Name: fluorescence in situ hybridizations (fish) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues
Uploaded: 2014-02-24T15:30:00
Description: Watch this Scientific Journal Video about Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues at JoVE.com

研究在加尼姆实验室是由研究经费不支持。 908-42.12/2006从德国和以色列基金会（GIF），不授予。 IS-4062-07从美国和以色列两个民族组成的农业研究和发展基金（BARD），以及研究经费没有。 884/07由以色列科学基金会（ISF），以MG

1。一般白粉虱，植物和病毒的准 ​​备FISH分析<ol><li>对棉花幼苗（ 陆地棉品种。阿卡拉）后B和Q型烟粉虱和里面的25±2℃，相对湿度60％标准条件下防虫笼和增长间保持，和一个14小时光照/ 10小时黑暗的光照。 </li><li>进行PCR与利用共生细菌特异性引物如前所述15-19内共生菌感染测试。 </li><li>从商业幼儿园或植物番茄种子购买番茄苗（ 期Zn2 +茄品种。牛排）。 </li><li>...

<ol>
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Biol. 140, 215-231 (2002).</li><li>Gottlieb, Y., 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+transmission+efficiency+of+Tomato+yellow+leaf+curl+virusby+the+whitefly+Bemisiatabaciis+correlated+with+the+presence+of+aspecificsymbiotic+bacterium+species.">The transmission efficiency of Tomato yellow leaf curl virusby the whitefly Bemisiatabaciis correlated with the presence of aspecificsymbiotic bacterium species.</a> J. Virol. 84, 9310-9317 (2010).</li><li>Brumin, M., Levy, M., Ghanim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transovarial+transmission+of+Rickettsia+spp.+and+organ-specific+infection+of+the+whitefly+Bemisia+tabaci.">Transovarial transmission of Rickettsia spp. and organ-specific infection of the whitefly Bemisia tabaci.</a> Appl. Environ. 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Methods. 159, 311-314 (2009).</li><li>Gotz, 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=Implication+of+Bemisia+tabaci+heat+shock+protein+70+in+Begomovirus-whitefly+interactions.">Implication of Bemisia tabaci heat shock protein 70 in Begomovirus-whitefly interactions.</a> J. Virol. 86, 13241-13252 (2012).</li><li>Nirgianaki, A., 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=Wolbachia+infections+of+the+whitefly+Bemisia+tabaci.">Wolbachia infections of the whitefly Bemisia tabaci.</a> Curr. Microbiol. 47, 93-101 (2003).</li><li>Baumann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+bacteriocyte-associated+endosymbionts+of+plant+sap-sucking+insects.">Biology of bacteriocyte-associated endosymbionts of plant sap-sucking insects.</a> Ann. Rev. Microbiol. 59, 155-189 (2005).</li><li>Gottlieb, Y., 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=Identification+and+localization+of+a+Rickettsia+sp.+in+Bemisia+tabaci+(Homoptera)+Aleyrodidae).">Identification and localization of a Rickettsia sp. in Bemisia tabaci (Homoptera) Aleyrodidae).</a> App. Environ. Microbiol. 72, 3646-3652 (2006).</li><li>Li, Z. X., Lin, H. Z., Guo, X. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+Wolbachia+infection+in+Bemisia+tabaci.">Prevalence of Wolbachia infection in Bemisia tabaci.</a> Curr. Microbiol. 54, 467-471 (2007).</li><li>Skaljac, M., Zanic, K., Hrncic, S., Radonjic, S., Perovic, T., Ghanim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+localization+of+bacterial+symbionts+in+three+whitefly+species+(Hemiptera:+Aleyrodidae)+from+the+east+coast+of+the+Adriatic.">Diversity and localization of bacterial symbionts in three whitefly species (Hemiptera: Aleyrodidae) from the east coast of the Adriatic.</a> Bull. Entomol. Res. 103, 48-59 (2013).</li></ol>

此处描述的植物病毒在其宿主植物和昆虫载体的本地化和共生细菌在其特定粉虱主机协议，可适于其它病毒在植物中，甚至在动物组织中的定位。此外，该协议可以用于本地化共生和致病性细菌和在植物和动物的系统的其他微生物。所描述的方法依赖于杂交的一个短的，荧光标记的寡核苷酸DNA探针与靶DNA或RNA分子在细胞之间简单的概念。的结果是一个特定的杂交，以最小的背景，并使用荧光或共?...

当研究其感染的植物宿主植物病毒和其他病原体之间的相互作用，它以可视化的病原体和它们各自的核酸在原位 ，而不论它们是否会造成对寄主的负面影响是非常重要的。内部和之间的植物细胞研究病原体的运动时，这是最重要的。 在病原体的基因产物原位定位是补充其他方法为研究致病过程中的重要一步。许多植物病原菌，尤其是病毒，是由昆虫传播的，有其复杂的向量和亲密的互动。这些病毒在它们的矢量定位，是研究的路径进行传输重要的，并且该载体内的可能的相互作用位点。某些昆虫向量植物病毒的传播是通过驻留这些昆虫内共生菌资助。为了更好地研究这些植物病毒B中的传输Ÿ他们的向量，它也是必不可少的，以可视化的一般内共生菌，以及参与病毒传播，尤其如此。因此，病毒和内共生细菌的共存需要调查在其昆虫宿主这些生物之间的可能关系。除了病毒传播，内共生细菌的影响昆虫媒介生物学等几个方面，这些细菌内昆虫因而空间定位较高的兴趣和重视的。 番茄黄化曲叶病毒 （TYLCV）（菜豆金色花叶病毒，Geminiviridae）是栽培番茄的1-4全世界最重要的病毒性疾病复杂。 TYLCV是一种韧皮部限制病毒和专门向量由粉虱 5,6。模型描述其向量粉虱双生的易位已经提出了6-9。两个具体的障碍正在积极越过DURing这个循环式传输：中肠/血淋巴和血淋巴/唾液腺障碍。这个传输过程中被假设由未知的受体能识别病毒衣壳来介导的。 TYLCV被认为是穿越B。粉虱肠6,10，并通过主唾液腺6吸收之前它被注入到植物。在粉虱血淋巴，TYLCV与由昆虫次级内共生菌Hamiltonella产生的GroEL的蛋白相互作用。这种相互作用保证了在血淋巴TYLCV的安全传输，并且由昆虫免疫系统11。Hamiltonella和Portiera，初级共生细菌免受攻击保护它粉虱的，都装在bacteriocytes，血淋巴和房子内共生菌11。B.发现昆虫细胞粉虱怀有额外的内共生菌，包括立克次氏体，Arsenophonus，沃尔巴克氏体和弗里奇EA，它可以内部或外部的bacteriocytes是局部的，并且对昆虫的生物学12不同的影响。 若干报告已经尝试通过使用费时和昂贵的协议，如透射电子显微镜（TEM），抗体和RNA 原位上微观厚部10,13来研究TYLCV的植物和粉虱定位，一项最近的研究中所述的定位通过使用一个简单的协议，14他们的工厂和矢量主机内的植物病毒。在这里，我们描述了一个简单的协议TYLCV在B中的定位烟粉虱解剖肠和唾液腺，并在部分从TYLCV感染的植物编制。我们进一步描述Portiera，B的主要共生细菌的本地化烟粉虱，其二次内共生菌Hamitonella，立克次氏体和Arsenophonus，该协议是基于使用短的DNA探针，即荧光标记在其5&#39;末端，并特异性杂交​​中病毒或细菌的基因序列互补的序列。样品处理是比较容易和得到的信号是高度特异性的。所描述的协议可以用来在他们的植物，动物和昆虫宿主本地化的病毒，细菌和其它病原体，并能进一步用于在任何给定的组织定位的mRNA。

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			<td style="width:212px;">Molecular Biology Grade</td>
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			<td height="21" style="height:21px;width:219px;">Glacial Acetic Acid</td>
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			<td>Ted Pella Inc.</td>
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fluorescence in situ hybridizations (fish) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues

荧光原位杂交（FISH）是考虑到了各种常用的用于可视化基因转录物在真核细胞中，并且可以被进一步修饰以可视化单元中的其他组分，如感染病毒和细菌的方法的名称。空间定位和感染过程中病毒和细菌的可视化的是，在应对不同的刺激补充表达谱实验，如芯片和RNAseq一个必不可少的步骤。了解时空感染这些药物补充生物实验旨在了解与细胞成分及其相互关系。几种技术用于可视化病毒和细菌等的报告基因系统或免疫组织化学的方法是耗时的，并且一些被限制与模型生物体的工作，并涉及复杂的方法。 FISH靶向的RNA或DNA的物种的细胞是相对容易和快速的我的ThOD用于研究基因的时空定位和诊断的目的。此法可强健和相对容易实现，当协议使用短的杂交，商业购买的探头，它并不昂贵。这是特别可靠时的样品制备，固定，杂交和显微可视化不涉及复杂的步骤。在这里，我们描述了一个协议，用于细菌和病毒在昆虫和植物组织定位。该方法是基于简单的制剂，固定，和昆虫整个支架并解剖器官或手工制作的植物部分杂交，用20个碱基对缀合的荧光染料在其5&#39;或3&#39;短的DNA探针末端。这个协议已经被成功地应用于许多昆虫和植物组织，可用于分析在细胞中的mRNA或其它RNA或DNA的物种中的表达。

研究了在该手稿的系统示于图1，并包括一个受感染的植株用TYLCV，成年人和粉虱B的若虫粉虱和白粉虱示出了用于在昆虫TYLCV易位路径的内部解剖结构， 图2示出了双FISH在成年烟粉虱的初级共生Portiera和次级共生Arsenophonus。 图3显示了双FISH为Portiera和Hamiltonella，并图4显示了双鱼的Portiera和立克次体，...

Watch this Scientific Journal Video about Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues at JoVE.com

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

荧光<em&gt;原位</em&gt;杂交（FISH）技术对病毒和植物和昆虫组织内共生细菌的本土化

我们在这里描述原位杂交简单的荧光（FISH）方法对病毒和细菌的昆虫和植物组织的定位。这个协议可以延长mRNA的整装可视化和显微切片。

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

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Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

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Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

协议侦办主机组织分布，传播模式和效果上浓核病毒的主机健身的棉铃虫

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

活细胞荧光显微镜研究细菌的亚细胞蛋白定位和细胞形态变化

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

免疫荧光和定量PCR对白蝇组织中白蝇病毒的定位和定量

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...