Name: a simple method for isolation of soybean protoplasts and application to transient gene expression analyses
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Diese Arbeit wurde von der Pflanze Genome Research Program von der National Science Foundation (NSF-PGRP-IOS-1339388) unterstützt.

1. Wachstum der Pflanzen
<ol><li>Säen von 5-10 Soja (Williams 82) in einem 13 cm-Topf im Gewächshaus unter Bedingungen der langen Tage (16 h Licht bei 1.500 µmol m-2 s-1) bei 25 ° C auf die benutzerdefinierte Erdmischung für Soja (1:1:1 Verhältnis von Erde, Perlite und Torpedo Sand).</li>
</ol>2. Vorbereitung von Plasmid DNA
<ol><li>Mit einem sterilen Pipettenspitze oder Zahnstocher, wählen Sie eine einzelne Kolonie oder gefrorenen Glycerin Lager von E. Coli</e...

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+constitutive+viral+promoters+in+transgenic+soybean+roots+and+nodules.">Evaluation of constitutive viral promoters in transgenic soybean roots and nodules.</a> Mol Plant Pathol. 21 (8), 1027-1035 (2008).</li><li>Nagamatsu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+soybean+genes+involved+in+flavonoid+biosynthesis+by+virus-induced+gene+silencing.">Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing.</a> Plant Biotechnol. J. 5 (6), 778-790 (2007).</li><li>Juvale, P. 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Dieses Protokoll für die Isolierung von Soja Protoplasten und die Anwendung auf transiente Expression Studien wurde gründlich getestet und funktioniert sehr gut in unserem Labor. Die Verfahren sind einfach und leicht und erfordern ordentliche Ausrüstung und minimalen Kosten. Unser Protokoll liefert große Mengen an gleichbleibende hohe Qualität Protoplasten im Vergleich zu bereits gemeldeten Methoden34,35,36,<sup ...

Protoplasten sind Pflanzenzellen, die Zellwände entfernt haben. Sie pflegen die meisten Funktionen und Aktivitäten von Pflanzenzellen, Protoplasten sind ein gutes Modellsystem zur Beobachtung und Auswertung der vielfältigen zelluläre Ereignisse, und wertvolle Tools für die somatische Hybridisierung1 zu studieren und Werk Regeneration2. Protoplasten wurden für Werk Verwandlung3,4,5, ebenfalls weit verbreitet genutzt, da Zellwände sonst den Durchgang von DNA in die Zelle blockieren würde. Protoplasten besitzen einige der physiologischen Reaktionen und zelluläre Prozesse der intakten Pflanzen, daher bietet Grundwert in der Grundlagenforschung, Protein subzelluläre Lokalisation6,7,8zu studieren, Protein-Protein Interaktionen9,10und Promotor-Aktivität11,12,13 in Leben Zellen.
Die Isolation der Pflanze Protoplasten wurde erstmals 196014 gemeldet und die Protokolle für Isolation und Transformation von Protoplasten entwickelt und optimiert wurden. Ein Standardverfahren der Protoplasten Isolation beinhaltet das Schneiden der Blätter und enzymatische Verdauung der Zellwände, gefolgt von Trennung von freigegebenen Protoplasten von Gewebe nicht verdaut Schutt. Transformationsstrategien umfasst Elektroporation15,16, Mikroinjektion17,18und Polyethylenglykol (PEG)4,5,19 Methoden. Eine Vielzahl von Arten gemeldet wurden erfolgreich für die Protoplasten Isolierung, einschließlich Zitrusfrüchte20, Brassica21, Solanaceae22 und anderen Zierpflanzen Familien23,24. Während verschiedene Gewebetypen in verschiedenen Arten verwendet werden, wurde ein System von transienten Expression in Arabidopsis Mesophyll Protoplasten (TEAMP) aus den Blättern der Modellpflanze Arabidopsis Thaliana isoliert gut etablierte25 und weit verbreiteten für vielfältige Anwendungen.
Sojabohne (Glycine max (L.) GESEG.) gehört zu den wichtigsten Protein und Öl Nutzpflanzen26. Im Gegensatz zu Arabidopsis und Reis transgene Sojapflanzen zu erhalten gilt eher schwierig und geringe Effizienz. Agrobacterium Tumefaciens-vermittelten Infiltration ist im Volksmund für transiente Genexpressionsstudien in der Epidermiszellen in Tabak27 und Sämlinge in Arabidopsis28,29, verwendet worden, während Transformation von behaarten Wurzeln in Soja30 Agrobacterium Rhizogenes bestritten. Virusbedingte Gen-Silencing-Ansätze wurden für Herabregulation von Ziel Gene31,32 und transiente Expression33 in einer systematischen Art und Weise genutzt. Protoplasten bieten eine wertvolle und vielseitige Alternative zu diesen Ansätzen. Protoplasten können Soja die oberirdische Materialien entnommen werden und ermöglichen schnelle und synchronisierte Transgene Ausdruck. Jedoch, seit die erste erfolgreiche Isolierung der Sojabohne Protoplasten in 198334gab es nur Berichte über die Anwendung von Protoplasten in Soja35,36,37, 38, in erster Linie aufgrund der relativ niedrigen Erträgen von Soja Protoplasten.
Hier beschreiben wir eine einfache und effiziente Protokoll für die Isolierung von Soja Protoplasten und seinen Antrag auf vorübergehende Genexpressionsstudien. Junge unifoliate Blätter aus Soja-Keimlingen konnten wir große Mengen an lebenswichtigen Protoplasten innerhalb weniger Stunden zu erhalten. Darüber hinaus haben wir einen PEG-Kalzium-vermittelten Transformationsmethode optimiert, die einfache und kostengünstige, DNA in Soja Protoplasten mit hohem Wirkungsgrad zu liefern ist.

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">MES</td>
			<td style="width:181px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">M8250-100G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulase CELF</td>
			<td>Worthington Biological Corporation</td>
			<td>LS002611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pectolyase Y-23</td>
			<td>BioWorld</td>
			<td>9033-35-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CELLULASE &#34;ONOZUKA&#34; R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACEROZYME R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td>ICN Biomedicals&#160;</td>
			<td>152540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher&#160;</td>
			<td>C79-500g&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>Sigma Aldrich&#160;</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma Aldrich&#160;</td>
			<td>S7653-1kg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher&#160;</td>
			<td>P217-500g&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma Aldrich&#160;</td>
			<td>M8266-100g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PEG4000</td>
			<td>Fluka</td>
			<td>81240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nylon mesh</td>
			<td>carolina</td>
			<td>652222N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Tissue Culture Plates&#160;</td>
			<td>USA Scientific</td>
			<td>CC7682-7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Razor Blades</td>
			<td style="width:181px;">Fisher</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hemacytometer</td>
			<td>hausserscientific</td>
			<td>1483</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:181px;">Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">EZNA plasmid miniprep kit</td>
			<td style="width:181px;">Omega</td>
			<td>D6942-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">GeneJET Plasmid Miniprep Kit</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>K0502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Centrifuge 5810</td>
			<td style="width:181px;">eppendorf</td>
			<td style="width:119px;">5811000827</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Centrifuge 5424</td>
			<td style="width:181px;">eppendorf</td>
			<td>22620401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Jencons Powerpette Plus Pipet Controller</td>
			<td style="width:181px;">Jencons</td>
			<td>14526-202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:256px;">Zeiss 710 Confocal Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:256px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td>
			<td style="width:181px;">Ambion</td>
			<td style="width:119px;">AM12450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">15 mL Centrifuge Tubes</td>
			<td style="width:181px;">Denville</td>
			<td style="width:119px;">C1018-P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">50 mL Centrifuge Tubes</td>
			<td style="width:181px;">Denville</td>
			<td style="width:119px;">C1060-P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Newborn Calf Serum</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:119px;">16010159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Soil</td>
			<td style="width:181px;">Ingram&#39;s Nursery</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">perlite</td>
			<td style="width:181px;">Vigoro</td>
			<td style="width:119px;">100521091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Torpedo Sand</td>
			<td style="width:181px;">JKS Ventures</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LB Broth, Lennox (Powder)</td>
			<td style="width:181px;">Fisher</td>
			<td style="width:119px;">BP1427-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple method for isolation of soybean protoplasts and application to transient gene expression analyses

Sojabohne (Glycine max (L.) GESEG.) ist eine wichtige Kulturarten und eine Hülsenfrucht-Modell für das Studium der genetischen und biochemischen Wege geworden. Daher ist es wichtig, eine effiziente transiente gen Expressionssystems in Soja zu etablieren. Hier berichten wir ein einfaches Protokoll für die Vorbereitung der Sojabohne Protoplasten und seinen Antrag auf vorübergehende Funktionsanalysen. Wir fanden, dass große Mengen an hochwertigen Protoplasten jungen unifoliate Blätter aus Soja-Keimlingen führte. Durch die Optimierung der PEG-Kalzium-vermittelten Transformationsmethode, erreichten wir hohe Transformationseffizienz mit Soja unifoliate Protoplasten. Dieses System bietet ein effiziente und vielseitige Modell für die Prüfung von komplexen regulatorischen und Signalisierung Mechanismen im live Soja Zellen und können helfen besser zu verstehen, diverse zelluläre, Entwicklungsstörungen und physiologische Prozessen von Leguminosen.

Verschiedene Organe des 10 - Tage alten Sojabohnen für Vorbereitung der Protoplasten (Abbildung 1) getestet wurden und Erträge beobachtet, unter dem Mikroskop (Abbildung 2). Zellwände aus Keimachse und Epicotyl waren kaum verdaut, und einige Zellen geblieben (Abbildung 2 b, 2 C) miteinander verbunden. Im Keimblatt (Abb. 2D) und Wurzel (<strong class="...

Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Wir entwickelten ein einfaches und effizientes Protokoll für die Herstellung großer Mengen von Soja Protoplasten, komplexe regulatorische und Signalisierung Mechanismen in lebenden Zellen zu studieren.

Eine einfache Methode zur Isolierung der Sojabohne Protoplasten und Anwendung auf vorübergehende Genexpressionsanalysen

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Research

JoVE Journal

Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Forschung

Biologie

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

RNA Isolation aus embryonalen Zebrafisch und cDNA-Synthese für die Genexpressionsanalyse

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

Hallo-C: eine Methode, um die dreidimensionale Architektur des Genoms Study.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Paraffin-Embedded-und Gefrierschnitte von Drosophila Adult Muskeln

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Rekonstitution der Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Analyse der globalen RNA-Synthese auf der Einzelzellebene folgende Hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Transiente Expression von Proteinen durch Hydrodynamische Gentransfer in Mäusen

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...