Name: measuring gene expression in bombarded barley aleurone layers with increased throughput
Uploaded: 2018-03-30T13:30:00
Description: Watch this Scientific Journal Video about Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput at JoVE.com

Forfatterne takke Greyson Butler og Margaret Barrett til hjælp ved udførelsen af eksperimenter, Judy Stone for rådgivning om korn homogenisering og Lynn Hannum for rådgivning om fluorometry. Dette arbejde blev støttet af National Science Foundation (IOB 0443676), en institutionel udvikling Award (idé) fra National Institute of General Medical Sciences af National Institutes of Health under grant nummer P20GM0103423, og tilskud fra Colby College Division af naturvidenskab.

1. forberedelse af effektor og Reporter gen konstruktioner
<ol><li>Bygge effektor konstruktioner, der beskæftiger en konstitutive promoter (f.eks. majs ubiquitin, UBI) at drive udtryk fra en ønskede åben behandling ramme. For eksempel konstruere et plasmid som indeholder UBI::TaABF1 til kørsel stærk konstitutiv udtryk af TaABF15.</li>
	<li>Bygge reporter konstruktioner, der indeholder arrangøren hvis aktivitet der skal måles, anbringes opstrøms på en åben læsning ram...

<ol>
	<li>Chen, K., An, Y. Q. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+responses+to+gibberellin+and+abscisic+acid+in+barley+aleurone.">Transcriptional responses to gibberellin and abscisic acid in barley aleurone.</a> J. Integ. Plant Biol. 48, 591-612 (2006).</li><li>Lanahan, M. B., Ho, T. H. D., Rogers, S. W., Rogers, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gibberellin+response+complex+in+cereal+alpha-amylase+gene+promoters.">A gibberellin response complex in cereal alpha-amylase gene promoters.</a> Plant Cell. 4, 203-211 (1992).</li><li>Shen, Q., Zhang, P., Ho, T. H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+nature+of+abscisic+acid+(ABA)+response+complexes;+composite+promoter+units+that+are+necessary+and+sufficient+for+ABA+induction+of+gene+expression.">Modular nature of abscisic acid (ABA) response complexes; composite promoter units that are necessary and sufficient for ABA induction of gene expression.</a> Plant Cell. 8, 1107-1119 (1996).</li><li>G&#243;mez-Cadenas, A., Zentella, R., Walker-Simmons, M. K., Ho, T. H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gibberellin/abscisic+acid+antagonism+in+barley+aleurone+cells:+site+of+action+of+the+protein+kinase+PKABA1+in+relation+to+gibberellin+signaling+molecules.">Gibberellin/abscisic acid antagonism in barley aleurone cells: site of action of the protein kinase PKABA1 in relation to gibberellin signaling molecules.</a> Plant Cell. 13, 667-679 (2001).</li><li>Johnson, R. R., Shin, M., Shen, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wheat+PKABA1-interacting+factor+TaABF1+mediates+both+abscisic+acid-suppressed+and+abscisic+acid-induced+gene+expression+in+bombarded+aleurone+cells.">The wheat PKABA1-interacting factor TaABF1 mediates both abscisic acid-suppressed and abscisic acid-induced gene expression in bombarded aleurone cells.</a> Plant Mol. Biol. 68, 93-103 (2008).</li><li>Harris, L. J., Martinez, S. A., Keyser, B. R., Dyer, W. E., Johnson, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+TaABF1+during+abscisic+acid+and+gibberellin+signaling+in+aleurone+cells+of+cereal+grains.">Functional analysis of TaABF1 during abscisic acid and gibberellin signaling in aleurone cells of cereal grains.</a> Seed Science Res. 23, 89-98 (2013).</li><li>Shen, Q., Casaretto, J., Zhang, P., Ho, T. H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+definition+of+ABA-response+complexes:+the+promoter+units+necessary+and+sufficient+for+ABA+induction+of+gene+expression+in+barley+(Hordeum+vulgare).">Functional definition of ABA-response complexes: the promoter units necessary and sufficient for ABA induction of gene expression in barley (Hordeum vulgare).</a> Plant Mol. Biol. 54, 111-124 (2004).</li><li>Ritchie, S., Gilroy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abscisic+acid+signal+transduction+in+the+barley+aleurone+is+mediated+by+phospholipase+D+activity.">Abscisic acid signal transduction in the barley aleurone is mediated by phospholipase D activity.</a> Proc. Natl. Acad. Sci. USA. 95, 2697-2702 (1998).</li><li>Takemiya, A., Shimazaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidic+acid+inhibits+blue+light-induced+stomatal+opening+via+inhibition+of+protein+phosphatase+1.">Phosphatidic acid inhibits blue light-induced stomatal opening via inhibition of protein phosphatase 1.</a> Plant Physiol. 153, 1555-1562 (2010).</li><li>Zou, X., Seeman, J. R., Neuman, D., Shen, Q. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+WRKY+gene+from+creosote+bush+encodes+an+activator+of+the+abscisic+acid+signaling+pathway.">A WRKY gene from creosote bush encodes an activator of the abscisic acid signaling pathway.</a> J. Biol. Chem. 279, 55770-55779 (2004).</li><li>Ishibashi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+are+involved+in+gibberellin/abscisic+acid+signaling+in+barley+aleurone+cells.">Reactive oxygen species are involved in gibberellin/abscisic acid signaling in barley aleurone cells.</a> Plant Physiol. 158, 1705-1714 (2012).</li><li>Piskurewicz, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gibberellic+acid+signaling+repressor+RGL2+inhibits+Arabidopsis+seed+germination+by+stimulating+abscisic+acid+synthesis+and+ABI5+activity.">The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and ABI5 activity.</a> Plant Cell. 20, 2729-2745 (2008).</li><li>Hong, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation-mediated+regulation+of+a+rice+ABA+responsive+element+binding+factor.">Phosphorylation-mediated regulation of a rice ABA responsive element binding factor.</a> Phytochemistry. 72, 27-36 (2011).</li><li>Lopez-Molina, L., Mongrand, S., McLachlin, D. T., Chait, B. T., Chua, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABI5+acts+downstream+of+ABI3+to+execute+an+ABA-dependent+growth+arrest+during+germination.">ABI5 acts downstream of ABI3 to execute an ABA-dependent growth arrest during germination.</a> Plant J. 32, 317-328 (2002).</li><li>Zhou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOS2-LIKE+PROTEIN+KINASE5,+an+SNF1-RELATED+PROTEIN+KINASE3-Type+protein+kinase,+is+important+for+abscisic+acid+responses+in+Arabidopsis+through+phosphorylation+of+ABSCISIC+ACID-INSESENSITIVE5.">SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-Type protein kinase, is important for abscisic acid responses in Arabidopsis through phosphorylation of ABSCISIC ACID-INSESENSITIVE5.</a> Plant Physiol. 168, 659-676 (2015).</li><li>Zong, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+regulation+of+ABA+signaling+and+biosynthesis+by+a+bZIP+transcription+factor+targets+drought-resistance-related+genes.">Feedback regulation of ABA signaling and biosynthesis by a bZIP transcription factor targets drought-resistance-related genes.</a> Plant Physiol. 171, 2810-2825 (2016).</li></ol>

Indførelsen af effektor gen konstruerer via partikel bombardement, efterfulgt af forbigående udtryk for reporter konstruktioner er en værdifuld strategi for dissekere rolle af særlige aktører i GA/ABA signalering og den resulterende hormon-reguleret gen udtryk. Dog er eksisterende protokoller for at udføre sådanne forsøg i Byg aleurone celler2,3,4,5,6</su...

Byg aleurone lag er en veletableret modelsystem for studiet af hormon-regulerede genekspression i planter1. Især aktiveres en række gener, der kræves til spiring eller tidlige sætteplante udvikling ved gibberellin (GA), mens gener forbundet med stress svar er aktiveret af abscisic syre (ABA). GA og ABA signalering veje er forbundet, som udtryk for nogle GA-aktiveret gener er hæmmet af ABA, og vice-versa1.
En værdifuld strategi for forståelse af særlige aktører i GA/ABA signalering rolle har været indførelsen af effektor gen konstruktioner via partikel bombardement, efterfulgt af forbigående udtryk for reporter konstruktioner, der giver den resulterende effekt på downstream genekspression skal fastlægges. Brug af reporter gener som GUS (β-glucuronidasesystemer) eller Luciferase tillader den følsomme og kvantitativ måling af genekspression specielt inden for de celler, der har modtaget effektor konstruktion. For eksempel, etableret indførelsen af en effektor konstruere kodning transkriptionsfaktor TaABF15,6 , at ABA-inducerede gener såsom HVA1 er induceret af TaABF1, mens GA-inducerede gener såsom Amy32b undertrykkes. Partikel bombardement som en eksperimentel strategi er blevet brugt af flere laboratorier til at undersøge forskellige aspekter af GA/ABA signalering. Dette arbejde har ført til identifikation af promotor elementer vigtig for aktivering af både GA-induceret2 og ABA-inducerede gener3og til opdagelsen af protein kinaser4 og transskription faktorer5 , der regulerer de udtryk for disse gener.
Den eksisterende protokoller2,3,4,5,6 til partikel bombardement og efterfølgende måling af forbigående genekspression er ganske arbejdsintensive, som hvert sæt bombarderet knap korn er homogeniseret i hånden i en morter og støder og enzym-assays skal foretages individuelt. Dette manuskript rapporterer en forbedret protokol, der delvist automatiserer og strømliner trinnet homogenisering og GUS-undersøgelser for at tillade betydeligt flere gennemløb, tillader et større antal behandlinger til testes i det samme eksperiment, og/eller den inddragelse af flere flergangsbestemmelser for hver behandling for at opnå mere statistisk solide resultater. Repræsentative resultater er vist for udtryk for HVA1 og Amy32b reporter konstruktioner, reguleret af transkriptionsfaktor TaABF1 samt af GA, ABA og andre regulerende molekyler.

<table>
	<tbody>
		<tr>
			<td>GeneElute HP plasmid Maxiprep kit</td>
			<td>Sigma</td>
			<td>NA0310-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-vis spectrophotometer</td>
			<td>Nanodrop</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Himalaya barley grains</td>
			<td>/</td>
			<td>/</td>
			<td>A variety of hulless barley (store in the dark at 4&#176; C)</td>
		</tr>
		<tr>
			<td>sodium succinate</td>
			<td>Sigma</td>
			<td>S2378</td>
			<td>Reagent for Imbibing Solution</td>
		</tr>
		<tr>
			<td>calcium chloride (dihydrate)</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td>Reagent for Imbibing Solution</td>
		</tr>
		<tr>
			<td>Imbibing Solution</td>
			<td>home made</td>
			<td>/</td>
			<td>20 mM sodium succinate, 20 mM calcium chloride, pH 5.0. Sterilize by autoclaving before use.</td>
		</tr>
		<tr>
			<td>chloramphenicol</td>
			<td>Sigma</td>
			<td>C0378</td>
			<td>Prepare a 10 mg/mL stock solution in 70% ethanol.</td>
		</tr>
		<tr>
			<td>vermiculite</td>
			<td>Fisher</td>
			<td>NC0430369</td>
			<td>Used for vermiculite plates.</td>
		</tr>
		<tr>
			<td>filter paper circles (90 mm)</td>
			<td>Whatman</td>
			<td>1001 090</td>
			<td>Used for vermiculite and for pre-bombardment grain preparation</td>
		</tr>
		<tr>
			<td>Vermiculite Plates</td>
			<td>home made</td>
			<td>/</td>
			<td>Add 50 mL of vermiculite to a glass petri dish. Place a 90 mm paper circle on top of the vermiculite. Autoclave.</td>
		</tr>
		<tr>
			<td>forceps (fine pointed)</td>
			<td>Fisher</td>
			<td>13-812-42</td>
			<td>Used for removing seed coat from barley grains.</td>
		</tr>
		<tr>
			<td>forceps (ultra fine point)</td>
			<td>Fisher</td>
			<td>12-000-122</td>
			<td>Used for removing seed coat from barley grains.</td>
		</tr>
		<tr>
			<td>gold microcarriers (1.6 &#956;m)</td>
			<td>BioRad</td>
			<td>1652264</td>
			<td></td>
		</tr>
		<tr>
			<td>macrocarriers</td>
			<td>BioRad</td>
			<td>1652335</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chloride (dihydrate)</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td>Prepare a 2.5 M stock solution and store 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>spermidine</td>
			<td>Sigma</td>
			<td>S0266</td>
			<td>Prepare a 100 mM stock solution and store 500 &#956;L aliquots at -20&#176; C (use within 2 months).</td>
		</tr>
		<tr>
			<td>rupture discs (1550 psi)</td>
			<td>BioRad</td>
			<td>1652331</td>
			<td></td>
		</tr>
		<tr>
			<td>stopping screens</td>
			<td>BioRad</td>
			<td>1652336</td>
			<td></td>
		</tr>
		<tr>
			<td>macrocarrier holders</td>
			<td>BioRad</td>
			<td>1652322</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolistic particle delivery system</td>
			<td>BioRad</td>
			<td>PDS-1000/He</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium phosphate monobasic monohydrate</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td>Reagent for 1M sodium phosphate pH 7.2</td>
		</tr>
		<tr>
			<td>sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S9763</td>
			<td>Reagent for 1M sodium phosphate pH 7.2</td>
		</tr>
		<tr>
			<td>1M sodium phosphate pH 7.2</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 6.9 g of sodium phosphate monobasic monohydrate with 7.1 g of sodium phosphate dibasic. Add water to 100 mL. Add NaOH to get pH 7.2.</td>
		</tr>
		<tr>
			<td>dithiothrietol (DTT)</td>
			<td>Sigma</td>
			<td>43819</td>
			<td>Dissolve in water to 1 M. Store at -20&#176; in 1 mL aliquots.</td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td>Dissolve in water to 10 mg/mL. Store at -20&#176; C.</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>Prepare a 50% solution in water.</td>
		</tr>
		<tr>
			<td>Grinding Buffer</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 10 mL of 1 M sodium phosphate pH 7.2, 500 &#956;L of 1 M DTT, 100 &#956;L of 10 mg/mL leupeptin, and 40 mL of 50% glycerol. Add water to 100 mL.</td>
		</tr>
		<tr>
			<td>stainlesss steel beads (5 mm)</td>
			<td>Qiagen</td>
			<td>69989</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL tubes</td>
			<td>Eppendorf</td>
			<td>22363352</td>
			<td>This specific model of tube is recommended for use with the homogenizer.</td>
		</tr>
		<tr>
			<td>bead homogenizer (TissueLyser)</td>
			<td>Qiagen</td>
			<td>85210</td>
			<td></td>
		</tr>
		<tr>
			<td>12mm x 75 mm glass test tubes</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>luciferin</td>
			<td>Goldbio</td>
			<td>LUCK-100</td>
			<td>Prepare a 25 mM stock solution and store 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A7699</td>
			<td>Prepare a 100 mM stock solution and store 250 &#956;L aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>Reagent for 1M Tris sulfate pH 7.7.</td>
		</tr>
		<tr>
			<td>sulfuric acid</td>
			<td>Sigma</td>
			<td>258105</td>
			<td>Reagent for 1M Tris sulfate pH 7.7.</td>
		</tr>
		<tr>
			<td>1M Tris sulfate pH 7.7</td>
			<td>home made</td>
			<td>/</td>
			<td>Dissolve 12.1 g Tris base in 100 mL of water. Adjust pH to 7.7 with sulfuric acid.</td>
		</tr>
		<tr>
			<td>magnesium chloride</td>
			<td>Sigma</td>
			<td>M9397</td>
			<td>Dissolve in water to 2 M.</td>
		</tr>
		<tr>
			<td>Luciferase Assay Buffer (LAB)</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 3 mL of 1 M Tris sulfate pH 7.7, 500 &#956;L of 2 M magnesium chloride, 1 mL of 1 M DTT, and 200 &#956;L of 0.5 M EDTA. Add water to 50 mL.</td>
		</tr>
		<tr>
			<td>Luciferase Assay Mixture</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 15 mL of LAB, 800 &#956;L of 25 mM luciferin, 200 &#956;L of 100 mM ATP, and 4 mL of water. This makes enough assay mixture (20 mL) for 100 luciferase assays.</td>
		</tr>
		<tr>
			<td>luminometer (Sirius)</td>
			<td>Berthold</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>4-methylumbelliferyl-&#946;-D-glucuronide (MUG)</td>
			<td>Goldbio</td>
			<td>MUG1</td>
			<td>Dissolve in DMSO to 100 mM.</td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Sigma</td>
			<td>S8032</td>
			<td>Prepare a 2% stock solution in water and store 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>96 well plates (standard)</td>
			<td>Fisher</td>
			<td>12565501</td>
			<td></td>
		</tr>
		<tr>
			<td>GUS assay buffer</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 2.5 mL of MUG, 5 mL of 1 M sodium phosphate pH 7.2, 400 &#956;L of 0.5 M EDTA, 1 mL of 1 M DTT, 100 &#956;L of 10 mg/ml leupeptin, 20 mL of methanol, and 1 mL of 2% sodium azide. Add water to 100 mL.</td>
		</tr>
		<tr>
			<td>TempPlate sealing film</td>
			<td>USA Scientific</td>
			<td>2921-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates (black)</td>
			<td>Costar</td>
			<td>3916</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium carbonate</td>
			<td>Sigma</td>
			<td>S7795</td>
			<td>Prepare a 200 mM solution in water.</td>
		</tr>
		<tr>
			<td>4-methylumbelliferone</td>
			<td>Sigma</td>
			<td>M1381</td>
			<td>Prepare a 100 &#956;M solution in water. Freeze 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>microplate fluouresence reader</td>
			<td>Bio-Tek</td>
			<td>FLX-800</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring gene expression in bombarded barley aleurone layers with increased throughput

Aleurone lag af Byg korn er en vigtig modelsystem for hormon-regulerede genekspression i planter. I aleurone celler, gener kræves for spiring eller tidlige sætteplante udvikling er aktiveret af gibberellin (GA), mens gener forbundet med stress svar er aktiveret af abscisic syre (ABA). Mekanismerne i GA og ABA signalering kan blive afhørt ved at indføre reporter gen konstruktioner i aleurone celler via partikel bombardement, med den deraf følgende forbigående udtryk målt ved hjælp af enzym-assays. En forbedret protokol er rapporteret som delvist automatiserer og strømliner korn homogenisering trin og enzym-assays, giver betydeligt mere hurtigere end eksisterende metoder. Homogenisering af korn prøver er udført ved hjælp af en automatiseret væv homogeniseringsapparat, og GUS (β-glucuronidasesystemer) assays udføres ved hjælp af en 96-brønd pladesystem. Repræsentative resultater ved hjælp af protokollen tyder på at fosfolipase D aktivitet kan spille en vigtig rolle i aktivering af HVA1 genekspression af ABA, gennem transkriptionsfaktor TaABF1.

Den teknik beskrevet her kan bruges til at indføre enhver test gen konstruktion i aleurone celler af Byg korn. Udtryk fra test-genet kan derefter være bekvemt målt (figur 2). Den højere overførselshastighed protokollen beskrevet her kraftigt øger effektiviteten af frø slibning og enzym assay trin. Denne metode har været brugt til at vurdere transskription faktor TaABF15,6 evne til at aktivere ...

Watch this Scientific Journal Video about Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput at JoVE.com

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

En forbedret protokol præsenteres for måling af forbigående genekspression fra reporter konstruktioner i Byg aleurone celler efter partikel bombardement. Kombinationen af automatiserede korn slibning med 96 brønde plade enzym-assays giver høj overførselshastighed for proceduren.

Måling af genekspression i bombarderede Byg Aleurone lag med øget gennemløb

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

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JoVE Journal

Genetics

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