Name: a method for characterizing embryogenesis in arabidopsis
Uploaded: 2017-08-04T10:00:00
Description: Watch this Scientific Journal Video about A Method for Characterizing Embryogenesis in Arabidopsis at JoVE.com

Vi takker Dr. Jessica Habashi for kritisk læsning af manuskriptet. Vi takker Dr. Xianyong Sheng Imaging Center, College of life Sciences, kapital Normal University (Beijing, Kina), til at udføre lokalisering af DR5-NGL assay. Dette arbejde blev støttet af tilskud fra Beijing Municipal regering Science Foundation (CIT &#38; TCD20150102) og fra den National Natural Science Foundation of China (31600248).

Bemærk: Denne protokol har tre dele: 1) observere mønster dannelsen i wild-type Arabidopsis embryoner ved hjælp af DIC mikroskopi; 2) karakteriserede af embryoer mønster dannelse via observation af markør protein udtryk ved hjælp af Konfokal laser scanning mikroskopi; og 3) bestemmelse betydning af et specifikt gen i embryogenese (Brug naa10 mutant som et eksempel).
1. ovule Clearing
<ol>
	<li>Plante materielle forberedelse Bemærk: Protok...

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	<li>Jenik, P. D., Gillmor, C. S., Lukowitz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+patterning+in+Arabidopsis+thaliana.">Embryonic patterning in Arabidopsis thaliana.</a> Annu Rev Cell Dev Biol. 23, 207-236 (2007).</li><li>Lau, S., Slane, D., Herud, O., Kong, J., Jurgens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+embryogenesis+in+flowering+plants:+setting+up+the+basic+body+pattern.">Early embryogenesis in flowering plants: setting up the basic body pattern.</a> Annu Rev Plant Biol. 63, 483-506 (2012).</li><li>Wendrich, J. R., Weijers, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+embryo+as+a+miniature+morphogenesis+model.">The Arabidopsis embryo as a miniature morphogenesis model.</a> New Phytol. 199 (1), 14-25 (2013).</li><li>ten Hove, C. A., Lu, K. J., Weijers, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+plant:+cell+fate+specification+in+the+early+Arabidopsis+embryo.">Building a plant: cell fate specification in the early Arabidopsis embryo.</a> Development. 142 (3), 420-430 (2015).</li><li>Feng, 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=Protein+N-terminal+acetylation+is+required+for+embryogenesis+in+Arabidopsis.">Protein N-terminal acetylation is required for embryogenesis in Arabidopsis.</a> J Exp Bot. 67 (15), 4779-4789 (2016).</li><li>Berleth, T., Jurgens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+monopteros+gene+in+organising+the+basal+body+region+of+the+Arabidopsis+embryo.">The role of the monopteros gene in organising the basal body region of the Arabidopsis embryo.</a> Development. 118, 575-587 (1993).</li><li>Luo, Y., 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=D-myo-inositol-3-phosphate+affects+phosphatidylinositol-mediated+endomembrane+function+in+Arabidopsis+and+is+essential+for+auxin-regulated+embryogenesis.">D-myo-inositol-3-phosphate affects phosphatidylinositol-mediated endomembrane function in Arabidopsis and is essential for auxin-regulated embryogenesis.</a> Plant Cell. 23 (4), 1352-1372 (2011).</li><li>Nodine, M. D., Yadegari, R., Tax, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPK1+and+TOAD2+are+two+receptor-like+kinases+redundantly+required+for+Arabidopsis+embryonic+pattern+formation.">RPK1 and TOAD2 are two receptor-like kinases redundantly required for Arabidopsis embryonic pattern formation.</a> Dev Cell. 12 (6), 943-956 (2007).</li><li>Ulmasov, T., Murfett, J., Hagen, G., Guilfoyle, T. 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Ovule clearance er en nyttig metode til påvisning af celledeling og vurdere morfologi under embryogenese i Arabidopsis; Brug af denne teknik, kan embryoner observeres direkte under DIC mikroskopi5,12. Det afgørende skridt for ovule clearance er trin 1.3.4. Den nødvendige tid til ovule clearance taktfast 1.3.4 er variabel. Embryoner på 2/4-celle stadium kan observeres klart efter 2 h i Hoyers løsning, mens de ser utydelig efter 12 timer. Således, de...

Embryogenese er den tidligste begivenhed i højere plante udvikling. En moden embryo formularer fra en zygote gennem celledeling og differentiering under streng genetiske kontrol1,2. Arabidopsis embryoner er en nyttig model til at studere kontrol af morfogenese, fordi rækkefølgen af celledelinger under embryogenese følger et forventet mønster3,4. Men embryonet Arabidopsis indeholdende én til flere celler er for lille til at være observeret og analyseret. Derudover kan mutation af visse gener forårsage embryo dødelighed5, der angiver den centrale rolle i disse gener i embryogenese. Karakterisering af mønster dannelse i fosteret-dødbringende mutanter giver et grundlag for at forstå de molekylære mekanisme, hvorved væsentlige gener regulere embryo udvikling.
En detaljeret metode er beskrevet her til karakterisering af embryo mønster dannelse i model planten Arabidopsis af direkte mikroskopiske observation. Metoden indebærer ovule clearance efterfulgt af mikroskopisk observation af et embryo. Karakterisering af et embryo-dødbringende mutant er også beskrevet.
Morfologi af embryoner på forskellige udviklingsstadier kan bestemmes direkte ved mikroskopi benytter de frøanlæg, der er blevet ryddet hos Hoyers løsning6,7. Disse æganlæg udviser en høj brydningsindeks; således, denne ovule clearing metode er særligt fordelagtigt for enheder, der skal overholdes af differential interferens kontrast (DIC) mikroskopi.
Derudover kan de gener, der er udtrykt i en bestemt celle eller et begrænset antal celler under embryogenese bruges som markører til at identificere cellerne i embryonet. Derfor, den celle identity specifikation under embryogenese kan påtegnes af overvågning udtryk for normal god landbrugspraksis-markeret datamærke proteiner ved hjælp af Konfokal laser scanning mikroskopi. Omvendt, observere udtryk mønster af en ukendt funktionel genet -normal god landbrugspraksis fusion under embryogenese kan give et link mellem embryo mønstre og udtryk for gen, og lette identifikationen af nye gener, der er nødvendige for embryogenese i Arabidopsis.
Metoden beskrevet her kan også bruges til at karakterisere Fænotypen af et embryo-dødbringende mutant og bestemme virkningen af de berørte gen på embryogenese på cellulært niveau. Desuden i kombination med en sammenligning af udtryk mønster af markørgener under embryogenese mellem wild-type og mutant planter, kan vil metoden give fingerpeg om de molekylære mekanismer og signalering veje involveret i embryogenese8,9. Metoden blev anvendt til naa10 planter, og det konstateredes, at de unormale division af hypofysen i muterede kan være forårsaget af en ændring i auxin fordelingen under embryogenese5.

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			<td height="21" style="height:21px;width:281px;">Chloral Hydrate</td>
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			<td>M5519</td>
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			<td height="21" style="height:21px;">Fluorescence microscope</td>
			<td>Zeiss Oberkochen</td>
			<td>Zeiss Image M2</td>
			<td>camera: AxioCam 506 color</td>
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			<td height="21" style="height:21px;">Confocal Laser Scanning Microscopy</td>
			<td>Zeiss Oberkochen</td>
			<td>Zeiss LSM 5</td>
			<td>filters: BP 495-555 nm</td>
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			<td height="21" style="height:21px;">Stereoscope</td>
			<td>Zeiss Oberkochen</td>
			<td>Axio Zoom V16</td>
			<td>camera: AxioCam MRc5</td>
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			<td height="21" style="height:21px;">nutrient-rich&#160;soil</td>
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			<td height="21" style="height:21px;width:281px;">10% sodium hypochlorite solution</td>
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			<td>&#160;Domestic analytical reagent</td>
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			<td height="21" style="height:21px;">sucrose</td>
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			<td>&#160;Domestic analytical reagent</td>
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			<td height="21" style="height:21px;">glycerol</td>
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			<td>&#160;Domestic analytical reagent</td>
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a method for characterizing embryogenesis in arabidopsis

Grund af deres udvikling meget forudsigelig er Arabidopsis embryoner blevet brugt som en model for studier af morfogenese i planter. Imidlertid tidlig fase plante embryoner er små og indeholder få celler, hvilket gør dem svære at observere og analysere. En metode er beskrevet her for kendetegner mønster dannelse i anlægget embryoner under et mikroskop ved hjælp af model organismen Arabidopsis. Efter afslutning af frisk æganlæg bruger Hoyers løsning, celle nummer i og morfologi af embryoner kunne konstateres, og deres udviklingsstadiet kunne bestemmes af differential interferens kontrast mikroskopi ved hjælp af en 100 X olie fordybelse linse. Desuden blev udtryk for specifikke markør proteiner markeret med grøn fluorescerende proteiner (NGL) overvåget for at anmærke celle identitet specifikation under embryo mønstre af Konfokal laser scanning mikroskopi. Således, denne metode kan bruges at observere mønster dannelse i wild-type plante embryoner på cellulære og molekylære niveau, og at karakterisere rolle af specifikke gener i fosteret mønstret ved at sammenligne mønster dannelse i embryoner fra wild-type planter og embryo-dødbringende mutanter. Metoden kan derfor anvendes til at karakterisere embryogenese i Arabidopsis.

Metoden i dette papir kan bruges til at observere embryogenese direkte under et mikroskop, anmærke celle identitet specifikation under embryogenese med specifikke markører og karakterisere et bestemt gen rolle under embryogenese.
Repræsentative resultater fra analysen af embryo mønster (fra den aflange zygote fase til den modne walking stick fase) i wild-type Arabidopsis er vist i figur 2</stron...

Watch this Scientific Journal Video about A Method for Characterizing Embryogenesis in Arabidopsis at JoVE.com

A Method for Characterizing Embryogenesis in Arabidopsis

Denne protokol beskriver en metode til at observere embryogenese i Arabidopsis via ovule clearance efterfulgt af inspektion af embryo mønster dannelse under et mikroskop.

En metode til kendetegner embryogenese i Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

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Developmental Biology

A Method for Characterizing Embryogenesis in Arabidopsis

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