Name: a simple method for isolation of soybean protoplasts and application to transient gene expression analyses
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Ce travail a été soutenu par le programme de recherche de génome de plante de la National Science Foundation (NSF-PGRP-IOS-1339388).

1. la croissance des plantes
<ol><li>Semer les graines de soja de 5-10 (82 Williams) dans un pot de 13 cm en serre dans des conditions de jours longs (16 h de lumière à 1 500 µmol m-2 s-1) à 25 ° C sur le mélange de sol personnalisé pour le soya (1:1:1 ratio de sable sol, de perlite et de torpilles).</li>
</ol>2. préparation d’ADN plasmidique
<ol><li>À l’aide d’une pointe de pipette stérile ou un cure-dent, choisir une seule colonie ou stock de glycérol c...

<ol>
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Ce protocole pour l’isolement de protoplastes de soja et de l’application aux études d’expression transitoire a été testé et fonctionne très bien dans notre laboratoire. Les procédures sont simples et faciles et nécessitent des équipements ordinaires et coût minimum. Notre protocole génère de grandes quantités de protoplastes uniforme et de grande qualité par rapport aux méthodes rapportées antérieurement34,35,36...

Les protoplastes sont des cellules végétales qui ont enlevé les parois cellulaires. Qu’ils conservent la plupart des fonctions et activités des cellules végétales, les protoplastes sont un système de bon modèle d’observer et d’évaluer les divers événements cellulaires et sont des outils précieux pour étudier l’hybridation somatique1 et2de la régénération des plantes. Protoplastes ont été également largement utilisés pour usine de transformation3,4,5, puisque les parois cellulaires bloquerait sinon le passage de l’ADN dans la cellule. Les protoplastes possèdent certaines des réponses physiologiques et des processus cellulaires des plantes intactes, offrant ainsi une valeur fondamentale dans la recherche fondamentale pour étudier les protéines subcellulaire localisation6,7,8, interactions de protéine-protéine9,10et promoteur activité11,12,13 en vivent les cellules.
L’isolement de protoplastes végétaux a été pour la première fois en 196014 et les protocoles d’isolement et de transformation des protoplastes ont été développés et optimisés. Une procédure normalisée d’isolement de protoplastes implique le découpage des feuilles et la digestion enzymatique des parois cellulaires, suivi par séparation des protoplastes libérés de débris tissulaires non digérés. Stratégies de transformation comprend électroporation15,16, microinjection17,18et à base de polyéthylène glycol (PEG)4,5,19 méthodes. Un large éventail d’espèces ont été signalées avec succès pour l’isolement de protoplastes, y compris les agrumes20, Brassica21, Solanaceae22 et autres plantes ornementales familles23,24. Tandis que les types de tissus différents sont utilisés dans diverses espèces, un système d’expression transitoire dans Arabidopsis protoplast de mésophylle (TEAMP) isolé des feuilles de la plante modèle Arabidopsis thaliana a été bien établi25 et largement adopté pour diverses applications.
Soja (Glycine max (L.) Merr.) est l’une des plus importantes protéines et huile cultures26. À la différence des Arabidopsis et le riz, obtention de plants de soja transgénique est connu pour être plutôt difficile et faible efficacité. Agrobacterium tumefaciens-médiation infiltration a été populairement utilisée pour des études d’expression génétique transitoire dans les cellules épidermiques en tabac27 et semis dans Arabidopsis28,29, alors que Agrobacterium rhizogenes a été utilisé pour la transformation des racines velues en soja30. Les silencieux approches gène induite par le virus ont été utilisés pour downregulation de cible gènes31,32 et transitoires expression33 de façon systémique. Protoplastes fournissent une alternative utile et polyvalente à ces approches. Les protoplastes peuvent provenir de matériaux hors sol de soja et permettent l’expression du transgène rapide et synchronisée. Cependant, depuis l’isolement succès initial des protoplastes de soja dans les 198334, on a signalé limitée sur l’application des protoplastes de soja35,36,37, 38, principalement en raison des rendements relativement bas de protoplastes de soja.
Nous décrivons ici un protocole simple et efficace pour l’isolement de protoplastes de soja et son application pour des études d’expression génétique transitoire. À l’aide de jeunes feuilles unifoliées de semis de soya, nous avons pu obtenir de grandes quantités de protoplastes vitales en quelques heures. En outre, nous avons optimisé une méthode de transformation PEG-calcium-négociée qui est simple et bon marché pour livrer l’ADN dans des protoplastes de soja à haut rendement.

<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

Soja (Glycine max (L.) Merr.) est une espèce de culture importante et est devenu un modèle de légumineuses pour l’étude des voies génétiques et biochimiques. Par conséquent, il est important de mettre en place un système d’expression de gène transitoire efficace chez le soja. Nous rapportons ici un protocole simple pour la préparation des protoplastes de soja et sa demande d’analyses fonctionnelles en régime transitoire. Nous avons constaté que les jeunes feuilles unifoliées de semis de soya résulte en grande quantité des protoplastes de haute qualité. En optimisant une méthode de transformation PEG-calcium-négociée, nous avons atteint l’efficacité de transformation élevés à l’aide de protoplastes unifoliées soja. Ce système fournit un modèle efficace et polyvalent pour l’examen des mécanismes de réglementation et signalisation complexes dans les cellules de soja direct et peut aider à mieux comprendre divers processus cellulaires, physiologiques et du développement des légumineuses.

Différents organes de soja vieux de 10 jours, ont été testés pour la préparation de protoplaste (Figure 1) et les rendements ont été observés au microscope (Figure 2). Les parois cellulaires de l’hypocotyle et épicotyles sont difficilement digérés, et certaines cellules sont restés attachés les uns aux autres (Figure 2 b, 2C). Cotylédon (Figure 2D...

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

Nous avons développé un protocole simple et efficace pour la préparation de grandes quantités de protoplastes de soja afin d’étudier les mécanismes réglementaires et signalisation complexes dans des cellules vivantes.

Une méthode Simple pour l’isolement de protoplastes de soja et Application aux Analyses d’Expression génétique transitoire

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

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

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

Recherche

Biologie

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

Isolement d&#39;ARN à partir de synthèse d&#39;ADNc embryonnaires Zebrafish et pour analyser l&#39;expression génique

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

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

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

Salut-C: une méthode d&#39;étude de l&#39;architecture tridimensionnelle des génomes.

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

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules Namalwa

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

Les articles inclus en paraffine et congelés de Drosophile</emMuscles adultes>

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

La reconstitution in vitro de l&#39;actif T. La télomérase castaneum

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

Isolement du ribosome Bound polypeptides naissants<em&gt; In vitro</em&gt; Pour identifier les sites de pause de translation le long d&#39;ARNm

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 de Global ARN de synthèse à la Cellule Plain suivante 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

Expression transitoire de protéines par hydrodynamique Gene livraison chez la souris

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