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

Gostaríamos de agradecer a Michelle Moon (Vanderbilt University) para obter assistência com a fotografia. Universidade de Vanderbilt fornecido suporte para PA e SSLB é suportada pela DGE-1445197.

1. planta culturas
<ol><li>Comprar sementes de qualquer fornecedor comercial ou coletar de plantas maduras no campo. Nota: Este protocolo é apropriado para a maioria das espécies de serralha comercialmente disponíveis (por exemplo, Asclepias incarnata, syriaca a., a. curassavica, Gomphocarpus physocarpus). Algumas sementes podem precisar ser frio-estratificado, e instruções do fornecedor de sementes que devem ser verificadas.</li>
	<li>
Plantar sementes em um solo fin...

<ol>
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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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Há muito tempo foi reconhecido que o aposemático a. nerii pode fornecer insights sobre os padrões e mecanismos de resistência às defesas vegetais e sequestro particularmente química18,37. Um número de recursos genômicos surgiram recentemente para a. nerii10, oferecendo novas oportunidades para estudos de genômicas funcionais e ecológicas que usam a. nerii como um modelo. Nós esboçamos protocolos bási...

Pulgões são insetos pequenos, hemimetabólicos que colonizam em famílias de diversas plantas em todo o mundo. Eles são distintos para vários recursos, mais notavelmente seus ciclos de vida complexos envolvendo partenogênese cíclica e polyphenisms discreta e sua simbiose nutricional obrigatórios com endossimbiontes bacterianos ou levedura que fornecem nutrientes faltando sua dieta da seiva de planta1. Enquanto a maioria dos pulgões são especialistas em planta de acolhimento, algumas espécies generalistas são pragas de cultura importante, infligindo danos económicos consideráveis nas culturas ou diretamente ou por meio dos agentes patogénicos e vírus eles vetor2. A publicação do primeiro genoma de pulgão em 2010, o pulgão ervilha pisum piolho3, marcado um marco importante no estudo da biologia de afídio porque forneceu os recursos genômicos para abordar questões sobre do inseto adaptações para os herbívoros estilos de vida, incluindo aqueles que possam levar a um melhor controle de estratégias4. Desde aquela época, recursos adicionais de genômicos acumularam-se com a publicação de um genoma anotado para o pulgão de soja glycines Aphis5e recursos do genoma inteiro publicamente disponíveis para outro afídio-três espécies (Myzus Cerasi (cereja preta afídio), Myzus persicae (pulgão da batata-pêssego), Rhopalosiphum padi (pulgão Azereiro-aveia)6. Valioso de novo transcriptomic recursos estão disponíveis, bem como para um número de outras espécies de pulgão (e.g.,Aphis gossypii (pulgão do algodão)7, Sitobion avenae (afídio grão)8, Cinara pinitabulaeformis (afídio pinho)9, Aphis nerii (pulgão da serralha-oleandro)10).
Pulgões também fizeram contribuições duradouras para nossa compreensão de interações inseto-planta e a ecologia da vida em plantas11. Uma área onde os pulgões fizeram contribuições particularmente importantes é em nossa compreensão da ecologia química nas interações planta do anfitrião. Expressa diversas adaptações de insetos herbívoros para superar as defesas da planta e alguns até mesmo cooptam defesas vegetais para seu próprio beneficiam12,13,14. Por exemplo, o pulgão da serralha-Oleandro, Aphis nerii, é um brilhante amarelo, invasivo pulgão encontrado em regiões temperadas e tropicais do mundo que coloniza em plantas da família serralha (Apocynaceae). Plantas da família Apocynaceae evoluíram diversas defesas químicas, incluindo látex leitoso e glicosídeos cardíacos, conhecidos como cardenolides, que ligam o transportador de cátion Na, K-ATPase e eficaz dissuasiva para generalista herbívoros15, 16. especialistas milkweed expressam vários modos de resistência ao cardenolides, e alguns seletivamente ou passivamente se acumulam ou modificar cardenolides em seus tecidos, como um meio de impedir a predação ou para outros benefícios de17. A. nerii é pensado para sequestrar cardenolides desta forma, embora os mecanismos e benefícios funcionais permanecem pouco claras10,18.
Tendo em conta os recursos genômicos em mãos, a. nerii fornece um excelente modelo experimental para o estudo dos mecanismos moleculares e genéticos envolvidos nas interações quimio-ecológicas entre plantas hospedeiras tóxicos e seu especialista herbívoros. É interessante notar que, enquanto alguns dos primeiros estudos de a. nerii focada em sequestro de cardenolides19, desde aquela época, estudos da . nerii forneceram introspecções em um amplo conjunto de questões evolutivas e ecológicas, incluindo a estrutura genética de insetos invasiva20 e a interação entre ascendente e descendente de regulamento sobre o herbívoro densidade21. A. nerii , portanto, é um bom candidato como modelo experimental para um especialmente amplo conjunto de estudos das interações inseto-planta. Crítico para o sucesso de qualquer estudo com a. nerii é a cultura cuidadosa de afídio populações, que inclui a cultura de plantas de que dependem os pulgões, assim como uma geração eficiente de dados de alta qualidade - omic. Nosso objetivo é orientar o leitor através de ambos. Descritos abaixo são os métodos para a geração e a manutenção das culturas vegetais e pulgão na estufa e laboratório, DNA e RNA extrações, análise de microssatélites, de novo transcriptome montagem e anotação, transcriptoma análise da expressão diferencial e qPCR verificação de diferencialmente expressos genes. Enquanto esses métodos são escritos pela . nerii, os métodos de cultivo, extração e análise gerais podem estender a uma variedade de espécies de pulgão.

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

Pulgões são excelentes modelos experimentais para uma variedade de questões biológicas, desde a evolução da simbiose e o desenvolvimento de polyphenisms a questões em torno as interações do inseto com suas plantas hospedeiras. Genômica recursos estão disponíveis para várias espécies de afídeos, e com os avanços no próxima geração de sequenciamento, transcriptomic estudos estão sendo estendidos para os organismos que não possuem genomas não-modelo. Além disso, culturas de pulgão podem ser coletadas do campo e criadas em laboratório para uso em experimentos de organismos e moleculares para colmatar o fosso entre os estudos ecológicos e genéticos. Por último, muitos pulgões podem ser mantidos no laboratório em suas plantas hospedeiras preferenciais perpétuos, parthenogenic ciclos de vida, permitindo comparações de genótipos assexuadamente. Aphis nerii, o pulgão da serralha-Oleandro, fornece um modelo deste tipo para estudar interações insetos com plantas tóxicas, usando as experiências dos e moleculares. Métodos para a geração e manutenção das culturas vegetais e pulgão na estufa e laboratório, extrações de DNA e RNA, análise de microssatélites, novo de transcriptoma montagem e anotação, transcriptoma expressão diferencial análise e verificação de qPCR de genes diferencialmente expressos são descritos e discutidos aqui.

Plantar culturas: Sementes levará cerca de duas a quatro semanas, dependendo da época, para que cresça o suficiente para ser re-envasadas (Figura 1A). Re-potted mudas levará outro duas a quatro semanas para crescer a um tamanho ideal para culturas de pulgão (Figura 1B).
Culturas de afídio: Adultos...

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

O pulgão Aphis nerii coloniza altamente defendido das plantas pertencente à família (Apocyanaceae) e oferece inúmeras oportunidades para estudar interações inseto-planta. Aqui, apresentamos uma série de protocolos para a manutenção da planta e pulgão, culturas e a geração e análise molecular e - omic dados pela . nerii.

Manutenção de culturas biológicas e medição de expressão gênica em Aphis nerii: um sistema Non-modelo de interações inseto-planta

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

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