Name: plant promoter analysis: identification and characterization of root nodule specific promoter in the common bean
Uploaded: 2017-12-23T12:30:00
Description: Watch this Scientific Journal Video about Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean at JoVE.com

Detta arbete var delvis stöds av Dirección General de Asuntos del personliga Académico, DGAPA/PAPIIT-UNAM (bevilja nr. IN219916 till M.L och IA205117 till M-K. (A) och Consejo Nacional de Ciencia y Tecnològia (CONACYT grant nr 240614 att M.L.).

1. identifiering, isolering och kloning av P. Vulgaris NIN arrangören
<ol>
	<li>Identifiera sekvensen promotorn för gen av intresse. Det finns flera arvsmassa databaser och analysverktyg för växter såsom Phytozome, häckning växter, NCBI, etc. A baljväxter arrangören P. vulgaris NIN (PvNIN; Phvul.009G115800) användes i denna studie20.</li>
	<li>Design oligos antingen för Gateway eller traditionella restriktionsenzym kloning beroende på vector systemet använ...

<ol>
	<li>Hern&#225;ndez-Garcia, C. M., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+validation+of+promoters+and+cis-actingregulatory+elements.">Identification and validation of promoters and cis-actingregulatory elements.</a> Plant Science. 217-218, 109-119 (2014).</li><li>Dhanapal, A. P., Govindaraj, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unlimited+Thirst+for+Genome+Sequencing,+Data+Interpretation,+and+Database+Usage+in+Genomic+Era:+The+Road+towards+Fast-Track+Crop+Plant+Improvement.">Unlimited Thirst for Genome Sequencing, Data Interpretation, and Database Usage in Genomic Era: The Road towards Fast-Track Crop Plant Improvement.</a> Genetics Research International. , 684321 (2015).</li><li>Nanjareddy, N., Arthikala, M. K., Blanco, L., Arellano, E. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+localization+of+a+symbiosis-specific+phosphate+transporter+is+mediated+by+a+transient+reorientation+of+secretion.">Polar localization of a symbiosis-specific phosphate transporter is mediated by a transient reorientation of secretion.</a> Proc Natl Acad Sci U S A. 109 (11), E665-E672 (2012).</li><li>Kryvoruchko, I. S., 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=MtSWEET11,+a+Nodule-Specific+Sucrose+Transporter+of+Medicago+truncatula.">MtSWEET11, a Nodule-Specific Sucrose Transporter of Medicago truncatula.</a> Plant Physiol. 171 (1), 554-565 (2016).</li><li>Suzaki, T., Yano, K., Ito, M., Umehara, Y., Suganuma, N., Kawaguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+and+negative+regulation+of+cortical+cell+division+during+root+nodule+development+in+Lotus+japonicus+is+accompanied+by+auxin+response.">Positive and negative regulation of cortical cell division during root nodule development in Lotus japonicus is accompanied by auxin response.</a> Development. 139 (21), 3997-4006 (2012).</li><li>Suzaki, T., 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=Endoreduplication-mediated+initiation+of+symbiotic+organ+development+in+Lotus+japonicus.">Endoreduplication-mediated initiation of symbiotic organ development in Lotus japonicus.</a> Development. 141 (12), 2441-2445 (2014).</li><li>Qin, L., 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+high-affinity+phosphate+transporter+GmPT5+regulates+phosphate+transport+to+nodules+and+nodulation+in+soybean.">The high-affinity phosphate transporter GmPT5 regulates phosphate transport to nodules and nodulation in soybean.</a> Plant Physiol. 159 (4), 1634-1643 (2012).</li><li>Wang, Z., Panfeng, L., Yan, Y., Chi, Y., Fan, B., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+Functional+Analysis+of+a+Novel+Group+of+Legume-specific+WRKY+and+Exo70+Protein+Variants+from+Soybean.">Expression and Functional Analysis of a Novel Group of Legume-specific WRKY and Exo70 Protein Variants from Soybean.</a> Sci Rep. 6, 32090 (2016).</li><li>Montiel, J., 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=A+Phaseolus+vulgaris+NADPH+oxidase+gene+is+required+for+root+infection+by+Rhizobia.">A Phaseolus vulgaris NADPH oxidase gene is required for root infection by Rhizobia.</a> Plant Cell Physiol. 53 (10), 1751-1767 (2012).</li><li>Arthikala, M. K., 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=PvRbohB+negatively+regulates+Rhizophagus+irregularis+colonization+in+Phaseolus+vulgaris.">PvRbohB negatively regulates Rhizophagus irregularis colonization in Phaseolus vulgaris.</a> Plant Cell Physiol. 54 (8), 1391-1402 (2013).</li><li>Arthikala, M. K., S&#225;nchez-L&#243;pez, R., Nava, N., Santana, O., C&#225;rdenas, L., Quinto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RbohB+a+Phaseolus+vulgaris+NADPH+oxidase+gene,+enhances+symbiosome+number,+bacteroid+size,+and+nitrogen+fixation+in+nodules+and+impairs+mycorrhizal+colonization.">RbohB a Phaseolus vulgaris NADPH oxidase gene, enhances symbiosome number, bacteroid size, and nitrogen fixation in nodules and impairs mycorrhizal colonization.</a> New Phytol. 202 (3), 886-900 (2014).</li><li>Barraza, A., 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=Down-regulation+of+PvTRE1+enhances+nodule+biomass+and+bacteroid+number+in+the+common+bean.">Down-regulation of PvTRE1 enhances nodule biomass and bacteroid number in the common bean.</a> New Phytol. 197 (1), 194-206 (2013).</li><li>Estrada-Navarrete, G., 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=An+autophagy-related+kinase+is+essential+for+the+symbiotic+relationship+between+Phaseolus+vulgaris+and+both+rhizobia+and+arbuscular+mycorrhizal+fungi.">An autophagy-related kinase is essential for the symbiotic relationship between Phaseolus vulgaris and both rhizobia and arbuscular mycorrhizal fungi.</a> Plant Cell. 28 (9), 2326-2341 (2016).</li><li>Nanjareddy, K., 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=A+Legume+TOR+Protein+Kinase+Regulates+Rhizobium+Symbiosis+and+Is+Essential+for+Infection+and+Nodule+Development.">A Legume TOR Protein Kinase Regulates Rhizobium Symbiosis and Is Essential for Infection and Nodule Development.</a> Plant Physiol. 172 (3), 2002-2020 (2016).</li><li>Fr&#252;hling, M., Schr&#246;der, G., Hohnjec, N., P&#252;hler, A., Perlick, A. M., K&#252;ster, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promoter+of+the+Vicia+faba+L.+gene+VfEnod12+encoding+an+early+nodulin+is+active+in+cortical+cells+and+nodule+primordia+of+transgenic+hairy+roots+of+Vicia+hirsuta+as+well+as+in+the+prefixing+zone+II+of+mature+transgenic+V.+hirsuta+root+nodules.">The promoter of the Vicia faba L. gene VfEnod12 encoding an early nodulin is active in cortical cells and nodule primordia of transgenic hairy roots of Vicia hirsuta as well as in the prefixing zone II of mature transgenic V. hirsuta root nodules.</a> Plant Science. 160 (1), 67-75 (2000).</li><li>Szabados, L., Ratet, P., Grunenberg, B., de Bruijn, F. J. <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+the+Sesbania+rostrata+leghemoglobin+glb3+gene+5'-upstream+region+in+transgenic+Lotus+corniculatus+and+Nicotiana+tabacum+plants.">Functional analysis of the Sesbania rostrata leghemoglobin glb3 gene 5'-upstream region in transgenic Lotus corniculatus and Nicotiana tabacum plants.</a> Plant Cell Online. 2 (10), 973-986 (1990).</li><li>Madsen, L. H., Tirichine, L., Jurkiewicz, A., Sullivan, J. T., Heckmann, A. B., Bek, A. S., Ronson, C. W., James, E. K., Stougaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+network+governing+nodule+organogenesis+and+infection+in+the+model+legume+Lotus+japonicus.">The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus.</a> Nat Commun. 12, 1-10 (2010).</li><li>Montiel, J., Arthikala, M. K., Quinto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phaseolus+vulgaris+RbohB+functions+in+lateral+root+development.">Phaseolus vulgaris RbohB functions in lateral root development.</a> Plant Signal Behav. 8 (1), 1-3 (2013).</li><li>Nanjareddy, K., Arthikala, M. K., G&#243;mez, B. M., Blanco, L., Lara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentially+expressed+genes+in+mycorrhized+and+nodulated+roots+of+common+bean+are+associated+with+defense,+cell+wall+architecture,+N+metabolism,+and+P+metabolism.">Differentially expressed genes in mycorrhized and nodulated roots of common bean are associated with defense, cell wall architecture, N metabolism, and P metabolism.</a> PLoS ONE. 12 (8), e0182328 (2017).</li><li>Karimi, M., Inz&#233;, D., Depicker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway+vectors+for+Agrobacterium-mediated+plant+transformation.">Gateway vectors for Agrobacterium-mediated plant transformation.</a> Trends Plant Sci. 7 (5), 193-195 (2002).</li><li>Broughton, W. J., Dilworth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+leghemoglobin+synthesis+in+snake+beans.">Control of leghemoglobin synthesis in snake beans.</a> Biochem J. 125 (4), 1075-1080 (1971).</li><li>Jefferson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+chimeric+genes+in+plants,+the+GUS+gene+fusion+system.">Assaying chimeric genes in plants, the GUS gene fusion system.</a> Plant Mol Biol Rep. 5 (4), 387-405 (1987).</li><li>Cho, H. J., Farrand, S. K., Noel, G. R., Widholm, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+induction+of+soybean+hairy+roots+and+propagation+of+the+soybean+cyst+nematode.">High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode.</a> Planta. 210 (2), 195-204 (2000).</li><li>Deng, Y., Mao, G., Stutz, W., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Composite+Plants+in+Medicago+truncatula+used+for+Nodulation+Assays.">Generation of Composite Plants in Medicago truncatula used for Nodulation Assays.</a> J. Vis. Exp. (49), e2633 (2011).</li><li>Kumagai, H., Kouchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+by+expression+of+hairpin+RNA+in+Lotus+japonicus+roots+and+root+nodules.">Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules.</a> Mol Plant Microbe Interact. 16 (8), 663-668 (2003).</li><li>Okamoto, S., Yoro, E., Suzaki, T., Kawaguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hairy+Root+Transformation+in+Lotus+japonicus.">Hairy Root Transformation in Lotus japonicus.</a> Bio-protocol. 3 (12), e795 (2013).</li><li>Jacobs, T. B., Martin, G. 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Under funktionell analys av gener spelar studiet av genen uttrycksmönster en avgörande roll i att förstå rumsliga och tidsmässiga regleringen av gener i vivo. En välkänd metod för att studera gen uttrycksmönster är att klona gen promotor regionen av intresse, uppströms till reporter gener som fluorescerande markörgener (GFP, RFP, etc.) eller β-glukuronidas. Häri, har vi valt en promotor regionen av roten knöl symbios (RNS) specifika genen, NIN, att studera plats och tid uttrycksmönster i...

Initiativtagare är viktiga molekylär biologiska verktyg som spelar en avgörande roll i förståelsen regleringen av uttrycket av gener av intresse. Initiativtagare är DNA-sekvenser som ligger uppströms av översättningen inledandet kodon av genen sekvenser och de bär den centrala föreskrifter av gener; sin rätt anteckning och karakterisering är därför avgörande för att förstå geners funktion. Beroende på uttrycket mönstren klassificeras anläggningen initiativtagare som konstituerande, vävnadsspecifika, eller utveckling-scenen-specifika och inducerbara1. Framsteg inom transcriptomic teknik, förbättringar i datormodellering och tillgängligheten av ökande antal genomet sekvenser för olika växtarter har underlättat storskaliga förutsägelse av promotorn sekvenser2.
Däremot, är det också avgörande för att fastställa verktyg för utvärdering av promotorn och genetisk transformation tekniker som är snabb, effektiv och reproducerbara. Till skillnad från de andra plantorna för modell hindras funktionell karakterisering av gemensamma bean baljväxter (P. vulgaris) gener främst på grund av hur motsträviga Phaseolus sp. för stabil genetisk transformation. Övergående omvandling system fungera som ett alternativ för snabb gen funktionell karakterisering studier3. I baljväxter symbios forskning är samspelet mellan baljväxter värdväxt och rhizobial bakterien en av de mest lätthanterlig modellsystem för den funktionella analysen av knöl-specifika gener och arrangören studier. Hittills har flera baljväxter initiativtagare relaterade till dessa Symbiosar har varit kännetecknas, nämligen, Medicago truncatula PT44, SWEET115, Lotus japonicus Cyclops, UBQ6, VAG17, Glycine max PT5 8, Exo70J9, P. vulgaris RbohB10,11,12, TRE113, PI3K14, TOR15, etc. CIS element direkt påverka genreglering. Transkriptionsfaktor ENBP1A binder till en Cis regulatoriska region (−692 bp) i tidig nodulin VfENOD12, och detta underlättar uttrycket av en reporter gen i knöl primordia Vicia faba16. Byte av Cis regulatoriska regioner (−161 till −48 bp) av den knöl-specifika promotorn leghemoglobin GLB3 med den heterologa trunkeras initiativtagare δ-p35S och δ-pNOS, resulterade i en förlust av knöl specificitet och reducerad arrangören aktivitet17 .
Tidigare rapporter visar att den transkriptionsfaktor NIN krävs för inledande av rhizobial infektion i roten hårcellerna och är också viktigt för knöl organogenes hos L. japonicus18. I föreliggande studie beskriver vi ett protokoll för identifiering, isolering, kloning och karakterisering av knöl-specifika promotorn i gemensamma bean hårig rötterna. För att uppnå detta, vi valde en rhizobial symbios-specifika NIN promotorn av P. vulgaris och klonade i en transkriptionell fusion till chimära reporter GUS-enhanced::GFP. Ytterligare, det här protokollet beskriver ett snabba och mångsidiga system för genetisk transformation i P. vulgaris med A. rhizogenes inducerad håriga rötter. Detta system genererar håriga rötter i mindre än 2 veckor efter transformation. Slutligen bedömde vi spatiotemporal uttrycket av NIN arrangören i kväveformer koloniserade rot knölar av GUS färgning.
Proceduren som beskrivs här kan vara användbart för studien av nodulationen och mycorrhization11 av baljväxter växter, men också för studier av promotorn uttrycksmönster i rötter19. Detta protokoll är dessutom lätt att använda i icke-specialiserade laboratorier.

<table border="0" cellpadding="0" cellspacing="0" width="1125">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Primers for qRT-PCR assay</td>
			<td style="width:253px;"></td>
			<td style="width:460px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pNIN Forward</td>
			<td>CACC ATA GCT CCC CAA AAT GGT AT</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pNIN Reverse</td>
			<td style="width:253px;">CAT CTT CCT TCC ACT AAC TAA C</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M13 Forward</td>
			<td>GTA AAA CGA CGG CCA G</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M13 Reverse</td>
			<td>CAG GAA ACA GCT ATG AC</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name</td>
			<td style="width:253px;">Company</td>
			<td style="width:460px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">REAGENTS</td>
			<td style="width:253px;"></td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pENTR/D-TOPO Cloning Kit</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">K243520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gateway LR Clonase II Enzyme Mix&#160;</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">11791100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">pBGWFS7.0&#160;</td>
			<td style="width:253px;">Plant systems biology</td>
			<td style="width:460px;">https://gateway.psb.ugent.be/vector/show/pBGWFS7/search/index/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum Taq DNA Polymerase</td>
			<td style="width:253px;">ThermoFisher Scientific</td>
			<td style="width:460px;">10966018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">DNeasy Plant Mini Kit</td>
			<td style="width:253px;">Qiagen</td>
			<td>69104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">PureLink Quick Gel Extraction Kit</td>
			<td style="width:253px;">ThermoFisher Scientific</td>
			<td style="width:460px;">K210012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Platinum Pfx DNA Polymerase</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">11708013</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:245px;">Certified Molecular Biology Agarose</td>
			<td style="width:253px;">Bio-Rad</td>
			<td>1613102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">One Shot TOP10 Chemically Competent E. coli</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">C404006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Nacl</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tryptone</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">T7293-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Yeast extract</td>
			<td style="width:253px;">Sigma-Aldrich</td>
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			<td height="21" style="height:21px;width:245px;">Nalidixic acid</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">N8878</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Magnesium sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">M7506</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Gel loading solution</td>
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			<td height="21" style="height:21px;width:245px;">Gel documentation unit</td>
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			<td height="42" style="height:42px;width:245px;">MaxQ SHKE6000 6000 Shaking Incubator - 115VAC</td>
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			<td style="width:460px;">EW-51708-70</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Plant growth chamber</td>
			<td style="width:253px;">MRC</td>
			<td>PGI-550RH&#160;</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Horizantal laminarair flow cabinate</td>
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			<td height="21" style="height:21px;width:245px;">Fluorescent microscope</td>
			<td style="width:253px;">Leica&#160;</td>
			<td style="width:460px;">DM4500 B</td>
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			<td height="21" style="height:21px;width:245px;">Petridish</td>
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			<td style="width:460px;">90X15</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Scalpel Blade&#160;</td>
			<td style="width:253px;">Fisher scientific</td>
			<td style="width:460px;">53223</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Falcon 15mL Conical Centrifuge Tubes</td>
			<td style="width:253px;">Fisher scientific</td>
			<td style="width:460px;">14-959-53A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">22 mL glass tubes</td>
			<td style="width:253px;">Thomas scientific</td>
			<td style="width:460px;">45048-16150</td>
			<td></td>
		</tr>
	</tbody>
</table>

plant promoter analysis: identification and characterization of root nodule specific promoter in the common bean

Uppströms sekvenser av gen kodande sekvenser kallas som arrangören sekvenser. Studera de uttrycksmönster av initiativtagare är mycket betydande i förståelsen av genreglering och spatiotemporal uttrycksmönster av målgener. Däremot, är det också avgörande för att fastställa verktyg för utvärdering av promotorn och genetisk transformation tekniker som är snabb, effektiv och reproducerbara. I denna studie undersökte vi spatiotemporal uttrycksmönstret av rhizobial symbios-specifika knöl starten (NIN) arrangören av Phaseolus vulgaris i transgena hårig rötterna. Använda växten arvsmassa databaser och analysverktyg som vi identifierat, isolerat och klonade P. vulgaris NIN arrangören i en transkriptionell fusion till chimära reporter β-glukuronidas (GUS) GUS-enhanced::GFP. Ytterligare, det här protokollet beskriver ett snabba och mångsidiga system för genetisk transformation i P. vulgaris med hjälp av Agrobacterium rhizogenes inducerad håriga rötter. Detta system genererar ≥2 cm håriga rötter på 10 till 12 dagar efter transformation. Nästa, vi bedömde spatiotemporal uttrycket av NIN arrangören i Rhizobium inokuleras håriga rötter med regelbundna mellanrum av efter inympningen. Våra resultat som skildras av GUS aktivitet visar att promotorn NIN var aktiva under processen av nodulationen. Tillsammans, visar detta protokoll hur till identifiera, isolera, klona och karakterisera en växt promotor i gemensamma bean hårig rötterna. Detta protokoll är dessutom lätt att använda i icke-specialiserade laboratorier.

Syftet med denna studie var att bedöma det spatiotemporal uttryck pattern knöl-specifika P. vulgaris NIN. Detta gör en 700 bp region uppströms i den översättning inledande Codonen av NIN gen valdes och en uppsättning oligos ritades som avbildas i figur 1A. Med en HiFi-polymeras, NIN arrangören fragmentet förstärktes, isolerade (figur 1B) och klonade in den destination binära vektor pBGWFS7.0 (<strong ...

Watch this Scientific Journal Video about Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean at JoVE.com

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

Anläggningen arrangören analys: Identifiering och karakterisering av roten knöl specifika promotorn i gemensamma bönan

Arrangören uttryck analyser är avgörande för att förbättra förståelsen av genreglering och spatiotemporal uttrycket av målgener. Här presenterar vi ett protokoll för att identifiera, isolera och klona en växt promotor. Vidare beskriver vi karakterisering av den knöl-specifika promotorn i gemensamma bean hårig rötterna.

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Hairy roots are used to obtain transformed roots in Phaseolus with a reporter construct. The resulting transgenic roots are used to examine the expression of the reporter gene. Promoter expression studies are crucial to understand the spatiotemporal expression patterns of genes.Today we are going to demonstrate the methodologies for hairy root induction and to study promoter expression patterns in transgenic hairy roots under root nodule symbiotic conditions in Phaseolus. Estrada-Navarrete and colleagues have previously established Agrobacterium rhizogenes induced hairy root system in Phaseolus. The current method will help accelerate the duration of hairy root induction and thereby improving the versatility of the system.Demonstrating the procedures will be my colleague Manoj-Kumar Arthikala and undergraduate students Alma Leticia and Brenda Mariana from my lab. To begin with, search the NIN gene sequence in Phaseolus vulgaris genome database from Phytozome, and select the required length of sequence upstream to ATG. Design the oligonucleotides following the appropriate promoter sequence analysis.Freshly prepared Phaseolus genomic DNA will serve as the template to PCR amplify the promoter sequence. Prepare the PCR master mix with high-fidelity Taq polymerase and standard PCR components, along with the prior-designed oligonucleotides. Aliquot the master mix into 2 mL PCR tubes, and perform the promoter fragment amplification in a thermocycler and with a suitable program.Separate the PCR product electrophoretically on a 1.2%agarose gel. Visualize the NIN fragment amplified at 700 base pairs under UV transillumination. Excise, elute, and clone the fragment into the Gateway entry vector pENTR/D-TOPO vector following the standard instructions.Transform two microliters of the reaction mix into competent E.coli cells at 42 degrees Celsius for 50 seconds, and plate the transformed E.coli on LB supplemented with 50 milligrams per liter kanamycin. Grow the plated cells at 37 degrees Celsius overnight. Perform a PCR reaction for plasmids isolated from randomly selected E.coli colonies using M13 oligonucleotides.The fragment at 1024 base pairs should indicate positive clones. Perform the LR reaction between the positive entry clone and destination binary vector. Transform two microliters of reaction mix into E.coli, and select the colonies on 100 milligrams per liter of spectinomycin at 37 degrees Celsius.Amplify the plasmids with NIN-specific oligonucleotides. The positive clone should amplify at 700 base pairs. Sequence and verify the correctness of the clone fragment.Mobilize the NIN promoter construct into electrocompetent K599 Agrobacterium rhizogenes cells. And once again, confirm the clone with promoter-specific oligonucleotides. Sulfur sterilize Phaseolus vulgaris seeds and germinate on sterile, wet filter papers for two days in dark at 28 degrees Celsius.Spread 150 microliters of Agrobacterium rhizogenes cells with NIN construct and control vector on LB plates with spectinomycin selection. Grow the culture overnight at 28 degrees Celsius. Decoat Phaseolus seedlings, and scrape out Agrobacterium rhizogenes cells with a bent, 200-microliters tip from the plate.And resuspend into Eppendorf Tube with one mL sterile, distilled water. Collect Agrobacterium rhizogenes cells into a three mL syringe, and place Phaseolus seedlings into previously prepared tube setup. Slightly prick the hypocotyls of germinated seeds with the needle tip, simultaneously injecting the Agrobacterium rhizogenes cells.Care should be taken that the needle do not pass through the hypocotyl. Maintain the injected seedlings in a growth chamber at 28 degrees Celsius with 16 hours light and eight hour dark photoperiod. Callus on the wounded hypocotyls should be visible by five to seven days.After two weeks, remove the primary root by cutting the stem two centimeters below the hairy roots, and transplant them into pots containing sterile vermiculite. Simultaneously, inoculate the plants with one ml Rhizobium tropici to induce nodulation. Make sure to dilute the Rhizobium culture to 6 at OD 600.Maintain the plants in a growth chamber at 28 degrees Celsius, 16 hours light, and eight hours dark photoperiod, and irrigate with B and D solution. To study spatiotemporal expression patterns of NIN promoter, sampling is done at different time points. Take out the plants without damaging the roots, and wash the roots without any specks of vermiculite.Randomly select the roots from NIN and control plants, and proceed to GUS histochemical staining by incubating the samples in GUS assay buffer at 37 degrees Celsius for 16 hours in dark. Observe the GUS-stained roots under stereo and compound microscope. This picture shows the Rhizobium-induced NIN promoter expression in the primary roots.NIN promoter expression could also be seen in Rhizobium-infected root hair cells. Further, the GUS expression can be documented at different stages of root nodule development. No GUS expression was seen in vector control roots.After watching this video, you should have a good understanding of designing your promoter constructs and studying the promoter expression patterns in transgenic hairy roots of Phaseolus. This system could be further extended to study the promoter expression patterns under other symbiotic and pathogen interactions in Phaseolus and also other eudicots.

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