Name: a simple method for isolation of soybean protoplasts and application to transient gene expression analyses
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Dit werk werd gesteund door de Plant Genome Research Program van de National Science Foundation (NSF-PGRP-IOS-1339388).

1. de groei van de planten
<ol><li>Zaaien 5-10 sojabonen (Williams 82) in een 13 cm pot in de serre omstandigheden lange-dag (16 h licht aan 1.500 µmol m-2 s-1) bij 25 ° C op de bodem van de aangepaste mix voor sojabonen (het 1:1:1 verhouding bodem, perliet en torpedo zand).</li>
</ol>2. voorbereiding van de plasmide DNA
<ol><li>Met behulp van een steriele Pipetteer tip of een tandenstoker, kies een één kolonie of bevroren glycerol voorraad van E. coli die de...

<ol>
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Dit protocol voor de isolatie van soja protoplasten en de toepassing op voorbijgaande expressie studies grondig is getest en werkt zeer goed in ons laboratorium. De procedures zijn eenvoudig en gemakkelijk en vereisen gewone apparatuur en minimale kosten. Ons protocol levert grote hoeveelheden van uniforme, hoge kwaliteit protoplasten in vergelijking met de eerder gemelde methoden34,35,,36,,37<...

Protoplasten zijn plantaardige cellen waarin celwanden verwijderd. Als ze beweert dat de meeste functies en activiteiten van plantencellen, protoplasten zijn een goed modelsysteem te observeren en evalueren van de verschillende cellulaire gebeurtenissen, en waardevolle hulpmiddelen te bestuderen van somatische hybridisatie1 en regeneratie2plant. Protoplasten hebben ook op grote schaal gebruikt voor plant transformatie3,4,5, aangezien celwanden zou anders het blokkeren van de passage van DNA in de cel. Protoplasten bezitten sommige van de fysiologische reacties en cellulaire processen van intact planten, vandaar het aanbieden van fundamentele waarde bij het basisonderzoek aan studie eiwit subcellular localisatie6,7,8, eiwit-eiwit interacties9,10, en promotor activiteit11,12,13 in levende cellen.
Het isolement van de plant protoplasten werd voor het eerst gemeld in 196014 en de protocollen voor zowel de isolatie en de transformatie van protoplasten zijn ontwikkeld en geoptimaliseerd. Een standaardprocedure voor isolatie van de protoplast impliceert het uitsnijden van bladeren en enzymatische vertering van celwanden, gevolgd door een scheiding van vrijgegeven protoplasten van niet-verteerde weefsels puin. Transformatie strategieën bevat electroporation15,16, microinjection17,18, en op basis van polyethyleenglycol (PEG)4,5,19 methoden. Een breed scala van soorten hebben gemeld succes voor isolatie van de protoplast, met inbegrip van Citrus20, Brassica21, Solanaceae22 en andere sierplanten gezinnen23,24. Terwijl verschillende weefseltypes (HLA) worden gebruikt in verschillende soorten, is een stelsel van meningsuiting van de voorbijgaande in Arabidopsis mesophyl protoplast (TEAMP) van de bladeren van de plant model Arabidopsis thaliana geïsoleerd goed ingeburgerd25 en algemeen aangenomen voor uiteenlopende toepassingen.
Sojabonen (Glycine max (L.) Merr.) is een van de belangrijkste eiwit en olie gewassen26. In tegenstelling tot Arabidopsis en rijst, is verkrijgen van transgene soja planten bekend nogal moeilijk en lage efficiëntie. Agrobacterium tumefaciens-gemedieerde infiltratie is volksmond gebruikt voor tijdelijke gen expressie studies in de epidermale cellen in tabak27 en zaailingen in Arabidopsis28,29, overwegende dat Agrobacterium rhizogenes is gebruikt voor transformatie van harige wortels in soja30. Virus-geïnduceerde gene silencing benaderingen hebben gebruikt voor Downregulatie van doel genen31,32 en voorbijgaande expressie33 met systemische. Protoplasten vormen een waardevolle en veelzijdig alternatief voor deze benaderingen. Protoplasten kunnen worden verkregen uit sojabonen van bovengrondse materialen en laat snel en gesynchroniseerde transgenic expressie. Echter sinds de eerste succesvolle isolatie van soja protoplasten in de 198334, zijn er beperkt verslagen over de toepassing van protoplasten in soja35,36,,37,, 38, voornamelijk te wijten aan de relatief lage opbrengst voor sojabonen protoplasten.
Hier beschrijven we een eenvoudig en efficiënt protocol voor isolatie van soja protoplasten en de toepassing ervan voor voorbijgaande gen expressie studies. Met behulp van jonge unifoliate bladeren van soja zaailingen, konden we krijgen van grote hoeveelheden van vitale protoplasten binnen een paar uur. Daarnaast hebben we een PEG-calcium-gemedieerde transformatie methode die is eenvoudig en low-cost te leveren van DNA in soja protoplasten met hoog rendement geoptimaliseerd.

<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

Sojabonen (Glycine max (L.) Merr.) is een belangrijk gewas soorten en geworden een peulvrucht model voor de studies van genetische en biochemische trajecten. Daarom is het belangrijk om een efficiënte voorbijgaande gen expressie systeem in soja. Wij rapporteren hier een eenvoudig protocol voor de bereiding van soja protoplasten en de toepassing ervan voor voorbijgaande functionele analyses. We vonden dat de jonge unifoliate bladeren van soja zaailingen in grote hoeveelheden hoogwaardige protoplasten resulteerde. We bereikt door het optimaliseren van een PEG-calcium-gemedieerde transformatie methode, hoge transformatie efficiëntie met behulp van soja unifoliate protoplasten. Dit systeem biedt een efficiënte en veelzijdige model voor onderzoek van complexe regelgeving en signalering mechanismen in levende soja cellen en kan helpen om beter begrijpen divers cellulaire-, ontwikkelings- en fysiologische processen van peulvruchten.

Verschillende organen van het 10 - dagen oude sojabonen werden getest voor protoplast voorbereiding (Figuur 1) en de opbrengsten werden waargenomen onder de Microscoop (Figuur 2). Celwanden van hypocotyl en epicotyl werden nauwelijks verteerd, en sommige cellen bleef vasthouden aan elkaar (figuur 2B, 2 C). In zaadlob (figuur 2D) en wortel (<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

We ontwikkelden een eenvoudige en efficiënte protocol voor de voorbereiding van grote hoeveelheden soja protoplasten om te studeren van de ingewikkelde regelgeving en signalering mechanismen in levende cellen.

Een eenvoudige methode voor het isoleren van soja protoplasten en toepassing op voorbijgaande gen expressie Analyses

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

De vervaardiging van primaire hematopoietische cellen culturen uit Murine Bone Marrow voor Elektroporatie

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

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

RNA isolatie van embryonale zebravis en cDNA synthese voor de analyse van genexpressie

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

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

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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

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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

Paraffine ingebedde en vriescoupes van Drosophila Adult Spieren

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 reconstitutie van de 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

Isolatie van Ribosoom Bound Nascent Polypeptiden<em&gt; In vitro</em&gt; Om Translationeel Pauze plaatsen langs mRNA identificeren

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 van Global RNA synthese in de Single Cell Level volgende 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

Voorbijgaande expressie van eiwitten door Hydrodynamic Gene Levering in Muizen

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