浏览次数: 10127

评审: Tie Liu

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The authors used this protocol in:

Cover of Plant Molecular Biology, featuring study using the protocol.

Oct 2012

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Abstract

Particle bombardment has been shown to be a useful method to study gene promoter regulatory elements by transient transformation of maize embryos with different constructions of gene promoters fused to a gene reporter. DNA to transfer is coated to high density gold microparticles and introduced into cells when accelerated by a helium pulse. This method allows a first rapid approach, avoiding time consuming stable transformation of maize plants and also allows quantitative promoter expression analysis by a histochemical or fluorometric assay.

Keywords: Particle bombardment (粒子轰击)

Maize embryos (玉米胚)

Promoter expression (启动子表达)

Quantitative analysis (定量分析)

Materials and Reagents

Plant Material: Maize plants from W64A pure inbred line
Ethanol absolute, gradient HPLC grade (Scharlau, S.A. UN1170)
1.0 μm Gold Microcarriers (Bio-Rad Laboratories, catalog number: 165-2263 )
Rupture Disks 900 psi (Bio-Rad Laboratories, catalog number: 165-2328 )
Macrocarriers (Bio-Rad Laboratories, catalog number: 165-2335 )
Stopping Screens (Bio-Rad Laboratories, catalog number: 165-2336 )
Murashige and Skoog medium (M&S) with vitamins (Duchefa, catalog number: M0222 )
Spermidine free base (Sigma-Aldrich, catalog number: S-2626 )
Sodium hypochlorite, solution 5-9% Cl active (Carlo Erba, catalog number: CH0223 )
Cell culture dishes 60 mm x 15 mm Style treated polystyrene (Corning Inc., catalog number: 430166 )
X-GlcA sodium trihydrate (Duchefa, catalog number: X-1406 )
X-glucuronide: 5 bromo-4-chloro-3-indolyl-β-D-glucoronic sodium salt 3H₂O
N,N-dimethylformamid for spectroscopy (Merck KGaA, catalog number: 102937 )
MUG: 4 methyl-umbelliferyl-B-D-glucuronide hydrate C (Sigma-Aldrich, catalog number: M9130 )
Potassium hexacyanoferrate (III) (Merck KGaA, catalog number: 104973 )
Potassium hexacyanoferrate (II) trihydrate (Merck KGaA, catalog number: 104984 )
4-Methylumbelliferone sodium salt (4-MU) (Sigma-Aldrich, catalog number: M-1508 )
Luciferase Assay Reagent (Promega Corporation, catalog number: E1500 )
CDTA: trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (Sigma-Aldrich, catalog number: D0922 )
Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
Histochemical analysis detection buffer (see Recipes)
Fluorometric lysis buffer (see Recipes)
Fluorometric analysis reaction buffer (see Recipes)
Fluorimetric analysis stopping buffer (see Recipes)
MSO medium (see Recipes)
1x Luciferase lysis buffer (see Recipes)

Equipment

BIORAD PDS-1000/He system (Biolistic Delivery Systems)
Laminar Flow Cabinet Telstar AH-100
Vacuum pump Telstar 50/60 Hz
Cylinder compressed helium gas (UN1046) 117 Kg (LYNDE)
RAYPA Bathwater sonicator 50 W
MINI-SECOEUR IKA MS2 minishaker with a support that allows simultaneous agitation of different Eppendorf tubs
MiniSpin eppendorf centrifuge
ND-1000 Spectrophotometer (NanoDrop)
Spectra Max M3 apparatus (bioNova cientifica S.L.)
Olimpus Stereomicroscope SZX16
Screw cylinder polypropylene microtube with attached O-Ring cap and conical base (Sarstedt, catalog number: 72.693.105 )