Name: a simple method for isolation of soybean protoplasts and application to transient gene expression analyses
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Dette arbeidet ble støttet av anlegget Genome Research Program fra National Science Foundation (NSF-PGRP-IOS-1339388).

1. vekst av planter
<ol><li>Så 5-10 soyabønner frø (Williams 82) i en 13 cm pott i drivhuset under lange dager forhold (16 h lys på 1500 µmol m-2 s-1) ved 25 ° C på egendefinerte jord blanding for soyabønner (1:1:1 ratio med jord, perlitt og torpedo sand).</li>
</ol>2. utarbeidelse av plasmider DNA
<ol><li>Bruke et sterilt pipette tips eller tannpirker, Velg en enkelt koloni eller frosne glyserol lager av E. coli bærer plasmider inneholder genet av inter...

<ol>
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Denne protokollen for isolering av soyabønner protoplasts og programmet forbigående uttrykk studier har blitt grundig testet og fungerer godt i vårt laboratorium. Fremgangsmåten er enkel og lett og krever vanlig utstyr og minimale kostnader. Våre protokollen gir store mengder ensartet, høy kvalitet protoplasts i forhold til tidligere rapportert metoder,34,,35,,36,,37,,<sup clas...

Protoplasts er anlegget celler som har cellevegger fjernet. De vedlikeholde det meste av funksjoner og aktiviteter i anlegget celler, protoplasts er en god modell å observere og vurdere ulike mobilnettet arrangementer, og er verdifulle verktøy å studere somatisk hybridiseringen1 og plante gjenfødelse2. Protoplasts har vært også mye benyttet for plante transformasjon3,4,5, siden cellevegger ellers blokkere passasjen av DNA i cellen. Protoplasts har noen av de fysiologiske responser og cellulære prosesser intakt planter, derfor tilbyr grunnleggende verdien i grunnleggende forskning til å studere subcellular protein lokalisering6,7,8, Protein-protein interaksjoner9,10og promoter aktivitet11,12,13 i lever celler.
Isolasjon av anlegget protoplasts ble først rapportert i 196014 og protokollene for både isolasjon og transformasjonen av protoplasts er utviklet og optimalisert. En standard prosedyre protoplast isolasjon innebærer skjæring av blader og enzymatiske fordøyelsen av cellevegger, etterfulgt av separasjon av utgitt protoplasts fra ikke-fordøyd vev rusk. Transformasjon strategier inkluderer electroporation15,16, microinjection17,18og polyetylenglykol-basert (PEG)4,5,19 metoder. En rekke arter har rapportert vellykket for protoplast isolasjon, inkludert sitrus20, Brassica21, Solanaceae22 og andre dekorative plant familier23,24. Mens ulike vev brukes i ulike arter, er et system av forbigående uttrykk i Arabidopsis mesophyll protoplast (TEAMP) isolert fra bladene av modellen anlegget Arabidopsis thaliana godt etablert25 og vidt vedtatt å forskjellige programmer.
Soyabønner (Glycine max (L.) Merr.) er en av de viktigste protein og olje avlinger26. I motsetning Arabidopsis og ris, er skaffe transgene soyabønner planter kjent for å være ganske vanskelig og lav effektivitet. Agrobacterium tumefaciens-mediert infiltrasjon har blitt populært brukt for forbigående gene expression studier i epidermal cellene i tobakk27 og planter i Arabidopsis28,29, mens Agrobacterium rhizogenes har blitt brukt for transformasjon av hårete røtter i soyabønner30. Forårsaket av virus genet stanse tilnærminger har blitt benyttet for downregulation målet gener31,32 og forbigående uttrykk33 i en systemisk måte. Protoplasts gir en verdifull og allsidig alternativ til disse tilnærmingene. Protoplasts kan fås fra soyabønner aboveground materialer og tillate rask og synkronisert transgene uttrykk. Men siden den første vellykkede isolering av soyabønner protoplasts i 198334, har det vært begrenset rapporter på anvendelse av protoplasts i soyabønner35,36,37, 38, hovedsakelig på grunn av relativt lav avkastning på soyabønner protoplasts.
Her beskriver vi en enkel og effektiv protokoll for isolering av soyabønner protoplasts og programmet for forbigående gene expression studier. Bruker unge unifoliate blader fra soybean frøplanter, kunne vi få store mengder av avgjørende protoplasts i noen timer. Dessuten, har vi optimalisert en PEG-kalsium-mediert transformasjon metode som er enkelt og rimelig å levere DNA i soyabønner protoplasts med høy effektivitet.

<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

Soyabønner (Glycine max (L.) Merr.) er en viktig avling og har blitt en legume modell for studier av genetiske og biokjemiske. Derfor er det viktig å etablere en effektiv forbigående gene expression system i soyabønner. Her rapporterer vi en enkel protokoll for utarbeidelse av soyabønner protoplasts og programmet for forbigående funksjonelle analyser. Vi fant at unge unifoliate blader fra soybean frøplanter resulterte i store mengder av høykvalitets protoplasts. Ved å optimalisere en PEG-kalsium-mediert transformasjon metoden, oppnådd vi høye transformasjon effektivitet ved hjelp soyabønner unifoliate protoplasts. Dette systemet gir en effektiv og allsidig modell for undersøkelse av komplekse regulatoriske og signalnettverk mekanismer i live soyabønner celler og kan bidra til å bedre forstå ulike mobilnettet, utviklingsmessige og fysiologiske prosesser av belgfrukter.

Ulike organer av 10 - dagers gammel soyabønner ble testet for protoplast forberedelse (figur 1) og gir ble observert under mikroskopet (figur 2). Cellevegger fra hypocotyl og epicotyl var knapt fordøyd, og noen celler ble festet til hverandre (figur 2B, 2 C). I cotyledon (figur 2D) og roten (figur 2A), ble cellevegger fjer...

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 utviklet en enkel og effektiv protokoll for utarbeidelse av store mengder soyabønner protoplasts å studere komplekse regulatoriske og signalnettverk mekanismer i levende celler.

En enkel metode for isolering av soyabønner Protoplasts og programmet forbigående Gene Expression 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

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