Name: polysome purification from soybean symbiotic nodules
Uploaded: 2022-07-01T15:00:00
Description: Watch this Scientific Journal Video about Polysome Purification from Soybean Symbiotic Nodules at JoVE.com

Denne forskning blev finansieret af CSIC I + D 2020-bevilling nr. 282, FVF 2017-bevilling nr. 210 og PEDECIBA (María Martha Sainz).

1. Plantevækst og rhizobia podning
<ol><li>For at generere de krævede knuder, så sojabønnefrøene efter eget valg i det valgte substrat i et vækstkammer under kontrollerede forhold. BEMÆRK: I denne protokol blev frø sået i en 0,5 L plastflaske fyldt med en blanding af sand:vermiculit (1:1). Vækstkammeret blev indstillet med en dag/nat-cyklustemperatur på henholdsvis 28 °C/20 °C og en lys/mørke-fotoperiode på henholdsvis 16 t/8 timer. Den fotosyntetisk aktive strålingsintensitet var 62...

<ol>
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At studere genekspressionsregulering på translationelt niveau er afgørende for bedre at forstå forskellige biologiske processer, da endepunktet for cellegenekspression er proteinmængde13,14. Dette kan vurderes ved at analysere translatomet af vævet eller organismen af interesse, for hvilket den polysomale fraktion skal oprenses, og dets associerede mRNA'er analyseres 3,4,34,35,36.<sup clas...

Bælgplanter, såsom sojabønner (Glycine max), kan etablere symbiose med specifikke jordbakterier kaldet rhizobia. Dette mutualistiske forhold fremkalder dannelsen af nye organer, de symbiotiske knuder, på plantens rødder. Knuderne er planteorganerne, der er vært for bakterierne og består af værtsceller, hvis cytoplasma koloniseres med en specialiseret form for rhizobia kaldet bakterioider. Disse bakteroider katalyserer reduktionen af atmosfærisk nitrogen (N2) til ammoniak, som overføres til planten til gengæld for kulhydrater 1,2.
Selvom denne kvælstoffikserende symbiose er en af de mest velundersøgte plantemikrobesymbioser, er der stadig mange aspekter, der skal forstås bedre, såsom hvordan planter, der udsættes for forskellige abiotiske stressforhold, modulerer deres interaktion med deres symbiotiske partner, og hvordan dette påvirker knudemetabolismen. Disse processer kunne bedre forstås ved at analysere knudetranslatomet (dvs. delmængden af messenger RNA'er [mRNA'er] aktivt oversat). Polyribosomer eller polysomer er komplekser af flere ribosomer forbundet med mRNA, der almindeligvis anvendes til at studere oversættelse3. Polysomprofileringsmetoden består af analysen af mRNA'erne forbundet med polysomer og er med succes blevet brugt til at studere de posttranskriptionelle mekanismer, der styrer genekspression, der forekommer i forskellige biologiske processer 4,5.
Historisk set har genomekspressionsanalyse primært fokuseret på at bestemme mRNA-overflod 6,7,8,9. Der er imidlertid en mangel på korrelation mellem transkription og proteinniveauer på grund af de forskellige stadier af posttranskriptionel regulering af genekspression, især translation10,11,12. Desuden er der ikke observeret nogen afhængighed mellem ændringerne på transkriptomniveauet og dem, der forekommer på translatomniveau13. Den direkte analyse af det sæt mRNA'er, der oversættes, muliggør en mere nøjagtig og komplet måling af cellegenekspressionen (hvis endepunkt er proteinoverflod) end den, der opnås, når kun mRNA-niveauer analyseres14,15,16.
Denne protokol beskriver, hvordan planteafledte polysomer renses fra intakte sojabønneknuder ved differentiel centrifugering gennem en tolags saccharosepude (figur 1). Men da bakteroidafledte ribosomer også er til stede i knuderne, renses en blanding af ribosomer og RNA-arter, selvom de eukaryote repræsenterer hovedfraktionen (90% -95%). Den efterfølgende RNA-isolering, kvantificering og kvalitetskontrol er også beskrevet (figur 1). Denne protokol skal i kombination med RNA-seq give eksperimentelle resultater om translationel regulering af eukaryote mRNA'er i et komplekst væv, såsom den symbiotiske knude.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64269/64269fig01.jpg"> Figur 1: Skematisk oversigt over den foreslåede metode til eukaryot polysomrensning fra symbiotiske knuder. Ordningen giver et overblik over de trin, der følges i protokollen fra (1) plantevækst og (2) knudehøst til (3) forberedelse af cytosolekstrakter, (3) opnåelse af TOTAL-prøver og (4) PAR-prøver og (5) RNA-ekstraktion og kvalitetskontrol. Forkortelser: PEB = polysomekstraktionsbuffer; RB = resuspension buffer; TOTAL = total RNA; PAR = polysom-associeret mRNA. <a href="https://www.jove.com/files/ftp_upload/64269/64269fig01large.jpg" target="_blank">Klik her for at se en større version af denne figur.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plant growth and rhizobia inoculation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Daihan Scientific</td>
			<td></td>
			<td>Model SHO-1D</td>
		</tr>
		<tr>
			<td>YEM-medium</td>
			<td>Amresco</td>
			<td>J850 (yeast extract) 0122 (mannitol)</td>
			<td></td>
		</tr>
		<tr>
			<td>Water deficit treatment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>Merck</td>
			<td>221295</td>
			<td></td>
		</tr>
		<tr>
			<td>Porometer</td>
			<td>Decagon Device</td>
			<td></td>
			<td>Model SC-1</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of cytosolic extracts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brij L23</td>
			<td>Sigma-Aldrich</td>
			<td>P1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sigma</td>
			<td></td>
			<td>Model 2K15</td>
		</tr>
		<tr>
			<td>Chloranphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>DOC</td>
			<td>Sigma-Aldrich</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA 360</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1.04936</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tissue grinder</td>
			<td>Fisher Scientific</td>
			<td>12649595</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>PTE</td>
			<td>Sigma-Aldrich</td>
			<td>P2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Invitrogen</td>
			<td>15504-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing dish&#160;</td>
			<td>Deltalab</td>
			<td>1911103</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of sucrose cushions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Invitrogen</td>
			<td>15503022</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 40 Ti rotor</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td>Optima L-100K</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman-Coulter</td>
			<td>344059</td>
			<td>13.2 mL tubes</td>
		</tr>
		<tr>
			<td>RNA extraction and quality control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Thermo scientific</td>
			<td>R0492</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td></td>
			<td>Model 2100. Eukaryote total RNA nano assay</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>DI</td>
			<td>41191</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Dorwil</td>
			<td>UN1170</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Mallinckrodt</td>
			<td>3032-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Sigma</td>
			<td>10814-010</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol LS</td>
			<td>Ambion</td>
			<td>102960028</td>
			<td></td>
		</tr>
		<tr>
			<td>Miscellaneous</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 15 mL</td>
			<td>Biologix</td>
			<td>10-0152</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 10 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>321-4050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 1000 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>361-1050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 20 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>341-4050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 200 &#181;L</td>
			<td>Tarsons</td>
			<td>528104</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5 mL</td>
			<td>Tarsons</td>
			<td>500010-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 2.0 mL</td>
			<td>Tarsons</td>
			<td>500020-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing company</td>
			<td>Macrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 250 mL flask</td>
			<td>Marienfeld</td>
			<td>4110207</td>
			<td></td>
		</tr>
	</tbody>
</table>

polysome purification from soybean symbiotic nodules

Formålet med denne protokol er at tilvejebringe en strategi til undersøgelse af det eukaryote translatom af sojabønnen (Glycine max) symbiotisk knude. Dette papir beskriver metoder optimeret til at isolere planteafledte polyribosomer og deres tilknyttede mRNA'er, der skal analyseres ved hjælp af RNA-sekventering. For det første opnås cytoplasmatiske lysater gennem homogenisering i polysom- og RNA-bevarende betingelser fra hele, frosne sojabønneknuder. Derefter ryddes lysater ved centrifugering ved lav hastighed, og 15% af supernatanten anvendes til total RNA (TOTAL) isolering. Det resterende ryddede lysat bruges til at isolere polysomer ved ultracentrifugering gennem en tolags saccharosepude (12% og 33,5%). Polysom-associeret mRNA (PAR) renses fra polysomale pellets efter resuspension. Både TOTAL og PAR evalueres ved meget følsom kapillærelektroforese for at opfylde kvalitetsstandarderne for sekventeringsbiblioteker for RNA-seq. Som et eksempel på en downstream-applikation kan standardrørledninger til genekspressionsanalyse efter sekventering anvendes til at opnå differentielt udtrykte gener på transkriptom- og translatomniveauerne. Sammenfattende tillader denne metode i kombination med RNA-seq undersøgelsen af den translationelle regulering af eukaryote mRNA'er i et komplekst væv såsom den symbiotiske knude.

Kvantitets- og kvalitetsvurderingen af TOTAL- og PAR-fraktionerne, der er oprenset med ovennævnte procedure, er nøglen til at bestemme dens succes, da prøver af høj kvalitet er grundlæggende for biblioteksforberedelse og sekventering for de fleste downstream-applikationer, såsom RNA-sekventering. Desuden tillader RNA-molekylernes integritet indfangning af et øjebliksbillede af genekspressionsprofilen på tidspunktet for prøveindsamling18. I denne sammenhæng opnås et RNA-integritetsnummer...

Watch this Scientific Journal Video about Polysome Purification from Soybean Symbiotic Nodules at JoVE.com

Polysome Purification from Soybean Symbiotic Nodules

Denne protokol beskriver en metode til eukaryot polysomrensning fra intakte sojabønneknuder. Efter sekventering kan standardrørledninger til genekspressionsanalyse bruges til at identificere differentielt udtrykte gener på transkriptom- og translatomniveauerne.

Polysomrensning fra sojabønne symbiotiske knuder

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

Denne protokol er vigtig, fordi den, så vidt vi ved, er den første, der beskriver et nyt centrifugebaseret metal til kinetisk polysomrensning fra sojabønnesymbiotiske knuder. Den store fordel ved denne teknik er, at i kombination med RNA-Seq tillader undersøgelsen af relationen og organiseret i et komplekst væv som den symbiotiske knude. Til at begynde med vejes ca. 0,2 gram intakte knuder i en vejeskål og overføres til et forkølet to milliliter rør.Tilsæt derefter 1,2 ml polysomekstraktionsbuffer, lad prøven tø op i to minutter og homogeniser med en vævssliber indtil fuldstændig forstyrrelse og homogenisering af knuderne. Derefter inkuberes prøverne på is med blid omrøring i 10 minutter, eller indtil alle prøver er behandlet, og centrifuger ved 16.000 gange G i 15 minutter ved fire grader Celsius for at pelletere affaldet. Gendan derefter supernatanten, og gentag centrifugetrinnet.Efter omhyggelig genvinding af det klargjorte cytosolekstrakt overføres en 200 mikroliter aliquot til et rent 1,5 ml mikrocentrifugerør til total isolering. Hæld derefter 4,5 ml af 33,5% saccharoselaget i centrifugerørene, og tilsæt 4,5 ml af 12%laget forsigtigt og langsomt med en P-1000 mikropipette. Sæt derefter rørene på is indtil tilsætningen af det afklarede cytosoliske ekstrakt.Læg en milliliter af det klargjorte cytosolekstrakt oven på saccharosepuden ved forsigtigt at pipettere på rørets sidevæg. Overfør derefter ultracentrifugerørene til forkølede spande og centrifuge ved 217, 874 G i to timer ved fire grader Celsius. Efter kassation af det resterende cytosolekstrakt og saccharosepude suspenderes polysomalpellet igen med 200 mikroliter forkølet re-suspensionsbuffer.Derefter inkuberes i 30 minutter på is, og overfør derefter den polysomale gensuspension til 1,5 ml forkølede rør for at fortsætte med RNA-ekstraktionen. Efter homogenisering inkuberes prøverne med 750 mikroliter af RNA-isolationsreagenset i fem minutter ved stuetemperatur. Tilsæt derefter 200 mikroliter kold chloroform, og ryst rørene kraftigt i 15 sekunder.Inkuberes derefter ved stuetemperatur i 10 minutter. Centrifuge ved 12.000 gange G i 15 minutter ved fire grader Celsius til faseadskillelse, og overfør 500 mikroliter af den øvre vandige fase til et rent rør uden at forstyrre den lyserøde organiske fase. Derefter tilsættes 375 mikroliter kold isopropanol og 0,5 mikroliter RNA-fri glykogen.Bland derefter grundigt ved at pipettere op og ned. Inkuber blandingen i 10 minutter ved fire grader Celsius og centrifuge ved 12.000 gange G i 15 minutter. Kassér derefter supernatanten, og vask RNA-bundfaldet med en milliliter kold, 75% ethanol.Bland ved kort hvirvel. Derefter centrifugeres ved 7.500 G i fem minutter ved fire grader Celsius. Kassér supernatanten, og lufttør RNA-pelleten.Derefter opløses pelleten i 50 mikroliter RNA-frit vand, og inkuberes ved 65 grader Celsius i fem minutter. Vurder RNA-koncentrationen og integriteten med meget følsom kapillær elektroforese og eller elektroforese på en 2% RNA-fri agarosegel. Efter estimering af prøvevolumenet tilsættes 0,1 volumener af tre molære natriumacetat, tre volumener kold ethanol og 0,5 mikroliter RNA'er, frit glykogen.Bland dem derefter grundigt. Kapillær elektroforese blev udført for flere totale og polysomale associerede RNA-prøvefraktioner, som viste skarpe ribosomale RNA-bånd uden noget udtværet udseende. Dette afspejles dog ikke i RIN-værdierne, da de ligger mellem 5,9 og 7,5, hvilket svarer til ikke-intakte prøver.Bioanalysatoren kunne ikke identificere 18S- og 25S-toppe som set i elektroferogrammerne, både for totale og polysomassocierede RNA-prøver. Der var ingen visuelle tegn på nedbrydning af prøver. Imidlertid blev en prøve analyseret for at visualisere resultatet af næsten helt nedbrudt prøve til sammenligning.Det vigtigste er at arbejde i polysom- og RNA-bevarende forhold under hele protokollen. Efter sekventering kan de samlede og par RNA-fraktioner standardmønstre for det analyserede genekspression bruges til at identificere differentielle ekspressgener på transkriptions- og oversættelsesniveauet.

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