Name: co-expression of multiple chimeric fluorescent fusion proteins in an efficient way in plants
Uploaded: 2018-07-01T12:30:00
Description: Watch this Scientific Journal Video about Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants at JoVE.com

Vi takker medlemmene av Wang laboratorium for nyttig diskusjoner og kommentarer. Dette arbeidet er støttet av National Natural Science Foundation av Kina (NSFC, grant nr. 31570001) og Natural Science Foundation av Guangdong-provinsen og Skopje (gi nr. 2016A030313401 og 201707010024) til HW

1. primer designstrategi og DNA amplifikasjon
<ol><li>Utforme primerne for molekylær kloning av DNA. Primerne består av 20 bp genet bestemt bindende sekvenser og 20 til 25 bp 5'-end overheng sekvenser, som er komplementære overlappende sekvensen av tilstøtende DNA molekyler (se tabell 1 for eksempel). Merk: Påfølgende montering av hver DNA fragmenter, kobling mellom annet protein uttrykk kassetter, og integrasjon med den endelige vektoren alle avhenger anerkjennelse av tilstøtende overlappend...

<ol>
	<li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu Rev Biochem. 67, 509-544 (1998).</li><li>Tsien, R. Y., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+machinery+of+live+cells.">Seeing the machinery of live cells.</a> Science. 280 (5371), 1954-1955 (1998).</li><li>Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+Plant+Cells+in+a+Brand+New+Way.">Visualizing Plant Cells in a Brand New Way.</a> Mol Plant. 9 (5), 633-635 (2016).</li><li>Zhang, J., Campbell, R. E., Ting, A. Y., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+new+fluorescent+probes+for+cell+biology.">Creating new fluorescent probes for cell biology.</a> Nat Rev Mol Cell Biol. 3 (12), 906-918 (2002).</li><li>Lippincott-Schwartz, J., Snapp, E., Kenworthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein+dynamics+in+living+cells.">Studying protein dynamics in living cells.</a> Nat Rev Mol Cell Biol. 2 (6), 444-456 (2001).</li><li>Tse, Y. C., 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=Identification+of+multivesicular+bodies+as+prevacuolar+compartments+in+Nicotiana+tabacum+BY-2+cells.">Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells.</a> Plant Cell. 16 (3), 672-693 (2004).</li><li>Wang, H., Zhuang, X., Cai, Y., Cheung, A. Y., Jiang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+F-actin-regulated+exocytic+targeting+of+NtPPME1+is+essential+for+construction+and+rigidity+of+the+pollen+tube+cell+wall.">Apical F-actin-regulated exocytic targeting of NtPPME1 is essential for construction and rigidity of the pollen tube cell wall.</a> Plant J. 76 (3), 367-379 (2013).</li><li>Wang, H., 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+Distinct+Pathway+for+Polar+Exocytosis+in+Plant+Cell+Wall+Formation.">A Distinct Pathway for Polar Exocytosis in Plant Cell Wall Formation.</a> Plant Physiol. 172 (2), 1003-1018 (2016).</li><li>Canto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+Expression+Systems+in+Plants:+Potentialities+and+Constraints.">Transient Expression Systems in Plants: Potentialities and Constraints.</a> Adv Exp Med Biol. 896, 287-301 (2016).</li><li>Ishida, Y., Hiei, Y., Komari, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-mediated+transformation+of+maize.">Agrobacterium-mediated transformation of maize.</a> Nat Protoc. 2 (7), 1614-1621 (2007).</li><li>Li, 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=Optimization+of+agrobacterium-mediated+transformation+in+soybean.">Optimization of agrobacterium-mediated transformation in soybean.</a> Front Plant Sci. 8, 246 (2017).</li><li>Manavella, P. A., Chan, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transformation+of+sunflower+leaf+discs+via+an+Agrobacterium-mediated+method:+applications+for+gene+expression+and+silencing+studies.">Transient transformation of sunflower leaf discs via an Agrobacterium-mediated method: applications for gene expression and silencing studies.</a> Nat Protoc. 4 (11), 1699-1707 (2009).</li><li>Martin, 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=Transient+expression+in+Nicotiana+benthamiana+fluorescent+marker+lines+provides+enhanced+definition+of+protein+localization,+movement+and+interactions+in+planta.">Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta.</a> Plant J. 59 (1), 150-162 (2009).</li><li>Miao, Y., Jiang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+expression+of+fluorescent+fusion+proteins+in+protoplasts+of+suspension+cultured+cells.">Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells.</a> Nat Protoc. 2 (10), 2348-2353 (2007).</li><li>Wang, H., Jiang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+expression+and+analysis+of+fluorescent+reporter+proteins+in+plant+pollen+tubes.">Transient expression and analysis of fluorescent reporter proteins in plant pollen tubes.</a> Nat Protoc. 6 (4), 419-426 (2011).</li><li>Yoo, S. D., Cho, Y. H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mesophyll+protoplasts:+A+versatile+cell+system+for+transient+gene+expression+analysis.">Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis.</a> Nat Protoc. 2 (7), 1565-1572 (2007).</li><li>Candat, 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=Experimental+determination+of+organelle+targeting-peptide+cleavage+sites+using+transient+expression+of+green+fluorescent+protein+translational+fusions.">Experimental determination of organelle targeting-peptide cleavage sites using transient expression of green fluorescent protein translational fusions.</a> Anal Biochem. 434 (1), 44-51 (2013).</li><li>Giepmans, B. N., Adams, S. R., Ellisman, M. H., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+toolbox+for+assessing+protein+location+and+function.">The fluorescent toolbox for assessing protein location and function.</a> Science. 312 (5771), 217-224 (2006).</li><li>Binder, 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=A+modular+plasmid+assembly+kit+for+multigene+expression,+gene+silencing+and+silencing+rescue+in+plants.">A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants.</a> PLoS One. 9 (2), e88218 (2014).</li><li>Engler, C., 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+golden+gate+modular+cloning+toolbox+for+plants.">A golden gate modular cloning toolbox for plants.</a> ACS Synth Biol. 3 (11), 839-843 (2014).</li><li>Forner, J., Binder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+red+fluorescent+protein+eqFP611:+Application+in+subcellular+localization+studies+in+higher+plants.">The red fluorescent protein eqFP611: Application in subcellular localization studies in higher plants.</a> BMC Plant Biol. 7, 28 (2007).</li><li>Wang, H., 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=Vacuolar+sorting+receptors+(VSRs)+and+secretory+carrier+membrane+proteins+(SCAMPs)+are+essential+for+pollen+tube+growth.">Vacuolar sorting receptors (VSRs) and secretory carrier membrane proteins (SCAMPs) are essential for pollen tube growth.</a> Plant J. 61 (5), 826-838 (2010).</li><li>Wang, H., Zhuang, X. H., Hillmer, S., Robinson, D. G., Jiang, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuolar+sorting+receptor+(VSR)+proteins+reach+the+plasma+membrane+in+germinating+pollen+tubes.">Vacuolar sorting receptor (VSR) proteins reach the plasma membrane in germinating pollen tubes.</a> Mol Plant. 4 (5), 845-853 (2011).</li><li>Chen, W. 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=Rapid+in+vitro+splicing+of+coding+sequences+from+genomic+DNA+by+isothermal+recombination+reaction-based+PCR.">Rapid in vitro splicing of coding sequences from genomic DNA by isothermal recombination reaction-based PCR.</a> Biotechnology &amp; Biotechnological Equipment. 30 (5), 864-868 (2016).</li><li>Zhu, Q. L., Yang, Z. F., Zhang, Q. Y., Chen, L. T., Liu, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+multi-type+plasmid+modifications+based+on+isothermal+in+vitro+recombination.">Robust multi-type plasmid modifications based on isothermal in vitro recombination.</a> Gene. 548 (1), 39-42 (2014).</li><li>Torella, J. P., 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=Rapid+construction+of+insulated+genetic+circuits+via+synthetic+sequence-guided+isothermal+assembly.">Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly.</a> Nucleic Acids Res. 42 (1), 681-689 (2014).</li><li>Siguret, V., Ribba, A. S., Cherel, G., Meyer, D., Pietu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+plasmid+size+on+transformation+efficiency+by+electroporation+of+Escherichia+coli+DH5+alpha.">Effect of plasmid size on transformation efficiency by electroporation of Escherichia coli DH5 alpha.</a> Biotechniques. 16 (3), 422-426 (1994).</li><li>Zhang, X., Henriques, R., Lin, S. S., Niu, Q. W., 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=Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana+using+the+floral+dip+method.">Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method.</a> Nat Protoc. 1 (2), 641-646 (2006).</li><li>Lam, S. K., Cai, Y., Hillmer, S., Robinson, D. G., Jiang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCAMPs+highlight+the+developing+cell+plate+during+cytokinesis+in+tobacco+BY-2+cells.">SCAMPs highlight the developing cell plate during cytokinesis in tobacco BY-2 cells.</a> Plant Physiol. 147 (4), 1637-1645 (2008).</li><li>Lam, S. 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=Rice+SCAMP1+defines+clathrin-coated,+trans-golgi-located+tubular-vesicular+structures+as+an+early+endosome+in+tobacco+BY-2+cells.">Rice SCAMP1 defines clathrin-coated, trans-golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells.</a> Plant Cell. 19 (1), 296-319 (2007).</li><li>Jiang, L., 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=Integral+membrane+protein+sorting+to+vacuoles+in+plant+cells:+evidence+for+two+pathways.">Integral membrane protein sorting to vacuoles in plant cells: evidence for two pathways.</a> J Cell Biol. 143 (5), 1183-1199 (1998).</li><li>Fernandez-Abalos, J. M., Fox, H., Pitt, C., Wells, B., Doonan, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-adapted+green+fluorescent+protein+is+a+versatile+vital+reporter+for+gene+expression,+protein+localization+and+mitosis+in+the+filamentous+fungus,+Aspergillus+nidulans.">Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans.</a> Mol Microbiol. 27 (1), 121-130 (1998).</li><li>Bruening, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+gene+silencing+regularized.">Plant gene silencing regularized.</a> Proc Natl Acad Sci U S A. 95 (23), 13349-13351 (1998).</li><li>Wassenegger, M., Pelissier, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+RNA-mediated+gene+silencing+in+higher+plants.">A model for RNA-mediated gene silencing in higher plants.</a> Plant Mol Biol. 37 (2), 349-362 (1998).</li><li>Dehio, C., Schell, 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+of+plant+genetic+loci+involved+in+a+posttranscriptional+mechanism+for+meiotically+reversible+transgene+silencing.">Identification of plant genetic loci involved in a posttranscriptional mechanism for meiotically reversible transgene silencing.</a> Proc Natl Acad Sci U S A. 91 (12), 5538-5542 (1994).</li><li>Zhong, G., Zhu, Q., Li, Y., Liu, Y., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Once+for+all:+A+novel+robust+system+for+co-expression+of+multiple+chimeric+fluorescent+fusion+proteins+in+plants.">Once for all: A novel robust system for co-expression of multiple chimeric fluorescent fusion proteins in plants.</a> Front Plant Sci. 8, 1071 (2017).</li><li>Li, X. T., Xie, Y. Y., Zhu, Q. L., Liu, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+genome+editing+in+genes+and+cis-regulatory+regions+improves+qualitative+and+quantitative+traits+in+crops.">Targeted genome editing in genes and cis-regulatory regions improves qualitative and quantitative traits in crops.</a> Molecular Plant. 10 (11), 1368-1370 (2017).</li><li>Zhu, Q. 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=Development+of+&amp;#34;purple+endosperm+rice''+by+engineering+anthocyanin+biosynthesis+in+the+endosperm+with+a+high-efficiency+transgene+stacking+system.">Development of &amp;#34;purple endosperm rice'' by engineering anthocyanin biosynthesis in the endosperm with a high-efficiency transgene stacking system.</a> Molecular Plant. 10 (7), 918-929 (2017).</li><li>Tang, H. 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=Multi-step+formation,+evolution,+and+functionalization+of+new+cytoplasmic+male+sterility+genes+in+the+plant+mitochondrial+genomes.">Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes.</a> Cell Research. 27 (1), 130-146 (2017).</li></ol>

Her har vi vist en ny metode for å robust co express chimeric fluorescerende fusion proteiner i planter. Den kan brukes for både forbigående uttrykk og genetisk transformasjon og er kompatibel med gjeldende fluorescerende protein-basert bio-imaging, molekylær og biokjemiske applikasjoner og teknologier9,10,13 . I tillegg overvinner det vanskelighetene av konvensjonelle metoder som bruker flere individuelle uttrykk plasmider ...

Chimeric fluorescerende fusion proteiner har vært betraktet som nyttige verktøy for å studere intracellulær dynamikk og subcellular lokalisering og videre forstå deres fysiologiske funksjoner og arbeider mekanismer1,2, 3 , 4. det er spesielt gunstig for co express kjente organelle reporter proteiner med protein aktuelle bedre illustrere dens spatiotemporal begrunnelse, distribusjon og funksjoner i endomembrane systemet i celler4 , 5 , 6 , 7 , 8.
Et chimeric fluorescerende fusion protein kan uttrykkes i planter via forbigående uttrykk og stabil genetisk transformasjon, som har sine respektive fordeler og begrensninger9,10,11. Forbigående uttrykk for et protein er en praktisk tilnærming som inkluderer biolistic bombardement-, polyetylenglykol (PEG)-, eller electroporation-mediert DNA forbigående uttrykk i protoplasts og Agrobacterium-mediert blad infiltrasjon i intakt anlegget celler, som vist i figur 1A, B12,13,14,15,16. Co uttrykk for flere chimeric fluorescerende fusion proteiner i en plante celle krever imidlertid en blanding av flere uavhengige uttrykk plasmider. Dermed ulempene ved å bruke flere plasmider protein co uttrykk i planter er lavere co uttrykk nivåer på grunn av dramatisk redusert sjanse for flere plasmider samtidig inn de samme cellene i forhold til en enkelt plasmider, og varianter av protein uttrykk nivåene skyldes ukontrollert tilfeldig beløpet av hver plasmider overføres til cellen17,18. I tillegg er det teknisk utfordrende å innføre flere uavhengige uttrykk plasmider i en enkelt Agrobacterium for protein co uttrykk9,10,11. Derfor Agrobacterium-mediert protein forbigående uttrykk av infiltrasjon av tobakk blader er bare i stand til å uttrykke en plasmider om gangen, som vist i figur 1B. Derimot er generasjon av transgene uttrykke fluorescerende fusion proteiner vanligvis oppnås ved Agrobacterium som bærer en binær transformasjon vektor. Binær vektoren som formidler genoverføring og innsetting anlegget genomer er imidlertid bare i stand til å uttrykke en enkelt fluorescerende fusion protein (figur 1B)9,10,12. Generere en transgene plante som uttrykker flere chimeric fluorescerende proteiner samtidig krever flere runder med genetisk krysset og screening, som kan ta fra måneder til år avhengig av antall gener skal co uttrykt.
Sysselsetting en enkelt utrykk vektor co uttrykk for flere proteiner i anlegg har blitt rapportert av flere tidligere studier19,20,21. Men er flere runder av enzymatisk fordøyelsen og DNA ligation av DNA molekyler og ryggraden vektorer vanligvis nødvendig for generering av den endelige plasmider protein co uttrykk eller over-uttrykk. Her har vi utviklet en ny og robust metode for å uttrykke co flere chimeric fluorescerende proteiner i planter. Det er en svært effektiv og praktisk metode som oppnår flere protein co uttrykk i planter for både forbigående uttrykk og stabil transformasjon i en hevdvunne måte. Den sysselsetter en enkelt vektor som inneholder flere funksjonelt uavhengig protein uttrykk kassetter for protein co uttrykk og dermed overvinner ulempene av konvensjonelle metoder. Videre er det en svært allsidig system i som DNA manipulasjoner og montering oppnås ved en enkel ett-trinns optimalisert reaksjon uten ekstra trinn DNA fordøyelsen og ligatur. The Arbeidsprinsipp er illustrert i figur 2. Videre er det fullt kompatibel med gjeldende mobilnettet, molekylær og biokjemiske tilnærminger som er basert på chimeric fluorescerende fusion proteiner.

<table>
	<tbody>
		<tr>
			<td>KOD-FX Polymerase</td>
			<td>TOYOBO</td>
			<td>KFX-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sma I</td>
			<td>NEB</td>
			<td>R0141L/S/V</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>BBI</td>
			<td>A600194-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>BBI</td>
			<td>A601336-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP</td>
			<td>NEB</td>
			<td>#N0447V</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>BBI</td>
			<td>C4H10O2S2</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>BBI</td>
			<td>A100159-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>NAD</td>
			<td>BBI</td>
			<td>A600641-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>T5 exonuclease</td>
			<td>Epicentre</td>
			<td>T5E4111K</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA polymerase</td>
			<td>NEB</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>NEB</td>
			<td>B9022S</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog Basal Salt Mixture(MS)</td>
			<td>Sigma</td>
			<td>M5524</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>BBI</td>
			<td>A500737-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>BBI</td>
			<td>A600560-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BBI</td>
			<td>A505255-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>BBI</td>
			<td>A614270-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold microcarrier particles</td>
			<td>Bio-Rad</td>
			<td>165-2263</td>
			<td>1.0 &#181;m</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>BBI</td>
			<td>CD0050-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrocarriers</td>
			<td>Bio-Rad</td>
			<td>165-2335</td>
			<td></td>
		</tr>
		<tr>
			<td>Rupture disk</td>
			<td>Bio-Rad</td>
			<td>165-2329</td>
			<td></td>
		</tr>
		<tr>
			<td>Stopping screen</td>
			<td>Bio-Rad</td>
			<td>165-2336</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>OXOID</td>
			<td>LP0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>OXOID</td>
			<td>LP0021</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BBI</td>
			<td>A610476-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>BBI</td>
			<td>A610440-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>BBI</td>
			<td>A600219-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Genview</td>
			<td>AH169-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Wortmannin</td>
			<td>Sigma</td>
			<td>F9128</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Sigma</td>
			<td>SML0975-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulphoxide (DMSO)</td>
			<td>BBI</td>
			<td>A600163-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Panasonic</td>
			<td>MLR-352H-PC</td>
			<td></td>
		</tr>
		<tr>
			<td>PSD-1000/He particle delivery system</td>
			<td>Bio-Rad</td>
			<td>165-2257</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Pulser</td>
			<td>Bio-Rad</td>
			<td>1652660</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvette</td>
			<td>Bio-Rad</td>
			<td>1652083</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Eppendorf</td>
			<td>5427000097</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>LSM 7 DUO (780&#38;7Live)</td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometers</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>EPS-300 Power Supply</td>
			<td>Tanon</td>
			<td>EPS 300</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Mshot</td>
			<td>MF30</td>
			<td></td>
		</tr>
		<tr>
			<td>Agrose</td>
			<td>BBI</td>
			<td>A600234</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>BBI</td>
			<td>A100339</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Diamine Tetraacetie Acid</td>
			<td>BBI</td>
			<td>B300599</td>
			<td></td>
		</tr>
	</tbody>
</table>

co-expression of multiple chimeric fluorescent fusion proteins in an efficient way in plants

Informasjon om den spatiotemporal subcellular localization(s) av et protein er avgjørende å forstå sine fysiologiske funksjoner i cellene. Fluorescerende proteiner og generasjon fluorescerende fusion proteiner har blitt vilt brukt som et effektivt verktøy å direkte visualisere protein lokalisering og dynamikk i celler. Det er spesielt nyttig å sammenligne dem med kjente organelle markører etter co uttrykk med protein av interesse. Likevel klassisk tilnærminger for protein co uttrykk i planter innebære vanligvis flere uavhengige uttrykk plasmider, og derfor har ulemper med lav co uttrykk effektivitet, uttrykk-nivå variasjon og høy utgifter til genetisk krysset og screening. I denne studien beskriver vi en robust og romanen metode for co uttrykk for flere chimeric fluorescerende proteiner i planter. Løser begrensningene av konvensjonelle metoder ved hjelp av en enkelt utrykk vektor som består av flere semi-uavhengige uttrykke kassetter. Hver protein uttrykk kassett inneholder sin egen funksjonelle protein uttrykkselementer, og derfor det kan fleksibelt justeres for å imøtekomme ulike uttrykk etterspørsel. Også det er enkelt og praktisk å utføre forsamlingen og manipulering av DNA fragmenter i uttrykket plasmider ved hjelp av en optimalisert ettrinns reaksjon uten ekstra fordøyelsen og ligatur skritt. Videre er det kompatible med dagens fluorescerende protein utvunnet bio-imaging teknologi og programmer, som bånd og BiFC. Som en validering av metoden ansatt vi dette nye systemet co express fluorescently smeltet mer sortering reseptor og sekretoriske carrier membran proteiner. Resultatene viser at deres perspektiv subcellular steder er de samme som tidligere studier både forbigående uttrykk og genetisk transformasjon i planter.

Vi har utviklet en robust og svært effektiv metode for co uttrykk for flere chimeric fluorescerende fusion proteiner i planter. Det bryter gjennom barrierene av den konvensjonelle tilnærminger bruke flere atskilt plasmider for protein co uttrykk, som vist i figur 1A, B, via forbigående uttrykk eller stabil genetisk transformasjon. I denne nye metoden generere vi en enkelt utrykk vektor som består av flere protein uttrykk kassette...

Watch this Scientific Journal Video about Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants at JoVE.com

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Vi har utviklet en ny metode for co uttrykke flere chimeric fluorescerende fusion proteiner i planter å overvinne vanskelighetene med konvensjonelle metoder. Det tar fordelen av benytter en enkelt utrykk plasmider som inneholder flere funksjonelt uavhengig protein uttrykke kassetter for å oppnå protein co uttrykk.

Co uttrykk for flere Chimeric fluorescerende Fusion proteiner på en effektiv måte i planter

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Research

JoVE Journal

Chemistry

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Polymalic Acid-baserte Nano Biopolymere for målretting av flere tumor markører: en mulighet for personlig medisin?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

Lettvinte og effektiv Utarbeidelse av Tri-komponent Fluorescent Glycopolymers via RAFT-kontrollerte Polymerisering

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Radiomerking og Kvantifisering av Cellular Nivåer av Phosphoinositides ved HPLC-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

In Situ Karakterisering av Hydratiserte Proteiner i vann ved Salvi og TOF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

Sekvensering av Plant Wall Heteroxylans Bruke enzymic, Chemical (Metylering) og fysiske (massespektrometri Nuclear Magnetic Resonance) Teknikker

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Syntese av Protein Bioconjugates<em&gt; via</em&gt; Cystein-maleimide kjemi

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Visualisere Lignification Dynamics i planter med Klikk kjemi: Dual merking er lykke!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

En effektiv utvalg forberedelse metode for å forbedre karbohydrater Ion signaler i Matrix-assistert Laser desorpsjon/ionisering massespektrometri

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of FÃƒÆ’Ã‚Â¢Ãƒâ€¹Ã¢â‚¬Â '

Photoelectron avbildning av anioner illustrert av 310 Nm avdeling av F−

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Bio-inspirert Polydopamine overflaten modifikasjon av Nanodiamonds og dens reduksjon av Silver nanopartikler

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...