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

Dette arbeidet ble delvis støttet av Dirección generelt de Asuntos del personlige Académico, DGAPA/PAPIIT-UNAM (gi nei. IN219916 M.L og IA205117 til M-K. A) og Consejo Nacional de Ciencia y Tecnològia (CONACYT grant nr. 240614 til M.L.).

1. identifikasjon, isolasjon og kloning av P. Vulgaris NIN arrangøren
<ol>
	<li>Identifisere promoter rekkefølgen for genet av interesse. Det er flere genomet databaser og analyseverktøy tilgjengelig for planter som Phytozome, Ensembl planter, NCBI, etc. A Belgfrukt promoter P. vulgaris NIN (PvNIN; Phvul.009G115800) ble brukt i denne studien20.</li>
	<li>Design oligos for Gateway eller tradisjonelle begrensning enzym kloning rarpd vektor brukes (<strong class="xfig"...

<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. S., 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=Protoplast+isolation,+transient+transformation+of+leaf+mesophyll+protoplasts+and+improved+Agrobacterium-mediated+leaf+disc+infiltration+of+Phaseolus+vulgaris:+Tools+for+rapid+gene+expression+analysis.">Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: Tools for rapid gene expression analysis.</a> BMC Biotechnol. 16 (1), 53 (2016).</li><li>Pumplin, N., Zhang, X., Noar, R. D., Harrison, 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=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 funksjonell analyse av gener spiller studiet av genet uttrykk mønstre en avgjørende rolle i å forstå romlige og tidsmessige regulering av gener i vivo. En velkjent metode å studere genet uttrykk mønstre er klone regionen genet arrangøren av interesse, oppstrøms til reporter gener som fluorescerende markør gener (GFP, RFP, etc.) eller β-glucuronidase. Her har vi valgt en promoter region av rot nodule symbiose (RNS) bestemte genet, NIN, å studere spatio-temporale uttrykk mønstre i P....

Arrangører er viktig molekylære biologiske verktøy som spiller en avgjørende rolle i å forstå regulering av uttrykket av gener av interesse. Arrangører er DNA-sekvenser ligger oppstrøms av oversettelsen innvielsen codon av genet sekvenser og de sentrale juridisk informasjon av gener; Derfor er deres riktig merknader og karakterisering avgjørende for å forstå gen funksjon. Avhengig av uttrykket mønstre, er plante arrangører klassifisert som konstituerende, vev-spesifikk, eller utvikling-scene-spesifikk og induserbart1. Fremskritt innen transcriptomic teknologier, forbedringer i datamodellering og tilgjengeligheten av økende antall genomet sekvenser for forskjellige plantearter har tilrettelagt store prediksjon av arrangøren sekvenser2.
På den annen side, er det også viktig å etablere promoter evaluering av verktøy og genetisk transformasjon teknikker som er rask, effektiv og reproduserbar. I motsetning til de andre modellen plantene, er funksjonelle karakterisering av felles bønne Belgfrukt (P. vulgaris) gener forhindret hovedsakelig på grunn av gjenstridige natur Phaseolus sp. for stabil genetisk transformasjon. Forbigående transformasjon systemer tjene som et alternativ for rask genet funksjonell karakteristikk studier3. I Belgfrukt symbiose forskning er samspillet mellom Belgfrukt verten anlegget og rhizobial bakterie en av de mest tett modell systemene for funksjonell analyse av nodule-spesifikke gener og promoter studier. Så langt, flere Belgfrukt arrangører knyttet til disse symbioses har vært preget, nemlig, Medicago truncatula PT44, SWEET115, Lotus japonicus Cyclops, UBQ6, VAG17, glysin max PT5 8, Exo70J9, P. vulgaris RbohB10,11,12, TRE113, PI3K14, TOR15, osv. CIS elementer direkte påvirke genet regulering. Den transkripsjon faktoren ENBP1A binder til en Cis regulatoriske region (−692 bp) av tidlig nodulin VfENOD12, og dette forenkler uttrykket med et reporter i nodule primordia vikker faba16. Utskifting av Cis regulatoriske regioner (−161 til −48 bp) til nodule-spesifikke promoter leghemoglobin GLB3 med de heterologous avkortet arrangører ses-p35S og ses-pNOS, resulterte i tap av nodule spesifisitet og redusert promoter aktivitet17 .
Tidligere rapporter viser at transkripsjon faktoren NIN kreves for oppstart av rhizobial infeksjon i roten hårcellene og er også viktig for nodule organogenesen L. japonicus18. I studien beskriver vi en protokoll for identifikasjon, isolasjon, kloning og karakterisering av nodule-spesifikke arrangøren i vanlige bønne hårete røttene. For å oppnå dette, vi valgte en rhizobial symbiose-spesifikke NIN promoter av P. vulgaris og klonet i en transcriptional fusion til chimeric reporteren GUS-enhanced::GFP. Videre, denne protokollen beskriver en rask og allsidig system av genetisk transformasjon i P. vulgaris bruker A. rhizogenes indusert hårete røtter. Dette systemet genererer hårete røtter i mindre enn 2 uker etter transformasjon. Til slutt, vi vurdert spatiotemporal uttrykket NIN formidler i rhizobia kolonisert rot knuter av GUS flekker.
Fremgangsmåten som er beskrevet her kan være nyttig ikke bare for studier av nodulation og mycorrhization11 Belgfrukt-planter, men også for studier av arrangøren uttrykk mønstre i røtter19. Videre er denne protokollen enkel å bruke i ikke-spesialiserte 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>
			<td style="width:460px;">Y1625-250G</td>
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			<td height="21" style="height:21px;width:245px;">Bacteriological agar</td>
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			<td height="21" style="height:21px;width:245px;">Kanamycin sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
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			<td height="21" style="height:21px;width:245px;">Spectinomycin sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
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			<td height="21" style="height:21px;width:245px;">Ethyl alcohol</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td>E7023</td>
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			<td height="21" style="height:21px;width:245px;">Bacteriological peptone</td>
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			<td height="21" style="height:21px;width:245px;">Calcium chloride</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">C1016</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Nalidixic acid</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">N8878</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Gel loading solution</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">G7654</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">EQUIPMENT</td>
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			<td height="21" style="height:21px;width:245px;">Thermocycler</td>
			<td style="width:253px;">Veriti Thermal Cycler</td>
			<td style="width:460px;">4375786</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Centrifuge</td>
			<td style="width:253px;">Sigma</td>
			<td style="width:460px;">Sigma 1-14K</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Gel documentation unit</td>
			<td style="width:253px;">Carestream&#160;</td>
			<td style="width:460px;">Gel Logic 212 PRO</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">MaxQ SHKE6000 6000 Shaking Incubator - 115VAC</td>
			<td style="width:253px;">Thermo scientific</td>
			<td style="width:460px;">EW-51708-70</td>
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		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Horizantal laminarair flow cabinate</td>
			<td style="width:253px;">Lumistell</td>
			<td style="width:460px;">LH-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Petridish</td>
			<td style="width:253px;">sym laboratorios</td>
			<td style="width:460px;">90X15</td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<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

Oppstrøms sekvenser av genet koding sekvenser er betegnet som formidler sekvenser. Studere uttrykk mønstre av arrangørene er svært viktig i å forstå gen regulering og spatiotemporal uttrykk mønstre av målet gener. På den annen side, er det også viktig å etablere promoter evaluering av verktøy og genetisk transformasjon teknikker som er rask, effektiv og reproduserbar. I denne studien undersøkte vi spatiotemporal uttrykksmønster av rhizobial symbiose-spesifikke nodule begynnelse (NIN) arrangøren av Phaseolus vulgaris i transgene hårete røttene. Bruk anlegget genomet databaser og analyseverktøy vi identifisert, isolert og klonet P. vulgaris NIN selskapet i en transcriptional fusion til chimeric reporteren β-glucuronidase (GUS) GUS-enhanced::GFP. Videre, denne protokollen beskriver en rask og allsidig system av genetisk transformasjon i P. vulgaris bruker Agrobacterium rhizogenes indusert hårete røtter. Dette systemet genererer ≥2 cm hårete røtter på 10 til 12 dager etter transformasjon. Neste, vi vurdert spatiotemporal uttrykket NIN formidler i Rhizobium inokulert hårete røtter ved periodiske intervaller av etter vaksinering. Våre resultater avbildet av GUS aktivitet viser at NIN arrangøren var aktiv under prosessen med nodulation. Sammen, demonstrerer nåværende protokollen hvordan å identifisere, isolere, Klon og karakterisere en plante promoter i vanlige bønne hårete røttene. Videre er denne protokollen enkel å bruke i ikke-spesialiserte laboratorier.

Målet med denne studien var å vurdere spatiotemporal uttrykk mønsteret av nodule-spesifikke P. vulgaris NIN. Gjør en 700 bp region oppstrøms av oversettelse innvielsen codon av NIN genet ble valgt og et sett med oligos var utformet som avbildet i figur 1A. Bruker en Hi-Fi-utvalg, NIN promoter fragmentet ble forsterket isolert (figur 1B) og klonet i destinasjon binære vektor pBGWFS7.0 (...

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

Anlegget Promoter analyse: Identifikasjon og karakterisering av rot Nodule bestemt Promoter i vanlige Bean

Arrangøren uttrykk analyser er avgjørende for å bedre forståelsen av genet regulering og spatiotemporal uttrykk for målet gener. Her presenterer vi en protokoll for å identifisere, isolere og klone en plante promoter. Videre beskriver vi karakterisering av nodule-spesifikke arrangøren i vanlige bønne hårete røttene.

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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