Name: maintaining biological cultures and measuring gene expression in aphis nerii: a non-model system for plant-insect interactions
Uploaded: 2018-08-31T14:00:00
Description: Watch this Scientific Journal Video about Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions at JoVE.com

Vorremmo ringraziare Michelle Moon (Vanderbilt University) per assistenza con la fotografia. Vanderbilt University fornito supporto per PA e SSLB è supportato da DGE-1445197.

1. pianta culture
<ol><li>Acquistare i semi da qualsiasi fornitore commerciale o raccogliere da piante mature nel campo. Nota: Questo protocollo è adatto per la maggior parte delle specie di euforbia commercialmente disponibili (ad es., Asclepias incarnata, a. syriaca, a. curassavica, Asclepias physocarpus). Alcuni semi potrebbero essere necessario essere stratificato al freddo, e le istruzioni direttamente dall'offerente seme dovrebbero essere controllate.</li>
	<li>
Pia...

<ol>
	<li>Brisson, J. A., Stern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pea+aphid,+Acyrthosiphon+pisum.:+an+emerging+genomic+model+system+for+ecological,+developmental+and+evolutionary+studies.">The pea aphid, Acyrthosiphon pisum.: an emerging genomic model system for ecological, developmental and evolutionary studies.</a> BioEssays. 28, 747-755 (2006).</li><li>Dixon, A. F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aphid Ecology: An Optimization Approach. , (1985).</li><li>Consortium, T. I. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Sequence+of+the+Pea+Aphid+Acyrthosiphon+pisum.">Genome Sequence of the Pea Aphid Acyrthosiphon pisum.</a> PLoS Biology. , 1000313 (2010).</li><li>Srinivasan, D. G., Brisson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aphids:+A+Model+for+Polyphenism+and+Epigenetics.">Aphids: A Model for Polyphenism and Epigenetics.</a> Genetics Research International. 2012, 1-12 (2012).</li><li>Wenger, J. 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=Whole+genome+sequence+of+the+soybean+aphid,+Aphis+glycines.">Whole genome sequence of the soybean aphid, Aphis glycines.</a> Insect Biochememistry and Molecular Biology. , 1-10 (2017).</li><li>Li, Z. -. Q., 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=Ecological+adaption+analysis+of+the+cotton+aphid+(Aphis+gossypii)+in+different+phenotypes+by+transcriptome+comparison.">Ecological adaption analysis of the cotton aphid (Aphis gossypii) in different phenotypes by transcriptome comparison.</a> PLoS ONE. 8, 83180 (2013).</li><li>Wang, D., Liu, Q., Jones, H. D., Bruce, T., Xia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+transcriptomic+analyses+revealed+divergences+of+two+agriculturally+important+aphid+species.">Comparative transcriptomic analyses revealed divergences of two agriculturally important aphid species.</a> BMC Genomics. 15 (1), 1023-1024 (2014).</li><li>Wu, 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=De+novo+characterization+of+the+pine+aphid+Cinara+pinitabulaeformis+Zhang+et+Zhang+transcriptome+and+analysis+of+genes+relevant+to+pesticides.">De novo characterization of the pine aphid Cinara pinitabulaeformis Zhang et Zhang transcriptome and analysis of genes relevant to pesticides.</a> PLoS ONE. 12, 0178496-0178517 (2017).</li><li>Birnbaum, S. S. L., Rinker, D. C., Gerardo, N. M., Abbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profile+and+differential+fitness+in+a+specialist+milkweed+insect+across+host+plants+varying+in+toxicity.">Transcriptional profile and differential fitness in a specialist milkweed insect across host plants varying in toxicity.</a> Molecular Ecology. , (2017).</li><li>Dixon, A. F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect Herbivore-Host Dynamics. , (2005).</li><li>Goggin, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-aphid+interactions:+molecular+and+ecological+perspectives.">Plant-aphid interactions: molecular and ecological perspectives.</a> Current Opinion in Plant Biology. 10, 399-408 (2007).</li><li>Will, T., Furch, A., Zimmermann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+phloem-feeding+insects+face+the+challenge+of+phloem-located+defenses.">How phloem-feeding insects face the challenge of phloem-located defenses.</a> Frontiers in Plant Science. 4, 1-12 (2013).</li><li>Webster, B. <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+olfaction+in+aphid+host+location.">The role of olfaction in aphid host location.</a> Physiological Entomology. 37, 10-18 (2012).</li><li>Agrawal, A. A., Petschenka, G., Bingham, R. A., Weber, M. G., Rasmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+cardenolides:+chemical+ecology+and+coevolution+of+specialized+plant-herbivore+interactions.">Toxic cardenolides: chemical ecology and coevolution of specialized plant-herbivore interactions.</a> New Phytologist. 194, 28-45 (2012).</li><li>Dobler, S., Petschenka, G., Pankoke, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coping+with+toxic+plant+compounds-+the+insect's+perspective+on+iridoid+glycosides+and+cardenolides.">Coping with toxic plant compounds- the insect's perspective on iridoid glycosides and cardenolides.</a> Phytochemistry. 72, 1593-1604 (2011).</li><li>Opitz, S. E. W., M&#252;ller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+chemistry+and+insect+sequestration.">Plant chemistry and insect sequestration.</a> Chemoecology. 19, 117-154 (2009).</li><li>Birnbaum, S. S. L., Abbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+adaptations+toward+plant+toxins+in+milkweed-herbivores+systems+-+a+review.">Insect adaptations toward plant toxins in milkweed-herbivores systems - a review.</a> Entomologia Experimentalis et Applicata. 58, 579-610 (2018).</li><li>Rothschild, M., von Euw, J., Reichstein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+glycosides+in+the+oleander+aphid,+Aphis+nerii.">Cardiac glycosides in the oleander aphid, Aphis nerii.</a> Journal of Insect Physiology. 16, 1141-1145 (1970).</li><li>Harrison, J. S., Mondor, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+an+invasive+aphid+'superclone':+extremely+low+genetic+diversity+in+oleander+aphid+(Aphis+nerii)+populations+in+the+southern+United+States.">Evidence for an invasive aphid 'superclone': extremely low genetic diversity in oleander aphid (Aphis nerii) populations in the southern United States.</a> PLoS ONE. 6, 17524 (2011).</li><li>Mooney, K. A., Halitschke, R., Kessler, A., Agrawal, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+trade-offs+in+plants+mediate+the+strength+of+trophic+cascades.">Evolutionary trade-offs in plants mediate the strength of trophic cascades.</a> Science. 327, 1642-1644 (2010).</li><li>Grabherr, M. 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=Full-length+transcriptome+assembly+from+RNA-Seq+data+without+a+reference+genome.">Full-length transcriptome assembly from RNA-Seq data without a reference genome.</a> Nature Biotechnology. 29, 644-652 (2011).</li><li>Haas, B. 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=De+novo+transcript+sequence+reconstruction+from+RNA-seq+using+the+Trinity+platform+for+reference+generation+and+analysis.">De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.</a> Nature Protocols. 8, 1494-1512 (2013).</li><li>Finn, R. D., 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+Pfam+protein+families+database:+towards+a+more+sustainable+future.">The Pfam protein families database: towards a more sustainable future.</a> Nucleic Acids Research. 44, 279-285 (2016).</li><li>The UniProt Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniProt:+the+universal+protein+knowledgebase.">UniProt: the universal protein knowledgebase.</a> Nucleic Acids Research. 45, 158-169 (2017).</li><li>Johnson, M., 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=NCBI+BLAST:+a+better+web+interface.">NCBI BLAST: a better web interface.</a> Nucleic Acids Research. 1 (36), 5-9 (2008).</li><li>Fu, L., Niu, B., Zhu, Z., Wu, S., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD-HIT:+accelerated+for+clustering+the+next+generation+sequencing+data.">CD-HIT: accelerated for clustering the next generation sequencing data.</a> Bioinformatics. 28 (23), 3150-3152 (2012).</li><li>Sim&#227;o, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V., Zdobnov, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BUSCO:+assessing+genome+assembly+and+annotation+completeness+with+single-copy+orthologs.">BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs.</a> Bioinformatics. 31, 3210-3212 (2015).</li><li>Bolger, A. M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30, 2114-2120 (2014).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. 9, 357-359 (2012).</li><li>Li, 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=The+Sequence+Alignment/Map+format+and+SAMtools.">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25, 2078-2079 (2009).</li><li>Love, M. I., Huber, W., Anders, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderated+estimation+of+fold+change+and+dispersion+for+RNA-seq+data+with+DESeq2.">Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.</a> Genome Biology. 15, 31 (2014).</li><li>Rieu, I., Powers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+RT-PCR:+design,+calculations,+and+statistics.">Real-time quantitative RT-PCR: design, calculations, and statistics.</a> Plant Cell. 21, 1031-1033 (2009).</li><li>Malcolm, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+defence+in+chewing+and+sucking+insect+herbivores:+plant-derived+cardenolides+in+the+monarch+butterfly+and+oleander+aphid.">Chemical defence in chewing and sucking insect herbivores: plant-derived cardenolides in the monarch butterfly and oleander aphid.</a> Chemoecology. 1, 12-21 (1990).</li><li>Agrawal, A. A., Underwood, N., Stinchcombe, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraspecific+variation+in+the+strength+of+density+dependence+in+aphid+populations.">Intraspecific variation in the strength of density dependence in aphid populations.</a> Ecological Entomology. 29, 521-526 (2004).</li><li>Zehnder, C. B., Hunter, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+maternal+effects+and+current+environment+on+vital+rates+of+Aphis+nerii,+the+milkweed-oleander+aphid.">A comparison of maternal effects and current environment on vital rates of Aphis nerii, the milkweed-oleander aphid.</a> Ecological Entomology. 32, 172-180 (2007).</li><li>Hartbauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+defense+of+Aphis+nerii+and+Uroleucon+hypochoeridis+(Homoptera,+Aphididae)+against+natural+enemies.">Collective defense of Aphis nerii and Uroleucon hypochoeridis (Homoptera, Aphididae) against natural enemies.</a> PLoS ONE. 5, 10417 (2010).</li></ol>

Lungamente è stato riconosciuto che l'aposematico a. nerii può fornire le comprensioni i modelli e meccanismi di resistenza per le difese della pianta e sequestro in particolare chimica18,37. Un numero di risorse genomiche è recentemente emerse per a. nerii10, offrendo nuove opportunità per studi di genomica funzionale ed ecologici che utilizzano a. nerii come un modello. Delineiamo protocolli di base nella c...

Gli afidi sono piccoli insetti hemimetabolous che colonizzano su famiglie di piante diverse in tutto il mondo. Sono distintivi per diverse funzioni, più in particolare i cicli di vita complessi che coinvolgono partenogenesi ciclica e discreti polyphenisms e loro simbiosi nutrizionali obbligano con endosimbionti batterici o lievito che forniscono le sostanze nutrienti mancanti da la loro dieta di pianta sap1. Mentre la maggior parte gli afidi sono specialisti di pianta ospite, alcune specie generalista sono parassiti delle colture importanti, infliggendo notevoli danni economici sulle colture sia direttamente o tramite gli agenti patogeni e virus hanno vector2. La pubblicazione del primo genoma afide nel 2010, l'afide di pisello Acyrthosiphon pisum3, segnò un'importante pietra miliare nello studio della biologia dell'afide perché ha fornito le risorse genomiche per questioni dell'insetto adattamenti agli erbivori stili di vita, comprese quelle che potrebbero portare a una migliore di strategie di controllo4. Da quel momento, ulteriori risorse genomiche hanno accumulato con la pubblicazione di un genoma con annotazioni per la soia afide glycines Aphis5e pubblicamente disponibile intero genoma risorse per un'altra specie di tre-afide (Myzus Cerasi (afide nero ciliegia), Myzus persicae (afide peach-patata), Rhopalosiphum padi (afide ciliegia di uccello-avena)6. Sono disponibili anche per un numero di altre specie di afide prezioso de novo Transcrittomica risorse (e.g.,Aphis gossypii (afide del cotone)7, Sitobion avenae (afide grano)8, Cinara pinitabulaeformis (afide pino)9, Aphis nerii (afide del milkweed-oleander)10).
Gli afidi hanno anche fatto duraturo contributo alla nostra comprensione delle interazioni pianta-insetto e l'ecologia della vita su piante11. Un'area dove gli afidi hanno dato contributi particolarmente importanti è nella nostra comprensione dell'ecologia chimica delle interazioni pianta dell'ospite. Adattamenti di esprimere diversi insetti erbivori per superare le difese della pianta e alcuni anche cooptare le difese della pianta per il proprio beneficio12,13,14. Ad esempio, l'afide del milkweed-oleander, Aphis nerii, è un brillante giallo, dilagante afide trovato nelle regioni temperate e tropicali in tutto il mondo che colonizza su piante appartenenti alla famiglia del milkweed (Apocynaceae). Piante della famiglia delle Apocynaceae si sono evolute diverse difese chimiche, tra cui lattice latteo e glicosidi cardiaci, noti come complessi, che legano il trasportatore di cationi Na, K-atpasi e sono efficaci deterrenti a generalista erbivori15, 16. specialisti milkweed express varie modalità di resistenza ai complessi, e alcuni in modo selettivo o passivamente si accumulano o modificare complessi nei loro tessuti come un mezzo per scoraggiare la predazione o per altri benefici17. A. nerii è pensato per sequestrare complessi in questo modo, anche se i meccanismi e i vantaggi funzionali rimangono poco chiari10,18.
Date le risorse genomiche a portata di mano, a. nerii fornisce un eccellente modello sperimentale per lo studio dei meccanismi molecolari e genetici coinvolti nelle interazioni chemio-ecologico tra piante tossiche e loro specialista erbivori. Vale la pena notare che, mentre alcuni dei primi studi di a. nerii incentrato sul sequestro di complessi19, da quel momento, gli studi di a. nerii hanno fornito le comprensioni in una vasta gamma di domande evolutive ed ecologiche, compresa la struttura genetica di insetti invasivi20 e l'interazione tra bottom up e top-down regolamento l'erbivoro densità21. A. nerii è così un buon candidato come un modello sperimentale per un insieme particolarmente vasto di studi delle interazioni insetto-pianta. Critico per il successo di qualsiasi studio con a. nerii è la cultura attenta dell'afide popolazioni, che comprende la coltura delle piante da cui dipendono gli afidi, come pure una generazione efficiente di dati di alta qualità - omic. Il nostro obiettivo è quello di guidare il lettore attraverso entrambi. Descritte di seguito sono i metodi per la generazione e la manutenzione delle colture vegetali e afide in serra e laboratorio, DNA ed estrazioni RNA, analisi dei microsatelliti, de novo del trascrittoma assemblaggio e annotazione, trascrittoma analisi di espressione differenziale e la verifica di qPCR di geni differenzialmente espressi. Mentre questi metodi vengono scritti per a. nerii, i metodi di coltura, estrazione e analisi generali possono estendersi ad una varietà di specie di afidi.

<table border="0" cellpadding="0" cellspacing="0" style="width:820px;" width="820">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:64px;">Sun Gro Fafard Germination Mix</td>
			<td style="width:160px;">Hummert International</td>
			<td style="width:184px;">10-0952-2</td>
			<td style="width:412px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:64px;">Sun Gro Fafard 3B/ Metro Mix</td>
			<td>Hummert International</td>
			<td style="width:184px;">10-0951-2</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:64px;">2x 4&#34; Round Standard Pot</td>
			<td>Anderson Pots</td>
			<td style="width:184px;">1503</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:64px;">DreamTaq DNA Polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">EP0701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:64px;">Trizol</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">15596026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript&#174; III First-Strand Synthesis kit</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">18080051</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:64px;">Power SYBR Green PCR Master Mix</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">4367659</td>
			<td></td>
		</tr>
	</tbody>
</table>

maintaining biological cultures and measuring gene expression in aphis nerii: a non-model system for plant-insect interactions

Gli afidi sono eccellenti modelli sperimentali per una varietà di domande biologiche che vanno dall'evoluzione delle simbiosi e lo sviluppo di polyphenisms a domande che circondano interazioni dell'insetto con le loro piante ospite. Risorse genomiche sono disponibili per diverse specie di afidi, e i progressi compiuti il sequenziamento di nuova generazione, trascrittomica studi sono stati estesi a organismi non-modello che non dispongono di genomi. Inoltre, afide culture possono essere raccolti dal campo e allevate in laboratorio per l'uso negli esperimenti organismi e molecolari per colmare il divario tra gli studi ecologici e genetici. Infine, molti afidi possono essere mantenuti in laboratorio sulle loro piante ospite preferito in perpetui, partenogenetiche cicli di vita consentendo confronti di riprodursi asessualmente genotipi. Aphis nerii, l'afide del milkweed-oleander, fornisce un modello per studiare le interazioni insetto con piante tossiche utilizzando esperimenti sia organismi che molecolari. Metodi per la generazione e la manutenzione delle colture vegetali e afide nell'espressione differenziale di serra e laboratorio, estrazioni di DNA e RNA, l'analisi del microsatellite, de novo del trascrittoma assemblaggio e annotazione, trascrittoma analisi e la verifica di qPCR di geni differenzialmente espressi sono descritte e discusse qui.

Colture della pianta: Semi prenderà circa due-quattro settimane, a seconda della stagione, per diventare grande abbastanza per essere ri-vaso (Figura 1A). Re-potted piantine porterà un altro due-quattro settimane per aumentare a una dimensione ottima per le culture di afide (Figura 1B).
Culture afide:...

Watch this Scientific Journal Video about Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions at JoVE.com

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

L'afide Aphis nerii colonizza sulle piante altamente difeso nella famiglia dogbane (Apocyanaceae) e offre numerose opportunità per studiare le interazioni pianta-insetto. Qui, presentiamo una serie di protocolli per la manutenzione di impianti e afide, culture e la generazione e l'analisi di molecolare e dati - omic per a. nerii.

Mantenimento di colture biologiche e misurazione dell'espressione genica in Aphis nerii: un sistema Non-modello per le interazioni pianta-insetto

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

This method describes general steps for aphid culturing and steps for the genetic analysis of aphids. These methods can be applied to answer key questions in the evolution and ecology of aphids and the moleculare mechanisms underlying aphid biology. The main advantage of this technique is it can be generally applied to most aphid species and performed at a relatively low cost.Begin by filling a standard seedling tray with germination mix soil, taking care that the soil reaches the top of the wells. When all of the wells have been filled, make a three centimeter deep hole in the soil of each well and place one seed into each hole. Water the cells until the soil covering the seeds is saturated and place the tray in a green house with daily to every other day watering, to maintain a moderate soil moisture level.When the seedlings have grown their first set of full leaves fill four inch round pots with potting soil, up to about five centimeters below their rims. Create a whole in the soil deep enough to reach the bottom of the pot and gently scoop the mature seedlings by hand to place them deep within the holes in the new pots. Then, cover the seedlings with soil and water the seedlings daily to every other day to maintain a moderate soil moisture level.When the plants have grown at least three to four sets of full leaves and are at least ten centimeters tall, manually inspect the plants for any unwanted pests and use a mouth pipette to carefully transfer a single reproducing field collected adult aphid onto the leaf of the first plant to create an iso-clonal line. When all of the aphids have been transferred, securely cover the plants with a custom made cup cage, and place the aphid infested plants in a controlled environment chamber. To maintain the stock populations, use a mouth pipette to safely transfer one to three second or third instar nymphs, and one adult aged aphid, to a new plant on a weekly basis.And cover each plant with a new cup cage for culture as demonstrated. For DNA extraction from a single adult aphid, place the aphid near the bottom of a sterile 1.5 milliliter microcentrifuge tube containing liquid nitrogen and grind the aphid with a pestle. Next, add 100 microliters of lysis buffer to the microcentrifuge tube and continue to grind the crushed aphid until the sample is visibly disintegrated.Wash the pestle with an additional 100 microliters of lysis buffer, and incubate the ground aphid at 65 degrees Celsius for 30 minutes. At the end of the incubation add 14 microliters of aemulor potassium acetate to the tube and invert to mix. After 30 minutes on ice collect the cellular debris by centrifugation and transfer the supernatant to a new 1.5 milliliter microcentrifuge tube without disturbing the pellet.Add 200 microliters of cold 100 percent molecular grade ethanol to the supernatant and invert the tube to mix. After 15 minutes at room temperature, collect the precipitated DNA by centrifugation, and remove the ethanol by pipette. Resuspend the pellet in 200 microliters of cold 70 percent molecular grade ethanol with flicking for a second centrifuge wash.And resuspend the pellet in 200 microliters of fresh, cold 100 percent molecular grade ethanol. After the third centrifugation, place the tube horizontally on a piece of tissue paper for air drying for five to ten minutes. And resuspend the DNA pellet in 80 microliters of low tris edta.Then, quantify the resuspended DNA in a spectrophotometer, and store the remaining sample at four degrees Celsius. After crushing up to five adult aphids in liquid nitrogen as demonstrated, transfer the sample to a fume hood and add 800 microliters of guanidinium thiocyanate-phenol-chloroform extraction reagent to the sample tube. Homogenize the sample with a pestle and incubate the homogenate for five minutes at room temperature.Next, add 160 microliters of chloroform to the sample, followed by 15 seconds of vigorous shaking by hand. Then, incubate the sample for two to three minutes at room temperature and centrifuge to collect the extracted RNA. To precipitate the RNA, transfer the aqueous phase to new RNS free microcentrifuge tube without disturbing the intermediate phase, and add 400 microliters of isopropanol to the RNA.After a ten minute incubation at negative 20 degrees Celsius, collect the precipitated RNA by centrifugation. Then, wash the sample two times with one milliliter of 75 percent ethanol in diethyl pyrocarbonate treated water, to the sample with slow vortexing and centrifugation to remove any phenol contaminants. After the second wash, mostly air dry the pellet for five to ten minutes on a sterile bench, and dissolve the RNA pellet in 30 microliters of ultra pure water with gentle pipetting before a ten to 15 minute incubation at 55 to 60 degrees Celsius.Seeds will take approximately two to four weeks, depending on the season, to grow large enough to be repotted. Repotted seedlings will take another two to four weeks to grow to an optimal size for aphid culture. Adult aye-near-ee-eye are distinguished by a darkened cauda, and may be unwinged or winged.Developing wing pads become visible when nymphs reach the third instar. Single adult aye-near-ee-eye will yield approximately 100 to 200 nanograms per microliter of DNA and 150 to 300 nanograms per microliter of RNA. The relative expression of a candidate gene under different treatment conditions can then be calculated.When attempting this procedure it's important to not contaminate you're iso-clonal stocks. Also, remember that the quality of your aphids is highly dependent upon the quality of your plants. Following this procedure, other extractions and sequencing techniques can be performed to answer question about the molecular mechanisms underlying aphid biology.

Research

JoVE Journal

Genetics

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Misura parallela di circadiano Clock espressione genica e la secrezione dell&#39;ormone in colture cellulari primarie umane

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Ricerca

Genetica

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

Perturbazioni di circolazione miRNA nella sindrome dell&#39;intestino irritabile rilevato utilizzando una piattaforma di espressione genica multiplex high-throughput

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Unicellulare profili di espressione genica Utilizzando FACS e qPCR con standard interni

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

Preparazione dei campioni e analisi dei dati di espressione genica basati su RNASeq dallo Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

Espressione genica in strati di Aleurone orzo bombardati con aumento della velocità di misura

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Produzione efficiente e identificazione di CRISPR/Cas9-generato Gene Ko nel sistema modello Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Un metodo di optogenetica per controllare e analizzare il pattern di espressione genica nelle interazioni della cellula--cellula

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Un ecdisone basati su recettori di interruttore di singolare Gene per deliberata espressione del Transgene con robustezza, reversibilità e permeabilità trascurabile

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

Esplorare il microbioma radice: estrazione dei dati di comunità batteriche dal suolo e la rizosfera radice Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Gene candidato test negli studi clinici di coorte con genotipizzazione multiplex e spettrometria di massa

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...