Name: a simple method for isolation of soybean protoplasts and application to transient gene expression analyses
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Detta arbete stöds av programmet växt Genome Research från National Science Foundation (NSF-PGRP-IOS-1339388).

1. tillväxt av växter
<ol><li>Så 5-10 sojabönor frön (Williams 82) i 13 cm kruka i växthuset under lång-dag förhållanden (16 h ljus på 1 500 µmol m-2 s-1) vid 25 ° C på den anpassade jord mix för sojabönor (1:1:1-förhållande av jord, perlit och torped sand).</li>
</ol>2. beredning av Plasmid DNA
<ol><li>Med hjälp av en steril pipettspetsen eller tandpetare, plocka en enda koloni eller frysta glycerol lager av E. coli transporterar plasmiden som ...

<ol>
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Detta protokoll för isolering av sojabönor protoplasts och ansökan till övergående uttryck studier har testats grundligt och fungerar mycket bra i vårt laboratorium. Förfaranden som är enkla och lätta och kräver vanlig utrustning och minsta möjliga kostnad. Våra protokoll ger stora mängder enhetlig, hög kvalitet protoplasts jämfört med tidigare rapporterade metoder34,35,36,37<su...

Protoplasts är växtceller som har cellväggar bort. De behålla de flesta av funktioner och aktiviteter av växtceller, protoplasts är en bra modell för att observera och utvärdera olika cellulära händelser och är värdefulla verktyg att studera somatisk hybridisering1 och växt regenerering2. Protoplasts har utnyttjats också allmänt för växt förvandling3,4,5, eftersom cellväggarna annars skulle blockera passagen av DNA i cellen. Protoplasts besitter några av fysiologiska svaren och cellulära processer av intakt växter, därför erbjuder grundläggande värde i grundforskning att studera subcellulär protein lokalisering6,7,8, protein-protein interaktioner9,10, och arrangören aktivitet11,12,13 i levande celler.
Isolering av växten protoplasts rapporterades först i 196014 och protokollen för både isolering och omvandling av protoplasts har utvecklats och optimerats. Ett standardförfarande av protoplast isolering innebär att skära av bladen och enzymatisk nedbrytning av cellväggar, följt av separation av släppt protoplasts från icke-smält vävnad skräp. Omvandling strategier inkluderar elektroporation15,16, Mikroskop17,18och polyetylenglykol (PEG)4,5,19 metoder. Ett stort antal arter har rapporterats framgångsrik för protoplast isolering, inklusive Citrus20, Brassica21, Solanaceae22 och andra prydnadsväxter familjer23,24. Medan olika vävnadstyper används i olika arter, har ett system av övergående uttryck i Arabidopsis mesophyll protoplast (TEAMP) isolerade från bladen på modell växten Arabidopsis thaliana varit väl etablerade25 och allmänt antagna till applikationsmöjligheter.
Sojabönor (Glycine max (L.) Merr.) är en av de viktigaste proteinet och olja grödor26. Till skillnad från Arabidopsis och ris, är att erhålla genmodifierade sojabönor växter kända för att vara ganska svårt och låga effektivitet. Agrobacterium tumefaciens-medierad infiltration har varit populärt används för övergående gen uttryck studier i de epidermala cellerna i tobak27 och plantor i Arabidopsis28,29, medan Agrobacterium rhizogenes har använts för omvandling av håriga rötter i sojabönor30. Virus-inducerade genen ljuddämpningssystemet metoder använts för nedreglering av mål gener31,32 och övergående uttryck33 i en systemisk sätt. Protoplasts ger en värdefull och mångsidigt alternativ till dessa synsätt. Protoplasts kan erhållas från sojabönors aboveground material och tillåter snabb och synkroniserade transgenens uttryck. Sedan första framgångsrika isolering av sojabönor protoplasts i den 198334, har det dock begränsat rapporter om tillämpningen av protoplasts i sojabönor35,36,37, 38, främst på grund av relativt låg avkastning av sojabönor protoplasts.
Här beskriver vi ett enkelt och effektivt protokoll för isolering av sojabönor protoplasts och dess tillämpning för övergående gen uttryck studier. Använder unga unifoliate blad från sojabönor plantor, kunde vi få stora mängder viktig protoplasts inom några timmar. Dessutom har vi optimerat en PEG-kalcium-medierad omvandling metod som är enkel och låg kostnad att leverera DNA in sojabönor protoplasts med hög verkningsgrad.

<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

Sojabönor (Glycine max (L.) Merr.) är en viktig gröda art och har blivit en baljväxt modell för studier av genetiska och biokemiska vägar. Det är därför viktigt att etablera en effektiv övergående gen uttryck system i sojabönor. Här rapporterar vi ett enkelt protokoll för beredning av sojabönor protoplasts och dess tillämpning för övergående funktionella analyser. Vi hittade att unga unifoliate blad från sojabönor plantor resulterade i stora mängder av hög kvalitet protoplasts. Genom att optimera en PEG-kalcium-medierad omvandling metod, uppnått vi hög förvandling effektivitet använder sojabönor unifoliate protoplasts. Detta system ger en effektiv och mångsidig modell för granskning av komplexa regelverk och signalering mekanismer i levande sojabönor celler och får hjälp att bättre förstå olika cellulär-, utvecklings- och fysiologiska processer av baljväxter.

Olika organ i 10 - dagar gamla sojabönor testades för protoplast beredning (figur 1) och avkastningen observerades under mikroskopet (figur 2). Cellväggar från hypocotyl och epicotyl var knappast rötas, och vissa celler stannade fästa vid varandra (figur 2B, 2 C). Hjärtblad (figur 2D) och rot (figur 2A), cellväggar t...

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

Vi utvecklade ett enkelt och effektivt protokoll för beredning av stora mängder sojabönor protoplasts att studera komplexa regelverk och signalering mekanismer i levande celler.

En enkel metod för isolering av sojabönor Protoplasts och ansökan till övergående gen uttryck analyser

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

Beredningen av primära hematopoietisk cell kulturer från Murine Bone Marrow för 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 ...

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

RNA Isolering från embryonala Zebrafish och cDNA syntes för Gene Expression Analysis

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

Använda Gene Pulser MXcell elektroporation System till transfektera primärceller med hög verkningsgrad

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

Hi-C: En metod för att studera den tredimensionella arkitektur för ledare.

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

Hjälp av ett automatiserat celltalsräknare att förenkla Studier Gene Expression: siRNA Knockdown av IL-4 beroende genuttryck i Namalwa Cells

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

Paraffininbäddade och frysta snitt av Drosophila Vuxen Muskler

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 Beredning av Active T. castaneum Telomeras

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

Isolering av ribosom Bundna framväxande Polypeptider<em&gt; In vitro</em&gt; För att identifiera Translationell Paus webbplatser Längs 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

Analys av global RNA syntes vid den inre cellnivå efter Hypoxi

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

Transient Expression av proteiner från Hydrodynamisk Gene Delivery hos möss

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