Name: immunofluorescent labeling of plant virus and insect vector proteins in hemipteran guts
Uploaded: 2021-05-14T15:00:00
Description: Watch this Scientific Journal Video about Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts at JoVE.com

这项工作得到了中国国家自然科学基金（31630058 X.W.和31772134 W.L.）的资助。

1. 非病毒昆虫饲养
<ol><li>从稻田收集WBPH，并在28°C的培养箱中用防虫网覆盖的1L玻璃烧杯中收集WBPH，16小时光照和8小时黑暗。由于SRBSDV不通过卵传播，因此新孵化的若虫不会产生病毒性。</li>
	<li>每周用毛笔轻轻地将烧杯中的昆虫刷入新鲜水稻幼苗的新烧杯中，直到WBPH若虫孵化。继续将这些孵化的非病毒若虫饲养到2或3龄。 注意：小心刷牙，以避免WBPH从烧杯中飞出或损坏它们。</li>
</ol><p class...

<ol>
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为了获得最佳结果，应考虑几个关键点。首先，病毒性昆虫在总种群中的高比例是必要的。虽然WBPH若虫和成虫对SRBSDV的最低AAP为5分17秒，但应允许昆虫以新鲜SRBSDV感染的水稻植物为食2天，以实现高达80%的采集效率。由于可以在80%的中肠18中检测到SRBSDV病毒粒子，因此我们在本协议中2 d AAP后2 d切除并标记了病毒性昆虫。其次，成虫和若虫的肠道与头部的唾液腺?...

大多数描述的植物病毒是由半翅目昆虫传播的，包括蚜虫、粉虱、叶蝉、飞虱和蓟马1，2。半翅目昆虫的刺吸口器刺穿植物组织以进食和分泌唾液，同时有效地传播病毒2。已经描述了植物病毒通过媒介昆虫的不同传播机制。这些包括非持久性、半持久性和持久性。持久性类型要么是非繁殖型，要么是繁殖型3，4，但对于这两种类型，传播的病毒必须在昆虫的整个身体中移动。在持续繁殖模式下，病毒最初感染并在昆虫肠道的上皮细胞中复制，然后传播到不同的组织中，最终进入唾液腺，然后从那里它们可以在昆虫进食期间通过唾液引入植物5，6。持续传播的病毒通过不同的器官并在其昆虫载体中复制，这需要病毒和载体成分在不同阶段的特定相互作用7，8。
病毒蛋白和昆虫蛋白必须相互作用，以促进病毒识别、感染、复制或传播的关键过程在媒介昆虫中9，10。虽然光学显微镜可用于观察昆虫的细胞结构，但它不能显示病毒粒子分布，病毒蛋白和昆虫蛋白的细胞定位或共定位，或昆虫组织和细胞的超微结构。免疫荧光标记首先由Coons等人通过标记特定的荧光素抗体在小鼠的吞噬细胞中进行，现在被广泛使用11。免疫荧光技术又称荧光抗体技术，是最早开发的免疫标记技术之一，是基于抗原与抗体之间的特异性结合反应11，12。已知抗体首先用荧光素标记，荧光素用作检测细胞或组织中相应抗原的探针13，14。荧光素标记的抗体与细胞或组织中的相应抗原结合后，探针在用激发波长照射并用荧光显微镜观察以定位抗原15时会发出明亮的荧光。
植物病毒的大多数媒介昆虫是半翅目。对植物病毒具有高传播效率的媒介昆虫种群密度较高，可导致病毒流行5。南方水稻黑纹矮病毒（SRBSDV，斐济病毒属，呼肠孤病毒科）是水稻最严重的病原菌之一，在东亚和东南亚的水稻种植区迅速蔓延，自2010年以来造成严重减产16，17。白背飞虱（WBPH，Sogatella furcifera Horváth）的成虫和若虫以持续繁殖的方式高效将SRBSDV传播给水稻。现场研究表明，SRBSDV诱发的水稻黑条纹侏儒病的暴发通常与WBPH的大规模长距离迁移同时发生，这是SRBSDV流行的关键因素7，8，18。囊泡相关膜蛋白7（VAMP7）是一种可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE），可通过囊泡融合介导物质的转运。VAMP7在体外与SRBSDV的外主要衣壳蛋白相互作用，这表明VAMP7可能与病毒传播密切相关16。
在这里介绍的方案中，我们从病毒性WBPH中切除肠道，作为标记中肠上皮细胞中SRBSDV病毒粒子和VAMP7的例子16。作为病毒的初始入侵部位，中肠上皮在病毒感染、复制和传播中起着至关重要的作用。首先，我们详细介绍了从若虫和WBPH成虫中切除肠道的步骤。其次，我们使用特定的荧光素标记抗体来标记肠道上皮细胞中的SRBSDV病毒粒子和VAMP7。然后，我们通过激光扫描共聚焦显微镜观察上皮细胞以及病毒粒子和VAMP7的细胞位置。结果表明，SRBSDV病毒粒子和VAMP7在中肠上皮细胞的细胞质中可共定位，提示VAMP7的特异性功能可能与中肠上皮细胞病毒粒子的传播有关。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3% Bull serum albumin (BSA)</td>
			<td>Coolaber</td>
			<td>SL1331</td>
			<td>Dilute antibodies</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Solarbio</td>
			<td>YA0771-18*18mm</td>
			<td>For slide making</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Beitja</td>
			<td>XTL-7045B1</td>
			<td>For insect dissection</td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss LSM880</td>
			<td>Observe fluorescence signal</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Solarbio</td>
			<td>ZBP-7105</td>
			<td>For slide making</td>
		</tr>
		<tr>
			<td>Mounting medium with 4&#39;6-diamidino-2-phenylindole (DAPI)</td>
			<td>Abcam</td>
			<td>AB104139</td>
			<td>Label cell necleus</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>For tissues fixation</td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>&#160;Invitrogen</td>
			<td>A22284</td>
			<td>Label actin of midgut epithiels</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Amresco</td>
			<td>0290C484</td>
			<td>For tissues permeation</td>
		</tr>
		<tr>
			<td>Tweezers (5-SA)</td>
			<td>AsOne</td>
			<td>6-7905-40</td>
			<td>For insect dissection</td>
		</tr>
	</tbody>
</table>

immunofluorescent labeling of plant virus and insect vector proteins in hemipteran guts

自然界中的大多数植物病毒都是通过半翅目昆虫从一种植物传播到另一种植物的。在病毒传播方面效率高的媒介昆虫的高种群密度在田间病毒流行中起着关键作用。研究病毒-昆虫媒介相互作用可以促进我们对病毒传播和流行病的理解，目的是设计控制植物病毒及其媒介昆虫的新策略。免疫荧光标记已被广泛用于分析病原体和宿主之间的相互作用，并用于白背飞虱（WBPH，Sogatella furcifera），它有效地传播南方水稻黑条纹侏儒病毒（SRBSDV，斐济病毒属，呼肠孤科），以定位中肠上皮细胞中的病毒粒子和昆虫蛋白。使用激光扫描共聚焦显微镜，我们研究了中肠上皮细胞的形态特征，昆虫蛋白的细胞定位以及病毒粒子和昆虫蛋白的共定位。该协议可用于研究昆虫中的病毒活动，昆虫蛋白质的功能以及病毒与媒介昆虫之间的相互作用。

图1说明了该协议中的所有步骤：昆虫饲养，病毒获取，肠道切除，免疫荧光标记和制作载玻片。
从成人中切除的WBPH内脏固定在4%（m / v）多聚甲醛中，用2%（v / v）Triton X-100透化，然后与Dylight 633鬼笔环肽10，18一起孵育。 图2 中的激光扫描共聚焦显微照片显示了用鬼笔环肽标记后切除的?...

Watch this Scientific Journal Video about Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts at JoVE.com

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

这种用于切除昆虫肠道中植物病毒蛋白和载体昆虫蛋白的免疫荧光标记的协议可用于研究病毒和载体昆虫之间的相互作用、昆虫蛋白功能和病毒传播的分子机制。

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

该协议可用于研究昆虫的各种运动，昆虫蛋白质的功能以及体内病毒与媒介昆虫之间的相互作用。这种方法高效且信息丰富。当用激光扫描共聚焦显微镜观察时，昆虫肠道和辅助细胞的结构清晰可见。演示该程序的将是来自我们实验室的张璐博士。首先，将玻璃烧杯中的非病毒性昆虫转移到新鲜的南方水稻黑条纹矮病毒SRBSDV上，受感染的水稻植物用防虫网覆盖，为期两天的病毒获取访问期。然后将昆虫收集在装有新鲜水稻幼苗的玻璃烧杯中。两天后，使用手动吸气器从玻璃烧杯中收集昆虫，以进行肠道解剖和切除。按照文本手稿中的说明准备 0.01 摩尔 PBS、4% 多聚甲醛和 2% Triton X-100。使用移液器将100微升PBS放在载玻片上，然后将载玻片放在光学显微镜的载物台上。使用手动吸引器从玻璃烧杯中收集感染SRBSDV的成虫，并将它们放在冰上的1.5毫升管中以麻痹昆虫。将瘫痪的成年人转移到载玻片上的100微升PBS中，腹部向上。用镊子夹住身体，然后用另一组镊子取下头部。用一组镊子夹住腹部两侧，用另一组夹住产卵器或尾巴的交配器官，然后小心地拉动一个腹部段的节间膜，慢慢暴露腹部的肠道。继续撕开膜，逐渐从腹部拉出完整的肠道。轻轻拉下连接到肠道末端的尾巴，以去除整个肠道。将切除的内脏放入 200 微升离心管中。在管中加入200微升PBS，并使用移液管彻底清洗内脏。使用移液器将100微升PBS放在载玻片上，然后将载玻片放在光学显微镜的载物台上。使用手动吸引器从玻璃烧杯中收集感染SRBSDV的若虫，并将它们放入放在冰上的1.5毫升管中以麻痹昆虫。将瘫痪的若虫转移到载玻片上的100微升缓冲液中，腹部朝上。拆下若虫的尾巴，然后夹住昆虫身体轻轻固定，用另一对夹住头部。轻轻地将头部从身体上拉开，同时仍然保持其与肠道的附着，使头部与身体分离，但肠道仍附着在胸部和腹部。在镊子仍然夹住身体的情况下，用另一对小心地移动头部并逐渐拉出肠道。将肠道从头部分离，以获得完整的肠道，而不会损坏身体。将切除的内脏放入管中。将PBS添加到管中，然后用移液管轻轻吸出释放溶液以彻底清洗内脏。按照文本手稿中的说明制备抗体和封片剂。将新鲜切除和PBS洗涤的WBPH肠放入室温下200微升离心管中的100微升4%多聚甲醛中。两小时后，用200微升PBS替换多聚甲醛溶液。10分钟后，取出PBS以消除任何多聚甲醛，并重复PBS洗涤步骤两次。两次PBS洗涤后，加入200微升非离子洗涤剂Triton X-100以在室温下透化样品。30 分钟后，取出 Triton X-100，用 PBS 洗涤三次 10 分钟，洗掉任何剩余的洗涤剂。用 50 微升公牛血清白蛋白将标记的抗体稀释 1 至 50。将稀释的抗体加入试管中，并将样品在 4 摄氏度下孵育过夜。在去除抗体后的早晨，用PBS洗涤三次10分钟，洗去剩余的抗体稀释剂。用50微升PBS稀释一微升DyLight 633-鬼笔环肽，并向管中加入50微升稀释的鬼笔环肽，在室温下孵育两小时。去除鬼笔环肽后，用PBS清洗三次10分钟，彻底洗掉剩余的鬼笔环肽。将一滴含有DAPI的封片剂放在显微镜载玻片上。将内脏转移到培养基上，轻轻地将盖玻片放在样品上，不要产生气泡。用鬼笔环肽标记后切除成人WBPH肠道的代表性激光扫描共聚焦显微照片显示前肠，中肠和后肠三个部分。其中，中肠是SRBSDV的初始感染部位。肠道的单层上皮细胞结构有助于研究昆虫蛋白的细胞定位以及病毒和昆虫蛋白的共定位。此处显示了带有 DyLight 488 标记的抗 VAMP-7 和带有 DyLight 549 标记的抗 SRBSDV 抗体的 SRBSDV 病毒粒子的切除 WBPH 内脏。VAMP-7和SRBSDV病毒粒子在细胞质中共定位，表明VAMP-7可能在体内病毒传播中发挥作用。透化的肠道在与荧光素偶联抗体孵育后应彻底清洁，以减少背景和非特异性结合。这是观察昆虫中病毒、其他病原体和昆虫蛋白定位的可靠方法。它为进一步研究病原体与昆虫之间的关系提供了基础。

Research

JoVE Journal

Biology

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

科研

生物学

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

ReAsH / FLASH标签和图像分析Tetracysteine​​传感器蛋白在细胞中

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

农杆菌介导的病毒诱导的基因沉默含量在棉花

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

生理录音和黄热病蚊子的味觉附属物RNA测序<em&gt;埃及伊蚊</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

细菌，昆虫细胞和植物系统：重组蛋白表达在不同的生物工厂的比较分析

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

注射重组狐尾花叶病毒和甘蔗花叶病毒传染性克隆直接农种玉米幼苗

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the ...

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

免疫荧光染色，用于 在果蝇 唾液腺中可视化异色素相关蛋白质

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila ...