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

写真についてミシェル月 (ヴァンダービルト大学) に感謝したいと思います。PA と SSLB にヴァンダービルト大学提供のサポートは、地区ガバナーエレクト ・ 1445197 によってサポートされます。

1. 植物培養
<ol><li>任意の商用ベンダーから種子を購入または分野で成熟した植物から収集します。 注: このプロトコルは、ほとんどの市販のトウワタ種 (例えば、トウワタ属 incarnata、 A. syriaca、 A. さんに感謝、 Gomphocarpus physocarpus) に適しています。冷たい成層をする必要がありますいくつかの種と種の製造者からの指示をチェックする必要があります。</li>
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<ol>
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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. 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. 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長いこと、ディアボルスA. neriiパターンと植物の防御、特に化学隔離18,37への抵抗のメカニズムに洞察力を提供できるが認識されています。ゲノム リソースの数は、 A. nerii10, A. neriiを使用して、モデルとして生態学的機能のゲノム研究の新しい機会を提供するため最近浮上しています。アブラムシ、植物文化の...

アブラムシは、世界中の多様な植物家族の植民地 hemimetabolous、小さな昆虫です。最も特にから不足している栄養素を供給する酵母や細菌の共生とその偏栄養共生と離散 polyphenisms 循環単為生殖を含む複雑なライフ サイクルのいくつかの機能の独特であります。sap の1工場の彼らの食事療法。ほとんどアブラムシはホスト植物の専門家、いくつか種がどちらか直接にある作物で、かなりの経済ダメージを与える重要な作物害虫や病原体やウイルスを介して彼らはベクトル2。最初アブラムシにおけるゲノム学 2010 年エンドウ アブラムシエンドウヒゲナガアブラムシ3、パブリケーション アブラムシの生物学の研究で重要なマイルス トーンをマークは、昆虫のアドレス指定についての遺伝子資源を提供されているため良いコントロールの戦略4につながる可能性のあるものを含む、草食性のライフ スタイルに適応します。それ以来、追加ゲノム リソース大豆アブラムシワタアブラムシ グリシン5注釈ゲノムと別の 3 アブラムシ種 (モモアカアブラムシの公に利用可能な全ゲノム リソースのパブリケーションと蓄積してきたcerasi (ブラック チェリー アブラムシ)、モモアカアブラムシ(桃ジャガイモ アブラムシ)した padi (鳥チェリー エンバク アブラムシ)6。貴重なデ novoトランスクリプトーム リソースが他のアブラムシの数も利用可能 (e.g.,Aphis gossypii (綿アブラムシ)7, Sitobion 褐(穀物アブラムシ)8, Cinarapinitabulaeformis (松アブラムシ)9、ワタアブラムシ nerii (トウワタ キョウチクトウ アブラムシ)10)。
アブラムシは植物11植物昆虫相互作用と生命の生態学の私達の理解に永続的な貢献をしたも。アブラムシが特に重要な貢献をした 1 つの領域は、宿主植物の化学生態学の私達の理解にあります。共同植物の防御、自分の選ぶも克服する植物の防御、およびいくつかの草食性昆虫表現多様な適応は利益12,13,14です。たとえば、トウワタ キョウチクトウ モモアカアブラムシ、ワタアブラムシ neriiは、明るい黄色、侵襲的なアブラムシ世界中の温帯、熱帯地域は、トウワタ家族 (キョウチクトウ科) 植物の定着です。キョウチクトウ科の植物は、乳状のラテックスとカチオン キャリア Na, K-atpase をバインドし、ジェネラ リスト草食動物15,に効果的な抑止力は、cardenolides として知られている強心配糖体などを含む、多様な化学防御を進化しています。16. トウワタ専門家エクスプレス、cardenolides に対する抵抗性のさまざまなモード、選択的または受動的蓄積や捕食を阻止するための手段としてまたは他の利点の17のための組織の cardenolides を変更あり。A. neriiはこの方法で cardenolides を隔離するメカニズムと機能上の利点のまま不明10,18です。
手で、ゲノム リソース、 A. neriiを提供します優れた実験モデル有毒な宿主植物との専門家の化学生態学的相互作用に関与する分子および遺伝のメカニズムの調査のため草食動物。A. nerii cardenolides19, その時以来の隔離に焦点を当てたの初期の研究のいくつか、 a. neriiの研究が広範な進化と生態学的な質問に洞察力を提供している注目に値する侵襲的な昆虫20と草食動物密度21のボトムアップとトップダウンの規則間の相互作用の遺伝的構造を含みます。A. neriiはこうして特に広範な昆虫植物相互作用の研究のための実験モデルとしての良い候補です。重要なA. neriiと任意の研究の成功に注意カルチャですアブラムシの植物のアブラムシが依存の文化を含む集団として高品質弔データの効率的な生成。私たちの目標は、両方を介してリーダーを指導することです。世代やメンテナンス、温室効果と研究所、DNA と RNA の抽出、マイクロ サテライト解析、 de novoトランスクリプトーム アセンブリおよび注釈、トランスクリプトームで植物とアブラムシの文化のための方法を以下に発現解析と qPCR 性遺伝子が発現。一方、これらのメソッドは、 A. neriiの書かれている、培養、抽出、および分析の一般的な方法をさまざまな種のアブラムシに拡張できます。

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		<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

アブラムシは宿主植物と昆虫の相互作用をめぐる質問に共生の進化と polyphenisms の開発に至る生物の質問の様々 な優れた実験的モデルです。アブラムシ数種の遺伝子資源があり、非モデル生物のゲノムを欠いているに延長されるトランスクリプトーム研究次世代シーケンシングの進化により、です。さらに、アブラムシの文化はフィールドから収集し、生態学的な遺伝学の間のギャップを埋めるための生物・分子の実験で使用する実験室で飼育できます。最後に、多くのアブラムシは無性再現遺伝子型の比較を可能にする永久的な単為のライフ サイクルでの優先ホスト植物研究所で維持できます。ワタアブラムシ nerii、トウワタ キョウチクトウ アブラムシは、生物分子の実験を使用して有毒な植物と昆虫の相互作用を研究するようなの 1 つのモデルを提供します。世代と温室効果と研究所、DNA および RNA の抽出、マイクロ サテライト解析、 de novoトランスクリプトーム アセンブリおよび注釈、トランスクリプトーム発現の植物とアブラムシの文化の維持の方法分析、および特異的発現遺伝子の qPCR 検証は概説し、ここで説明します。

植物の文化:種子は季節に応じて約 2 〜 4 週間の時間がかかります、大きく十分に鉢植え (図 1A)。再鉢植え苗は、アブラムシの文化 (図 1B) の最適なサイズに成長する別の 2 〜 4 週間かかります。
アブラムシ文化:大人A. nerii</e...

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

アブラムシワタアブラムシ nerii付いているバシクルモン家族 (Apocyanaceae) で強く擁護した植物の定着植物昆虫の相互作用を研究する多くの機会を提供しています。ここでは、我々 の文化と生成分子の解析植物とアブラムシの保守のためのプロトコルのシリーズの紹介とA. nerii用プロファイルー データ。

生物学的文化とワタアブラムシ neriiの遺伝子発現を測定を維持する: 植物・昆虫間の相互作用の非モデル系

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

ヒト初代細胞培養物における概日時計遺伝子発現やホルモン分泌のパラレル測定

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

遺伝学

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

過敏性腸症候群におけるmiRNA循環の摂動は多重化ハイスループット遺伝子発現プラットフォームを使用して検出します

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

内部標準でFACSおよび定量PCRを用いた単一細胞遺伝子発現プロファイリング

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

サンプル準備およびゼブラフィッシュから RNASeq による遺伝子発現データの解析

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

スループットを向上させると衝撃されたオオムギ糊粉層における遺伝子発現を測定

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

効率的な生産とモデル システムの動脈分布に CRISPR 識別/Cas9 生成遺伝子のノックアウト

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

制御、細胞間相互作用における遺伝子発現パターンを分析して光遺伝学的方法

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

脱皮ホルモン受容体を介した特異遺伝子スイッチ意図的な堅牢性、可逆性、ごくわずかな水漏れしたためと遺伝子発現の

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

探索ルート マイクロバイ: 土、根、およびルート Endosphere から細菌群集のデータを抽出

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

多重遺伝子型及び質量分析法と臨床コホート研究でテスト候補遺伝子

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