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

我们要感谢米歇尔. 月亮 (范德比尔特大学) 为摄影提供帮助。范德比尔特大学向 PA 和 SSLB 提供支持, DGE-1445197 支持。

1. 植物文化
<ol><li>从任何商业供应商购买种子或从该领域的成熟植物中收集。 注: 本协议适用于大多数商业上可用的马利筋物种 (如马利 incarnata、 syriaca、 curassavica、 Gomphocarpus physocarpus)。有些种子可能需要冷分层, 并检查种子供应商的指示。</li>
	<li>
植物种子在一个美好的发芽土壤 (60–70% 细泥煤青苔, 珍珠岩, 蛭石, 石灰石)。
	<ol>
<li>用发芽混合土填充?...

<ol>
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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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长期以来, 人们已经认识到, aposematic A. nerii可以提供对抵抗植物防御的模式和机制的洞察力, 特别是化学封存18,37。一些基因组资源最近出现在. nerii10, 为生态和功能基因组研究提供了新的机会, 利用nerii作为模型。我们概述了蚜虫和植物培养的基本协议, 以及分子/基因组技术, 假设未来这一物种的工作可能涉及利用基...

蚜虫是小型的, hemimetabolous 昆虫, 在世界各地的不同的植物家庭殖民。它们是独特的几个特点, 最明显的是他们复杂的生命周期涉及周期性的孤雌和离散 polyphenisms, 和他们的义务营养共生与细菌或酵母 endosymbionts 提供营养缺乏从他们的饮食植物汁液1。虽然大多数蚜虫都是寄主植物专家, 但一些通才物种是重要的农作物害虫, 直接或通过其载体2的病原体和病毒对农作物造成相当大的经济损失。2010年的第一个蚜虫基因组的出版, 豌豆蚜虫Acyrthosiphon 豌豆3, 标志着蚜虫生物学研究的一个重要里程碑, 因为它提供了基因组资源, 以解决有关昆虫的问题对草食生活方式的适应, 包括那些可能导致更好的控制策略4。自那时以来, 额外的基因组资源已经积累了为大豆蚜虫蚜线虫5的注明基因组, 和公开提供的全基因组资源为另三-蚜虫种类 (桃cerasi (黑樱桃蚜虫),桃蚜(桃马铃薯蚜虫),蚜配弟(鸟樱桃燕麦蚜虫)6。有价值的 transcriptomic 资源, 以及其他一些蚜虫种 (例如,棉蚜 (棉花蚜虫)7,麦管蚜管蚜(谷物蚜虫)8, Cinarapinitabulaeformis (松蚜)9,蚜虫 nerii (马利筋-夹竹桃蚜虫)10)。
蚜虫也为我们对植物-昆虫相互作用和植物生命生态学的理解提供了持久的贡献11。蚜虫作出特别重要贡献的一个领域是我们对寄主植物相互作用的化学生态学的理解。草食昆虫为克服植物防御能力提供了多样的适应, 一些甚至为自己的利益选择了植物防御12,13,14。例如, 马利筋-夹竹桃蚜虫, nerii, 是一个明亮的黄色, 侵入性蚜虫发现在全球温带和热带地区, 殖民植物在马利筋家庭 (夹竹桃科)。夹竹桃科家族中的植物进化出多样的化学防御, 包括乳白色乳胶和心苷 cardenolides, 它结合了阳离子载体 Na, K atp 酶, 是对通才草食动物15的有效威慑,16. 马利筋专家表达了各种抵抗 cardenolides 的方式, 有些人有选择地或被动地积累或修改其组织中的 cardenolides, 以此来阻止捕食或其他好处17。nerii被认为以这种方式封存 cardenolides, 虽然机制和功能效益仍然不清楚10,18。
鉴于目前的基因组资源, nerii提供了一个优秀的实验模型, 用于研究有毒寄主植物与其专门草食动物之间的化学-生态相互作用的分子和遗传机制。值得注意的是, 虽然一些早期的研究nerii集中于 cardenolides19的封存, 但从那时起, 对一. nerii的研究提供了对一系列进化和生态学问题的洞察力,包括入侵昆虫的遗传结构20以及自底向上和自上而下调节对草食动物密度的相互作用21。因此, nerii是一个很好的候选者, 作为实验模型, 对昆虫-植物相互作用进行了特别广泛的研究。对任何与nerii的研究的成功至关重要的是蚜虫群体的细致文化, 其中包括蚜虫赖以生存的植物的文化, 以及高效生成高质量涨价可能数据。我们的目标是通过两者来引导读者。下面概述了在温室和实验室, DNA 和 RNA 提取, 微卫星分析, 转录组装和注释的产生和维持植物和蚜虫文化的方法转录差异表达分析和差异表达基因的 qPCR 验证。虽然这些方法是为. nerii编写的, 但一般的培养、提取和分析方法可以扩展到各种蚜虫种类。

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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

蚜虫是一个优秀的实验模型的各种生物学问题, 从进化的共生和发展 polyphenisms 的问题周围昆虫与他们的寄主植物的相互作用。基因组资源可用于几种蚜虫物种, 随着下一代测序的进展, transcriptomic 研究正在扩展到缺乏基因组的非模型生物。此外, 蚜虫的培养可以从田间收集, 并在实验室饲养, 用于生物体和分子实验, 以弥合生态和遗传学研究之间的差距。最后, 许多蚜虫可以保持在实验室对他们的首选寄主植物在永久, 孤雌生殖生命周期允许比较无性繁殖基因型。蚜虫nerii, 马利筋-夹竹桃蚜, 提供一个这样的模型, 研究昆虫与有毒植物的相互作用, 利用生物体和分子实验。温室植物和蚜虫培养方法及实验室、DNA 和 RNA 提取、微卫星分析、转录组装和注释、转录差异表达本文对差异表达基因的分析和 qPCR 验证进行了概述和讨论。

植物文化:种子将需要大约四周, 取决于季节, 增长足够大, 以重新盆栽 (图 1)。再盆栽苗将需要另外四周的时间才能长成蚜虫培养的最佳大小 (图 1B)。
蚜虫文化:成人A. nerii的区别是一些黑暗的马尾, 可能是无翅 (apterous,<strong cla...

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

蚜虫蚜虫nerii殖民在罗布麻家族 (Apocyanaceae) 高度防卫的植物上, 为研究植物-昆虫相互作用提供了许多机会。在这里, 我们提出了一系列维护植物和蚜虫文化的协议, 以及对nerii的分子和涨价可能数据的生成和分析。

nerii 蚜生物培养和基因表达的维持--植物-昆虫相互作用的非模型系统

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

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

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

科研

遗传学

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

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

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

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

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

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

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

提高吞吐量的轰击大麦糊粉层层中基因表达的测定

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

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

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

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 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

一种基于昆虫蜕皮激素受体的奇异基因开关, 用于对转基因进行有鲁棒性、可逆性和可忽略的泄漏的刻意表达

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

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

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

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

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