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 takke medlemmerne af Wang laboratorium for nyttige diskussioner og kommentarer. Dette arbejde er støttet af National Natural Science Foundation of China (NSFC, bevilling nr. 31570001) og Natural Science Foundation i Guangdong provinsen og Guangzhou City (Giv nr. 2016A030313401 og 201707010024) til H.W.

1. primer Design strategi og DNA amplifikation
<ol><li>Design primere for Molekylær kloning af DNA-fragmenter. Primere omfatter 20 bp gen specifikke bindende sekvenser og 20 til 25 bp 5'-enden overhæng sekvenser, som er den komplementære overlappende sekvens af tilstødende DNA molekyler (Se tabel 1 for eksempel). Bemærk: Den efterfølgende samling af hver DNA fragmenter, sammenkobling af forskellige protein udtryk kassetter, og integration med den endelige udtryk vektor alle afhænge af anerken...

<ol>
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Her har vi vist en roman metode håndfast Co udtrykke kimære fluorescerende fusion proteiner i planter. Det kan bruges til både forbigående udtryk og genetiske transformation og er kompatible med aktuelle fluorescerende proteiner-baseret bio-imaging, molekylære og biokemiske applikationer og teknologier9,10,13 . Derudover overvinder det vanskelighederne af de konventionelle metoder, der bruger flere individuelle udtryk plasm...

Kimære fluorescerende fusion proteiner har været betragtet som nyttige redskaber til at studere intracellulære dynamics og subcellulært lokalisering og yderligere at forstå deres fysiologiske funktioner og arbejder mekanismer1,2, 3 , 4. det er især gavnligt for Co express velkendte organelle reporter proteiner med protein til bedre illustrere sine spatiotemporelle rationale, distribution og funktioner inden for endomembrane systemet i celler4 , 5 , 6 , 7 , 8.
En kimære fluorescerende fusion protein kan udtrykkes i planter via forbigående udtryk og stabil genetiske transformation, som har deres respektive fordele og begrænsninger9,10,11. Forbigående udtryk for et protein er en praktisk tilgang, der omfatter biolistic bombardement-, polyethylenglycol (PEG)-, eller elektroporation-medieret DNA forbigående udtryk i protoplasts og Agrobacterium-medieret blad infiltration i intakt planteceller, som vist i figur 1A, B12,13,14,15,16. Co udtryk for flere kimære fluorescerende fusion proteiner i en enkelt plante celle kræver imidlertid en blanding af flere uafhængige udtryk plasmider. Ulemperne ved beskæftiger flere plasmider for protein Co udtryk i planter er således lavere Co udtryk niveauer på grund af den dramatisk reduceret chance for flere plasmider samtidigt ind i de samme celler i forhold til en enkelt plasmid, og den variationer af udtryk Proteinniveau forårsaget af ukontrolleret tilfældige mængden af hver slags plasmid overføres til den celle17,18. Det er endvidere teknisk udfordrende at indføre flere uafhængige udtryk plasmider i en enkelt Agrobacterium for protein Co udtryk9,10,11. Derfor Agrobacterium-medieret protein forbigående udtryk af infiltration af tobak efterlader er kun i stand til at udtrykke et plasmid ad gangen, som vist i figur 1B. Derimod er generation af transgene planter at udtrykke fluorescerende fusion proteiner normalt opnås ved Agrobacterium , der bærer en binær transformation vektor. Den binære vektor, der medierer genoverførsel og indsættelse i anlægget genomer er imidlertid kun i stand til at udtrykke en enkelt fluorescerende fusion protein (figur 1B)9,10,12. Generering af en genetisk modificeret plante, som udtrykker flere kimære fluorescerende proteiner samtidig kræver flere runder af genetiske passage og screening, som kan tage fra måneder til år afhængigt af antallet af generne at være co udtrykt.
Den beskæftigelse, et enkelt udtryk vektor for Co udtryk for flere proteiner i planter er blevet rapporteret af flere tidligere undersøgelser19,20,21. Dog er flere runder af enzymatisk fordøjelse og DNA ligatur af DNA-molekyler og rygraden vektorer normalt kræves til generering af den endelige plasmid for protein Co udtryk eller overdrevne udtryk. Her, har vi udviklet en ny og solid metode til co udtrykker flere kimære fluorescerende proteiner i planter. Det er en meget effektiv og praktisk metode, der opnår flere protein Co udtryk i planter til både forbigående udtryk og stabil transformation i en hævdvundne mode. Det beskæftiger en enkelt vektor, der indeholder flere funktionelt uafhængige protein udtryk kassetter til protein Co udtryk og dermed overvinder ulemperne ved de konventionelle metoder. Derudover er det et meget alsidigt system i hvilke DNA manipulationer og montage er opnået ved en simpel et-trins optimeret reaktion uden ekstra trin af DNA fordøjelse og ligatur. Funktionsprincip er illustreret i figur 2. Derudover er det fuldt ud kompatible med aktuelle cellulære, molekylære og biokemiske metoder, der er baseret på kimære 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

Oplysninger om den spatiotemporelle subcellulært localization(s) af en protein er afgørende for at forstå dens fysiologiske funktioner i celler. Fluorescerende proteiner og generation af fluorescerende fusion proteiner har været vildt bruges som et effektivt redskab til direkte visualisere protein lokalisering og dynamik i celler. Det er især nyttigt at sammenligne dem med velkendte organelle markører efter Co interessetilkendegivelse med protein. Ikke desto mindre, klassiske tilgange for protein Co udtryk i planter som regel involverer flere uafhængige udtryk plasmider, og derfor har ulemper, der omfatter lav Co udtryk effektivitet, udtryk-niveau variation og høj tid udgifter i genetiske krydser og screening. I denne undersøgelse beskriver vi en robust og roman metode for Co udtryk for flere kimære fluorescerende proteiner i planter. Det overvinder begrænsningerne af konventionelle metoder ved hjælp af et enkelt udtryk vektor, der er sammensat af flere semi-uafhængig udtrykker kassetter. Hver protein udtryk kassette indeholder sin egen funktionelle protein udtrykselementer, og derfor det kan fleksibelt tilpasses for at imødekomme forskellige udtryk efterspørgsel. Også, det er nemt og bekvemt at udføre forsamlingen og manipulation af DNA fragmenter i udtryk plasmid ved hjælp af en optimeret one-step reaktion uden yderligere fordøjelse og ligatur trin. Derudover er det fuldt ud kompatible med aktuelle fluorescerende protein afledt bio-imaging teknologier og applikationer, såsom FRET og BiFC. Som en validering af metoden ansat vi dette nye system til co express fluorescently sammenvokset vakuolære sortering receptor og sekretoriske carrier Membranproteiner. Resultaterne viser, at deres perspektiv subcellulært lokaliseringer er den samme som i tidligere undersøgelser af både forbigående udtryk og genetiske transformation i planter.

Vi har udviklet en robust og yderst effektiv metode til fælles udtryk for flere kimære fluorescerende fusion proteiner i planter. Det bryder gennem barriererne af de konventionelle metoder bruger flere adskilte plasmider for protein Co udtryk, som vist i figur 1A, B, via enten forbigående udtryk eller stabil genetiske transformation. I denne nye metode genererer vi en enkelt udtryk vektor, der er sammensat af flere protein udtryk ...

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 udviklet en ny metode til at co udtrykke flere kimære fluorescerende fusion proteiner i planter til at overvinde vanskelighederne af konventionelle metoder. Det tager fordel ved at bruge en enkelt udtryk plasmid som indeholder flere funktionelt uafhængige protein udtrykker kassetter for at opnå protein Co udtryk.

Co udtryk for flere kimære fluorescerende Fusion proteiner på en effektiv måde 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?

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

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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

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

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

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

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Visualisere Lignification Dynamics i planter med Klik på kemi: dobbelt mærkning er LYKSALIGHED!

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

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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

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