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

Vi vil gjerne takke Michelle Moon (Vanderbilt University) for hjelp med fotografering. Vanderbilt University gitt støtte til PA og SSLB støttes av DGE-1445197.

1. anlegget kulturer
<ol><li>Kjøpe frø fra en kommersiell leverandør eller samle fra modne planter i feltet. Merk: Denne protokollen er egnet for de fleste kommersielt tilgjengelige milkweed arter (f.eks Asclepias incarnata, A. syriaca, A. curassavica, Gomphocarpus physocarpus). Noen frø må være kaldt-lagdelt, og instruksjoner fra frø leverandør bør sjekkes.</li>
	<li>
Plante frø i en fin germinating jord (60-70% fine torv moss, perlitt, vermikulitt, kalkstein).<...

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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. 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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. 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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. 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Det har lenge vært anerkjent som aposematic A. nerii kan gi innsikt i mønstre og mekanismer for resistens mot anlegget forsvar og spesielt kjemiske lagring18,37. En rekke genomisk ressurser har nylig kommet i A. nerii10, gir nye muligheter for økologiske og funksjonelle genomisk studier som bruker A. nerii som modell. Vi skissere grunnleggende protokoller i bladlus og plant kultur og molekylære/genomisk tekni...

Bladlus er liten, hemimetabolous insekter som koloniser på ulike anlegg familier over hele verden. De er karakteristiske for flere funksjoner, mest spesielt komplekse livssyklusen med syklisk Partenogenese og diskret polyphenisms, og deres forplikte ernæringsmessige symbioses med bakteriell eller gjær endosymbionts som leverer næringsstoffer mangler kostholdet anlegget sap1. Mens de fleste bladlus vert anlegg spesialister, noen generalist arter er viktig avling skadedyr, påførte betydelig økonomisk skade på avlinger enten direkte eller via patogener og virus vektor de2. Utgivelsen av det første bladlus genomet i 2010, ert bladlus Acyrthosiphon erter3, markerte en viktig milepæl i studiet av bladlus biologi fordi det ga genomisk ressursene for adressering spørsmål om insekt tilpasninger til planteetende livsstil, inkludert de som kan føre til en bedre kontroll strategier4. Siden den gangen akkumulert genomisk tilleggsressurser med utgivelsen av en annotert genomet for soyabønner bladlus Aphis glycines5og offentlig tilgjengelig hele genomet ressurser for en tre-bladlus Art (Myzus cerasi (black cherry bladlus), Myzus persicae (fersken-potet bladlus), Rhopalosiphum padi (Hegg-katt bladlus)6. Verdifulle de novo transcriptomic ressurser er tilgjengelige i tillegg til en rekke andre bladlus arter (e.g.,Aphis gossypii (bomull bladlus) Sitobion avenae (korn bladlus)8, Cinara pinitabulaeformis (furu bladlus)9, Aphis nerii (milkweed-oleander bladlus)10).
Bladlus har også gjort varig bidrag til vår forståelse av plante-insekt interaksjoner og økologi av livet på planter11. Et område der bladlus har gjort spesielt viktig bidrag er i vår forståelse av kjemiske økologi av verten anlegget interaksjoner. Planteetende insekter uttrykke ulike tilpasninger fordel for overvinne plante forsvar, og noen enda co-opt anlegget forsvar for sin egen12,13,14. Milkweed-oleander bladlus, Aphis nerii, er for eksempel en lys gul, invasiv bladlus i tempererte og tropiske regioner over hele verden som colonizes på planter i milkweed familien (Apocynaceae). Planter i familien Apocynaceae utviklet seg ulike kjemiske forsvar, inkludert melkeaktig latex og cardiac glykosider kjent som cardenolides, som binder kasjon transportøren Na, K-ATPase og effektiv deterrents å generalist planteetere15, 16. milkweed spesialister express forskjellige moduser av motstand mot cardenolides og noen selektivt eller passivt samle eller endre cardenolides av vev å avskrekke predasjon eller andre fordeler17. A. nerii er tenkt å beslaglegge cardenolides på denne måten, selv om de mekanismer og funksjonelle fordelene fortsatt uklart10,18.
Gitt genomisk ressursene for hånden, gir A. nerii en utmerket eksperimentell modell for studiet av molekylære og genetiske mekanismer involvert i kjemoterapi-økologiske samspillet mellom giftige vert planter og deres spesialist planteetere. Det er verdt å merke seg at mens noen av de tidligste studiene av A. nerii fokusert på lagring cardenolides19, siden den gangen har studier av A. nerii har gitt innsikt i et bredt spekter av evolusjonære og økologiske spørsmål, inkludert den genetiske strukturen av invasiv insekter20 og samspillet mellom opp- og topp-ned regulering på herbivore tetthet21. A. nerii er således en god kandidat som en eksperimentell modell for en spesielt lang rekke studier av insekt-plant interaksjoner. Kritisk for at enhver studie med A. nerii er forsiktig kultur bladlus befolkninger, som inkluderer kultur planter som bladlus avhenger, samt en effektiv generasjon av høy kvalitet - omic data. Vårt mål er å lede leseren gjennom både. Skissert nedenfor er metoder for generasjon og vedlikehold av anlegg og bladlus kulturer i drivhus og laboratorium, DNA og RNA utdrag, mikrosatellittmarkør analyse, de novo transcriptome montering og merknaden, transcriptome differensial uttrykk analyse og qPCR verifikasjon av ulikt uttrykt gener. Mens disse metodene er skrevet for A. nerii, generell dyrking, utvinning, og analyseteknologi kan utvide til en rekke bladlus arter.

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

Bladlus er utmerket eksperimentelle modeller for en rekke biologiske spørsmål fra utviklingen av symbioses og utvikling av polyphenisms til spørsmål omkring insekt interaksjon med deres Salix. Genomic ressurser er tilgjengelige for flere bladlus arter, og med fremskritt innen neste generasjons sekvensering, transcriptomic studier utvides til ikke-modellen organismer som mangler genomer. Bladlus kulturer kan videre, samlet fra feltet og oppdratt i laboratoriet for bruk i organismebiologi og molekylære eksperimenter for å bygge bro mellom økologiske og genetiske studier. Sist, mange bladlus kan opprettholdes i laboratoriet på foretrukne vert plantene i evigvarende, parthenogenic sykluser slik at sammenligninger av asexually gjengivelse genotyper. Aphis nerii, milkweed-oleander bladlus, gir en slik modell for å studere insekt interaksjon med giftige planter ved hjelp av både organismebiologi og molekylære eksperimenter. Metoder for generasjon og vedlikehold av anlegg og bladlus kulturer i drivhus og laboratory, DNA og RNA utdrag, mikrosatellittmarkør analyse, de novo transcriptome montering og merknaden, transcriptome differensial uttrykket analyse og qPCR verifikasjon av ulikt uttrykt gener er beskrevet og diskutert her.

Plante kulturer: Frø vil ta omtrent to til fire uker, avhengig av sesongen, for å vokse stort nok til å være re-puttet (figur 1A). Re-Potted planter vil ta ytterligere to til fire uker å vokse til en optimal størrelse for bladlus kulturer (figur 1B).
Bladlus kulturer: Voksen A. nerii kjen...

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

Bladlus Aphis nerii colonizes på svært forsvarte planter i familien dogbane (Apocyanaceae) og gir mange muligheter til å studere plante-insekt interaksjoner. Her presenterer vi en rekke protokoller for vedlikehold av anlegg og bladlus kulturer, og generasjon og analyse av molekylære og - omic data for A. nerii.

Opprettholde biologisk kulturer og måle byggkorn under Aphis nerii: en ikke-modellsystem for plante-insekt interaksjoner

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.

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Genetics

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

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