Name: expression of recombinant cellulase cel5a from trichoderma reesei in tobacco plants
Uploaded: 2014-06-13T15:00:00
Description: Watch this Scientific Journal Video about Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants at JoVE.com

Dette arbejde blev støttet af BMBF Forschungsinitiative &quot;BioEnergie 2021 - Forschung für die Nützung von Biomasse&quot;, og Cluster of Excellence &quot;Skræddersyede brændstoffer&quot;, der finansieres gennem Excellence-initiativet af de tyske føderale og statslige regeringer til at fremme videnskab og forskning på tyske universiteter.

1.. Vækst af vildtype N. tabacum Planter <ol><li> At vokse N. tabacum L. cv. Petit Havana SR1 planter på jorden, skal du placere tobak frø på passende krukker fyldt med normal pottemuld for spiring. Efter 2 uger, overføre kimplanter til individuelle potter. Inkubér planter i et drivhus med konstant 22 ° C (mørk periode), eller 25 ° C (lys periode) og 70% relativ luftfugtighed. Giv belysning i en 16/8 timers lys / mørke-cyklus (f.eks Philips IP65 400 W lamper, 180 pmol · sek -...

<ol>
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Rekombinant ekspression af cellulaser er et område af stor interesse på grund af ønsket om mere effektive biobrændstoffer produktionssystemer 1. Planter byder på mange muligheder for en vellykket cellulase produktion, med funktioner såsom økonomisk høst teknikker og autohydrolysis metoder bliver meget ønskelige egenskaber for storstilet produktion af biobrændstoffer. Yderligere brug af forskellige lokaliseringer til fremstilling af proteiner tillader, om nødvendigt, posttranslationelle modifikation...

<html> Nedbrydning af lignocellulose biomasse og dets omdannelse til flydende brændstof er blevet planlagt som en metode til at mindske afhængigheden af ​​fossile brændstoffer. En væsentlig forhindring i at etablere økonomisk gennemførlige biomasse systemer er i den enzymatiske nedbrydning af cellulose og hemicellulose 1. Plant ekspressionssystemer stort potentiale til fremstilling af enzymer i industriel skala. Landbrugssystemer til at høste planter økonomisk og volumen er allerede etableret, da de har været i tusinder af år. Produktionen af cellulaser i planter er ønskeligt på grund af faktorer som den lethed autohydrolysis 2, og potentialet for at maksimere brugen af lavere enzym niveau ved at øge fordøjeligheden af plante cellevægge 1. Endelig tillader plantesystemer målretning af rekombinante proteiner til specifikke områder af planteceller og er i stand til at posttranslationelt ændre enzymer, hvor det er påkrævet 3. ve_content &quot;&gt; Nicotiana tabacum (tobak) er meget almindeligt anvendt som en model organisme til heterologe protein udtryk studier i planter, på grund af sin hurtige vækst og biomasse akkumulerende har 4. Transient ekspression af rekombinante proteiner er en teknik, som giver proteinproduktion i korte perioder 5, samtidig med at fleksibilitet med hensyn til lokalisering og dermed posttranslationelle modifikationer af de udvalgte proteiner, dvs cellulaser. Dette muliggør produktionen af ​​cellulase (r) til grundlæggende analyse, mens også om grundlaget for yderligere strategier ekspression i planter. Ved hjælp af en sådan forbigående udtryk strategi, er en endoglucanase fra glycosylhydrolase familie 5, Cel5A, der stammer fra svampeværten Trichoderma reesei (Hypocrea jecorina) 6. produceres (i det følgende benævnt TrCel5A). TrCel5A er et 42 kDa protein, som er indbygget glycosyleret og er yderst aktive i hydroylzing cellulose kæder 7. Transient ekspression her beskrevne teknik er baseret på et relativt almindeligt anvendte system, infiltrere planternes blade med Agrobacterium, der bærer genet af interesse i en passende ekspressionsvektor. At give mulighed for hurtig analyse af succes i planta udtryk, kan TrCel5A også udtrykkes som et fusionsprotein med mCherry, en monomer fluorescerende protein oprindeligt stammer fra Discosoma sp. Protein dsRed 8, med yderligere seks histidinrester (His-tag) fusioneret til C-terminalen (TrCel5A-mCherry). Kan således monitoreres ekspression af det heterologe fusionsprotein TrCel5A-mCherry inden for den voksende plante ved hjælp af grønt lys at undersøge mCherry spredning. Hvis det ønskes, kan tynde snit af plantematerialet undersøges i grønt lys mikroskopisk at indføre den specifikke lokalisering af proteinet. For dette arbejde, signal-og transidde peptider blev indarbejdet i konstruktionen, at lokalisere det heterologe protein eksport til det endoplasmatiske reticulum 9. For at analysere aktiviteten af ​​planteprodukter udtrykt cellulaser, herunder TrCel5A en række af cellulase aktivitet test kan køres. Efter ekstraktion af de samlede opløselige vegetabilske proteiner kan TrCel5A delvist oprenset under anvendelse af en termisk inkubation teknik. Protein størrelse er etableret ved hjælp af SDS-PAGE efterfulgt af Western blotting. Zymografi kan anvendes til at analysere aktivitet mod substrater, fx cellulose, som har været carboxy-methylerede (hvilket carboxymethylcellulose: CMC) opløselighed 10. Cellulase og glucosidase-aktivitet kan overvåges ved hjælp af fluoroforen 4-methylumbelliferyl-(4-MU) i forbindelse med β-D-cellobiosid (kombineret: 4-MUC). En anden metode til at vurdere endoglucanaseaktivitet involverer spektrometriske analyse af CMC, som er blevet forbundet med et azo-dI (Remazolbrilliant Blue R) 11.. Derudover kan protein-aktivitet i begge endoglucanaser og en række cellulaser overvåges ved anvendelse af en sukker-analyse test, såsom p-hydroxy-benzoesyre-hydrazid (PAHBAH)-assay 12. Disse teknikker kan anvendes til at belyse og kvantificere aktiviteten af ​​det udtrykte cellulase af interesse.

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:308px;">4-methylumbelliferyl &#946;-D-cellobioside</td>
			<td style="width:127px;">SIGMA-ALDRICH</td>
			<td style="width:156px;">M6018</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>ROTH</td>
			<td>3738</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetosyringon (concentrated)</td>
			<td>ROTH</td>
			<td>6003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - kanamycin</td>
			<td>ROTH</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - rifampicin</td>
			<td>ROTH</td>
			<td>4163</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - carbenicillin</td>
			<td>ROTH</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibody - rabbit anti His(6) pAb</td>
			<td>ROCKLAND</td>
			<td>600-401-382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibody - goat anti rabbit AP, FC pAb</td>
			<td>DIANOVA</td>
			<td>111-055-008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Azo-carboxymethyl cellulose</td>
			<td>MEGAZYME</td>
			<td>S-ACMC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-cyclodextrin</td>
			<td>SIGMA-ALDRICH</td>
			<td>C4805</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carboxymethyl cellulose</td>
			<td>ROTH</td>
			<td>3333.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellulase from Trichoderma reesei ATCC 26921</td>
			<td>SIGMA-ALDRICH</td>
			<td>C2730</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Congo red</td>
			<td>SIGMA-ALDRICH</td>
			<td>75768</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coomasie brilliant blue G 250</td>
			<td>ROTH</td>
			<td>9598.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D(+)-glucose</td>
			<td>ROTH</td>
			<td>8337</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>ROTH</td>
			<td>K928</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter paper - Rotilabo blotting paper</td>
			<td>ROTH</td>
			<td>CL67</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NBT/BCIP</td>
			<td>SIGMA-ALDRICH</td>
			<td>72091</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES buffer</td>
			<td>ROTH</td>
			<td>4256</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>ROTH</td>
			<td>8388</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium citrate</td>
			<td>ROTH</td>
			<td>3580</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecylsulfate</td>
			<td>SERVA</td>
			<td>20765</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>ROTH</td>
			<td>6771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Soil, Einheitserde classic&#160;</td>
			<td>PATZER GMBH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectrometer - Infinite M200</td>
			<td>TECAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>SIGMA-ALDRICH</td>
			<td>S-5390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Hydroxy benzhydrazide&#160;</td>
			<td>SIGMA-ALDRICH</td>
			<td>H9882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>ROTH</td>
			<td>1058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV light source - BLAK RAY</td>
			<td>UVP</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vibratome - VT1000S</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

expression of recombinant cellulase cel5a from trichoderma reesei in tobacco plants

Cellulose nedbrydende enzymer, cellulaser, er mål for både forskning og industrielle interesser. Overvægten af ​​disse enzymer i vanskelige-til-kultur organismer, såsom hyfer-building svampe og anaerobe bakterier, har fremskyndet brug af rekombinante teknologi på dette område. Plant ekspressionsmetoder er en ønskelig for stor-skala produktion af enzymer og andre industrielt anvendelige proteiner. Heri er fremgangsmåder til transient ekspression af en svampeinfektion endoglucanase, Trichoderma reesei Cel5A i Nicotiana tabacum påvist. Vellykket proteinekspression vist overvåges ved fluorescens under anvendelse af et mCherry-enzym-fusionsprotein. Derudover er et sæt af grundlæggende test bruges til at undersøge aktiviteten af forbigående udtrykt T. reesei Cel5A, herunder SDS-PAGE, Western blotting, zymografi, samt fluorescens og farvestofbaserede substrat nedbrydnings assays. Det her beskrevne system kan anvendes til at fremstille en aktiv celleulase i en kort periode, for at vurdere potentialet for yderligere produktion i planter gennem konstitutive eller inducerbare ekspressionssystemer.

TrCel5A og TrCel5A-mCherry blev udtrykt med succes i tobaksplanter ved hjælp af den forbigående ekspression-systemet (figur 1A og 1B). Undersøgelse af ekspressionsmønsteret af TrCel5A-mCherry fusionsproteinet viste sunde blade (figur 1C), der viste udbredt protein ekspression under grønt lys (figur 1D). Ekstraktion af de samlede opløselige proteiner blev udført på tobaksplanter, der havde blade, der indeholdt udtrykt...

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Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobaksplanter blev anvendt til at fremstille en fungal cellulase, TrCel5A via et transient ekspressionssystem. Udtrykket kan overvåges ved hjælp af et fluorescerende fusionsprotein, og proteinet aktivitet blev karakteriseret efter ekspression.

Ekspression af rekombinant Cellulase Cel5A fra<em&gt; Trichoderma reesei</em&gt; I tobaksplanter

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

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Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Forbigående genekspression i Tobacco hjælp Gibson forsamling og Gene Gun

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Anvendelsen af ​​høj opløsning Infrarød termografi (HRIT) for Studiet af isnukleering og Ice Formering i Plants

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Protokol for at producere tredimensionale infrarød Video af frysning i planter

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The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...