Name: a simple method for isolation of soybean protoplasts and application to transient gene expression analyses
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Este trabajo fue apoyado por el programa de investigación de genoma de planta de la National Science Foundation (NSF-PGRP-IOS-1339388).

1. crecimiento de las plantas
<ol><li>Sembrar semillas de soja de 5-10 (Williams 82) en una maceta de 13 cm en el invernadero bajo condiciones de día largo (16 h de luz en 1.500 μmol m-2 s-1) a 25 ° C en la mezcla de suelo personalizados para soja (el 1: relación 1:1 de arena de tierra, perlita y torpedo).</li>
</ol>2. preparación de plásmido ADN
<ol><li>Utilizando una pipeta estéril o palillo de dientes, elige una sola Colonia o stock de glicerol congelados de <em...

<ol>
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Este protocolo para el aislamiento de protoplastos de soja y la aplicación a los estudios de expresión transitoria ha sido completamente probado y funciona muy bien en nuestro laboratorio. Los procedimientos son simples y fáciles y requieren equipo ordinario y mínimo coste. Nuestro protocolo produce grandes cantidades de protoplastos de uniforme y de alta calidad en comparación con métodos previamente divulgados34,35,36<su...

Protoplastos son células vegetales que han quitado las paredes celulares. Como sostienen la mayoría de características y actividades de las células vegetales, protoplastos son un buen modelo de sistema para observar y evaluar diversos eventos celulares y son herramientas valiosas para el estudio de hibridación somática1 y2de la regeneración de la planta. Protoplastos han sido también ampliamente utilizados para planta transformación3,4,5, ya que las paredes celulares lo contrario bloquearía el paso de DNA en la célula. Protoplastos poseen algunas de las respuestas fisiológicas y los procesos celulares de las plantas intactas, ofreciendo por lo tanto, valor fundamental en la investigación básica para el estudio de proteína subcelular localización6,7,8, de9,de las interacciones proteína-proteína10y promotor actividad11,12,13 en viven las células.
El aislamiento de protoplastos de plantas primero fue divulgado en 196014 y los protocolos de aislamiento y transformación de protoplastos se han desarrollado y optimizado. Un procedimiento estándar de aislamiento protoplasto implica el corte de hojas y digestión enzimática de las paredes celulares, seguido por separación de protoplastos liberados de restos de tejido no-digerido. Estrategias de transformación incluye electroporación15,16, microinyección17,18y polietilenglicol (PEG)4,5,19 métodos basados. Una amplia gama de especies se han divulgado éxito para el aislamiento del protoplasto, incluyendo cítricos20, Brassica21, Solanaceae22 y otras plantas ornamentales familias23,24. Tipos de tejidos diversos se utilizan en varias especies, un sistema de expresión transitoria en el protoplasto de mesófilo de Arabidopsis (TEAMP) aislado de hojas de la planta modelo Arabidopsis thaliana ha sido bien establecida25 y ampliamente adoptado para diversas aplicaciones.
Soja (Glycine max (L.) Merr.) es una de las más importantes proteínas y aceite de cultivos de26. A diferencia de Arabidopsis y arroz, obtención de plantas transgénicas de soja se sabe para ser algo difícil y baja eficiencia. Tumefaciens de la agrobacteria-mediada por la infiltración ha sido popularmente utilizada para estudios de expresión transitoria del gen en las células epidérmicas en tabaco27 y plántulas de Arabidopsis28,29, mientras que Agrobacterium rhizogenes ha sido utilizado para la transformación de las raíces pilosas en soja30. Enfoques de silenciamiento génico inducido por virus han sido utilizados para la desregulación de los genes del blanco31,expresión32 y transitorio33 de manera sistémica. Protoplastos proporcionan una alternativa versátil y valiosa a estos enfoques. Protoplastos pueden obtenerse materiales sobre tierra de soja y permiten la expresión del transgen rápida y sincronizada. Sin embargo, desde el éxito inicial aislamiento de protoplastos de soja en el 198334, ha habido informes limitados en el uso de protoplastos en soja35,36,37, 38, principalmente debido a la relativamente baja producción de protoplastos de soja.
Aquí, describimos un protocolo sencillo y eficaz para el aislamiento de protoplastos de soja y su aplicación para estudios de expresión génica transitoria. Utilizando hojas unifolioladas jóvenes de plántulas de soja, hemos sido capaces de obtener grandes cantidades de protoplastos vitales dentro de unas horas. Además, hemos optimizado un método de transformación mediada por PEG-calcio que es simple y de bajo costo para entregar ADN en protoplastos de soja con alta eficiencia.

<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.) es una especie de cultivo importante y se ha convertido en un modelo de leguminosas para los estudios de vías genéticas y bioquímicas. Por lo tanto, es importante establecer un sistema de expresión eficaz de genes transitorios en soja. Aquí, Divulgamos un protocolo simple para la preparación de protoplastos de soja y su aplicación para el análisis funcionales transitorios. Hemos encontrado que jóvenes unifolioladas hojas de plántulas de soja resultaron en grandes cantidades de protoplastos de alta calidad. Mediante la optimización de un método de transformación mediada por PEG-calcio, logramos eficiencia alta transformación mediante protoplastos unifolioladas soja. Este sistema proporciona un modelo eficiente y versátil para el examen de mecanismos complejos de regulación y señalización en células de soya vivo y puede ayudar a mejor entender diversos procesos celulares, fisiológicos y de desarrollo de las leguminosas.

Diferentes órganos de soja 10 - viejo fueron probados para la preparación del protoplasto (figura 1) y rendimientos fueron observados bajo el microscopio (figura 2). Las paredes celulares de hipocótilo y epicotyl apenas fueron digeridas, y algunas células se quedaron conectadas entre sí (figura 2B, 2C). En el cotiledón (Figura 2D) y raíz (<strong ...

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

Hemos desarrollado un protocolo sencillo y eficaz para la preparación de grandes cantidades de protoplastos de soja para el estudio de complejos mecanismos de regulación y señalización en células vivas.

Un método Simple para el aislamiento de protoplastos de soja y aplicación al análisis de expresión génica transitoria

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 preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

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

Investigación

Biología

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

Aislamiento del ARN de síntesis embrionarias pez cebra y el cDNA de análisis de expresión génica

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

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

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

Hi-C: Un método para estudiar la arquitectura tridimensional de los genomas.

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

El uso de un contador de células automatizado para simplificar estudios de expresión génica: siRNA Derribo de IL-4 la expresión génica en células dependientes 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

Las secciones incluidas en parafina y congeladas de Drosophila Los músculos de Adultos

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

En la reconstitución in vitro de los activos T. castaneum telomerasa

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

El aislamiento de los ribosomas polipéptidos nacientes Bound<em&gt; In vitro</em&gt; Para la identificación de sitios de traslación pausa a lo largo del 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

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

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

Expresión transitoria de proteínas por hidrodinámico entrega de genes en ratones

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