Name: detecting virus and salivary proteins of a leafhopper vector in the plant host
Uploaded: 2021-09-14T14:00:00
Description: Watch this Scientific Journal Video about Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host at JoVE.com

这项工作得到了中国国家自然科学基金（31772124和31972239）和福建农林大学（KSYLX014）的资助。

非病毒性成虫叶蝉在中国福建农林大学媒介传播病毒研究中心繁殖。
1. 非病毒昆虫饲养
<ol><li>将成虫饲养在40厘米x 35厘米x 20厘米（长x宽x高）的立方体笼中。保持笼子的一侧盖上防虫网，以便通风。<ol><li>将带有叶蝉的笼子保存在包含内置湿度控制器的培养箱中，在26°C下，相对湿度为60-75%，光周期为16小时光照和8小时黑暗。</li>
	</ol></li>
	<li>使用吸气器轻轻地将所有成虫从...

<ol>
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Series B, Biological Sciences. 374 (1767), 20180313 (2019).</li><li>Omura, T., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+outer+capsid+proteins+in+transmission+of+phytoreovirus+by+insect+vectors.">Role of outer capsid proteins in transmission of phytoreovirus by insect vectors.</a> Advances in Virus Research. 54, 15-43 (1999).</li><li>Chen, Q., Liu, Y., Long, Z., Yang, H., Wei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+release+threshold+in+the+salivary+gland+of+leafhopper+vector+mediates+the+intermittent+transmission+of+rice+dwarf+virus.">Viral release threshold in the salivary gland of leafhopper vector mediates the intermittent transmission of rice dwarf virus.</a> Frontiers in Microbiology. 12, 639445 (2021).</li><li>Fukushi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+studies+on+the+dwarf+disease+of+rice+plant.">Further studies on the dwarf disease of rice plant.</a> Journal of the Faculty of Agriculture, Hokkaido Imperial University. 45 (3), 83-154 (1940).</li><li>Miyazaki, N., 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+functional+organization+of+the+internal+components+of+rice+dwarf+virus.">The functional organization of the internal components of rice dwarf virus.</a> Journal of Biochemistry. 147, 843-850 (2010).</li><li>Wei, T., Shimizu, T., Hagiwara, K., Kikuchi, A., Omura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pns12+protein+of+rice+dwarf+virus+is+essential+for+formation+of+viroplasms+and+nucleation+of+viral-assembly+complexes.">Pns12 protein of rice dwarf virus is essential for formation of viroplasms and nucleation of viral-assembly complexes.</a> Journal of General Virology. 87, 429-438 (2006).</li><li>Chen, Q., Zhang, L., Chen, H., Xie, L., Wei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonstructural+protein+Pns4+of+rice+dwarf+virus+is+essential+for+viral+infection+in+its+insect+vector.">Nonstructural protein Pns4 of rice dwarf virus is essential for viral infection in its insect vector.</a> Virology Journal. 12, 211 (2015).</li><li>Chen, Q., 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=Nonstructural+protein+Pns12+of+rice+dwarf+virus+is+a+principal+regulator+for+viral+replication+and+infection+in+its+insect+vector.">Nonstructural protein Pns12 of rice dwarf virus is a principal regulator for viral replication and infection in its insect vector.</a> Virus Research. 210, 54-61 (2015).</li><li>Chen, Q., Zhang, L., Zhang, Y., Mao, Q., Wei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubules+of+plant+reoviruses+exploit+tropomodulin+to+regulate+actin-based+tubule+motility+in+insect+vector.">Tubules of plant reoviruses exploit tropomodulin to regulate actin-based tubule motility in insect vector.</a> Scientific Reports. 7, 38563 (2017).</li><li>Wei, T., 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+spread+of+Rice+dwarf+virus+among+cells+of+its+insect+vector+exploits+virus-induced+tubular+structures.">The spread of Rice dwarf virus among cells of its insect vector exploits virus-induced tubular structures.</a> Journal of Virology. 80 (17), 8593-8602 (2006).</li><li>Mao, Q., 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=Insect+bacterial+symbiont-mediated+vitellogenin+uptake+into+oocytes+to+support+egg+development.">Insect bacterial symbiont-mediated vitellogenin uptake into oocytes to support egg development.</a> mBio. 11 (6), 01142 (2020).</li><li>Tufail, M., Takeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characteristics+of+insect+vitellogenins.">Molecular characteristics of insect vitellogenins.</a> Journal of Insect Physiology. 54 (12), 1447-1458 (2008).</li><li>Sappington, T. 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刺吸昆虫唾液腺直接分泌的唾液起着举足轻重的作用，因为它将宿主组织和载体的跨王国生物因子预先消化和解毒到宿主1，3，4中。跨王国的生物因子，包括诱发子、效应子和小RNA，对于昆虫-宿主通讯至关重要14，15，16。因此，揭示唾液成分的更多种类和?...

虫媒病毒的媒介-宿主传播方式是一个基本问题，处于生物科学的前沿。许多具有农业重要性的植物病毒通过昆虫媒介横向传播1。超过一半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。这些昆虫具有独特的特征，使它们能够有效地传播植物病毒1。它们拥有刺吸口器，以韧皮部和木质部的汁液为食，分泌唾液1，2，3，4。随着技术的发展和改进，唾液成分的鉴定和功能分析正在成为深入研究的新焦点。唾液中已知的唾液蛋白包括许多酶，例如果胶酯酶、纤维素酶、过氧化物酶、碱性磷酸酶、多酚氧化酶和蔗糖酶等5，6，7，8，9，10，11，12，13.唾液中的蛋白质还包括触发宿主防御反应的诱发子，从而改变昆虫的性能，以及抑制宿主防御的效应子，其增强昆虫适应性和诱导宿主病理反应的成分14，15，16，17。因此，唾液蛋白是昆虫与宿主之间交流的重要材料。在病毒传播过程中，刺吸病毒昆虫唾液腺分泌的唾液也含有病毒蛋白。病毒成分利用唾液的流动将它们从昆虫释放到植物宿主。因此，昆虫唾液弥合了病毒-载体-宿主的三营养相互作用。研究含病毒昆虫分泌的唾液蛋白的生物学功能有助于了解病毒-载体-宿主的关系。
对于动物病毒，据报道，蚊子的唾液介导西尼罗河病毒（WNV）和登革热病毒（DENV）的传播和致病性。唾液蛋白AaSG34通过激活自噬促进登革热-2病毒复制和传播，而唾液蛋白AaVA-1通过激活自噬来促进登革热和寨卡病毒（ZIKV）传播18，19。蚊子的唾液蛋白D7可以通过与DENV病毒粒子和重组DENV包膜蛋白20的直接相互作用，在体外和体内抑制DENV感染。在植物病毒中，白栗病毒番茄黄叶卷曲病毒（TYLCV）诱导粉虱唾液蛋白Bsp9，抑制植物宿主WRKY33介导的免疫力，增加粉虱的偏好和性能，最终增加病毒的传播21。由于对昆虫唾液蛋白在植物宿主中的作用的研究落后于动物宿主，因此迫切需要一个稳定可靠的系统来检测植物宿主中的唾液蛋白。
被称为水稻矮病毒（RDV）的植物病毒由叶蝉Nephotettix cincticeps（半翅目：Cicadellidae）高效且持续繁殖22，23传播。RDV首先被报道通过昆虫媒介传播，并在亚洲引起严重的水稻病害24，25。病毒粒子为二十面体和双层球形，外层含有P8外衣壳蛋白22。RDV在蝴蝶奈瑟蝇中的循环传播周期为14天26，27，28，29，30。当RDV到达唾液腺时，病毒粒子通过胞吐样机制释放到唾液腺中储存唾液的腔中23。卵黄原蛋白（Vg）是雌性昆虫卵母细胞发育所必需的卵黄蛋白前体31，32，33。大多数昆虫物种至少有一个6-7 kb的Vg转录本，其编码约220 kDa的前体蛋白。Vg的蛋白质前体在进入卵巢之前通常可以裂解成大的（140至190 kDa）和小的（<50 kDa）片段18，19。先前的蛋白质组学分析显示，叶蝉Recilia dorsalis分泌的唾液中存在来自Vg的肽，尽管它们的功能尚不清楚（未发表的数据）。新报道，飞虱口服分泌的Vg起到效应作用，破坏植物的防御能力34。目前尚不清楚猪笼草的Vg是否也可以随唾液流释放到植物宿主，然后在植物中发挥作用干扰植物防御。为了解决猪笼草是否利用唾液蛋白（如Vg）来抑制或激活植物防御的问题，第一步是识别在摄食过程中释放到植物中的蛋白质。了解鉴定植物中存在的唾液蛋白的方法对于解释唾液蛋白的功能以及半翅目与植物之间的相互作用至关重要。
在这里介绍的协议中，以N . cincticeps 为例，提供一种方法来检查通过昆虫喂养引入的植物宿主中唾液蛋白的存在。该方案主要详细说明了唾液蛋白的收集和检测，有助于对大多数半翅目动物的进一步研究。

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	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Roche</td>
			<td>D609K69032</td>
			<td>For 5&#215;Tris-glycine buffer and 10&#215;TBS buffer preparation</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Sigma-Aldrich</td>
			<td>WXBD0677V</td>
			<td>For 5&#215;Tris-glycine buffer preparation</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>SLCB4394</td>
			<td>For 5&#215;Tris-glycine buffer preparation</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10019318</td>
			<td>For 10&#215;TBS buffer preparation</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10016318</td>
			<td>For 10&#215;TBS buffer preparation</td>
		</tr>
		<tr>
			<td>&#223;-mercaptoethanol</td>
			<td>Xiya Reagent</td>
			<td>B14492</td>
			<td>For 4&#215; protein sample buffer preparation</td>
		</tr>
		<tr>
			<td>bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>SHBL3668</td>
			<td>For 4&#215; protein sample buffer preparation</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10010618</td>
			<td>For 4&#215; protein sample buffer preparation</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10014118</td>
			<td>For transfer buffer preparation</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Coolaber SCIENCE&#38;TeCHNoLoGY</td>
			<td>CT30111220</td>
			<td>For TBST preparation</td>
		</tr>
		<tr>
			<td>non-fat dry milk</td>
			<td>Becton.Dickinso and company</td>
			<td>252038</td>
			<td>For membrane blocking, antibodies dilution</td>
		</tr>
		<tr>
			<td>goat anti-rabbit IgG</td>
			<td>Sangon Biotech</td>
			<td>D110058-0001</td>
			<td>Recognization of the primary andtibody</td>
		</tr>
		<tr>
			<td>ECL Western kit</td>
			<td>ThermoFisher Scientific</td>
			<td>32209</td>
			<td>Chemiluminescent substrate</td>
		</tr>
		<tr>
			<td>nitrocellulose membrane</td>
			<td>Pall Corporation</td>
			<td>25312915</td>
			<td>For proteins transfer</td>
		</tr>
		<tr>
			<td>Buffers and Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer</td>
			<td>Composition</td>
			<td></td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>&#160;5&#215;Tris-glycine buffer</td>
			<td>15.1 g Tris base 
			94 g glycine 
			&#160;5 g SDS in 1 L sterile water</td>
			<td></td>
			<td>&#160;Stock solution</td>
		</tr>
		<tr>
			<td>1&#215;Tris-glycine buffer</td>
			<td>200 mL of 5&#215;Tris-glycine buffer 
			800 mL sterile water</td>
			<td></td>
			<td>Work solution, for SDS-PAGE</td>
		</tr>
		<tr>
			<td>10&#215;Tris-buffered saline (TBS) buffer</td>
			<td>80 g NaCl 
			30 g Tris base 
			2 g KCl 
			in 1 L sterile water</td>
			<td></td>
			<td>Stock solution</td>
		</tr>
		<tr>
			<td>TBS with Tween 20 (TBST) solution</td>
			<td>100 mL 10&#215;TBS solution 
			3 mL Tween 20 
			900 mL sterile water</td>
			<td></td>
			<td>Work solution</td>
		</tr>
		<tr>
			<td>4&#215; protein sample buffer</td>
			<td>8 g SDS 
			4 mL &#223;-mercaptoethanol 
			0.02 g bromophenol blue 
			40 mL glycerol 
			in 40 mL 0.1 M Tris-HCl (pH 6.8)</td>
			<td></td>
			<td>For protein extraction</td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td>800 mL Tris-glycine buffer 
			200 mL methanol</td>
			<td></td>
			<td>For protein transfer</td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>SHBL3668</td>
			<td>For 4x protein sample buffer preparation</td>
		</tr>
		<tr>
			<td>Constant temperature incubator</td>
			<td>Ningbo Saifu Experimental Instrument Co., Ltd.</td>
			<td>PRX-1200B</td>
			<td>For rearing leafhoppers</td>
		</tr>
		<tr>
			<td>Electrophoresis</td>
			<td>Tanon Science &#38; Technology Co.,Ltd.</td>
			<td>Tanon EP300</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>Electrophoretic transfer core module</td>
			<td>BIO-RAD</td>
			<td>1703935</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10010618</td>
			<td>For 4x protein sample buffer preparation</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Sigma-Aldrich</td>
			<td>WXBD0677V</td>
			<td>For 5x Tris-glycine buffer preparation</td>
		</tr>
		<tr>
			<td>goat anti-rabbit IgG</td>
			<td>Sangon Biotech</td>
			<td>D110058-0001</td>
			<td>Recognization of the primary andtibody</td>
		</tr>
		<tr>
			<td>High-pass tissue grinding instrument</td>
			<td>Shanghai Jingxin Industrial Development Co., Ltd.</td>
			<td>JXFSIPRP-24</td>
			<td>For grinding plant tissues</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10016318</td>
			<td>For 10x TBS buffer preparation</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10014118</td>
			<td>For transfer buffer preparation</td>
		</tr>
		<tr>
			<td>Mini wet heat transfer trough</td>
			<td>BIO-RAD</td>
			<td>1703930</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10019318</td>
			<td>For 10x TBS buffer preparation</td>
		</tr>
		<tr>
			<td>nitrocellulose membrane</td>
			<td>Pall Corporation</td>
			<td>25312915</td>
			<td>For proteins transfer</td>
		</tr>
		<tr>
			<td>non-fat dry milk</td>
			<td>Becton.Dickinso and company</td>
			<td>252038</td>
			<td>For membrane blocking, antibodies dilution</td>
		</tr>
		<tr>
			<td>Pierce ECL Western kit</td>
			<td>ThermoFisher Scientific</td>
			<td>32209</td>
			<td>Chemiluminescent substrate</td>
		</tr>
		<tr>
			<td>Protein color instrument</td>
			<td>GE Healthcare bio-sciences AB</td>
			<td>Amersham lmager 600</td>
			<td>For detecting proteins</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>SLCB4394</td>
			<td>For 5x Tris-glycine buffer preparation</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Roche</td>
			<td>D609K69032</td>
			<td>For 5x Tris-glycine buffer and 10&#215;TBS buffer preparation</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Coolaber SCIENCE&#38;TeCHNoLoGY</td>
			<td>CT30111220</td>
			<td>For TBST preparation</td>
		</tr>
		<tr>
			<td>Vertical plate electrophoresis tank</td>
			<td>BIO-RAD</td>
			<td>1658001</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Shanghai Jinghong Experimental equipment Co., Ltd.</td>
			<td>XMTD-8222</td>
			<td>For boil the protein samples</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Xiya Reagent</td>
			<td>B14492</td>
			<td>For 4x protein sample buffer preparation</td>
		</tr>
	</tbody>
</table>

detecting virus and salivary proteins of a leafhopper vector in the plant host

昆虫媒介水平传播许多具有农业重要性的植物病毒。超过一半的植物病毒是由具有刺穿吸口器的半翅目昆虫传播的。在病毒传播过程中，昆虫唾液桥接病毒载体宿主，因为唾液载体病毒和昆虫蛋白触发或抑制植物从昆虫到植物宿主的免疫反应。唾液蛋白的鉴定和功能分析正在成为虫媒病毒-宿主相互作用研究领域的新热点。该协议提供了一个使用植物宿主检测叶蝉唾液中蛋白质的系统。感染水稻矮病毒（RDV）的叶蝉载体Nephotettix cincticeps就是一个例子。在以辛克二头肌猪笼草为食的水稻植株中，可以同时检测到由辛克二头肌奈瑟菌唾液载体的RDV的卵黄原蛋白和主要外衣壳蛋白P8。该方法适用于检测昆虫摄食后暂时滞留在植物宿主中的唾液蛋白。相信这种检测系统将有利于半翅目-病毒-植物或半翅目-植物相互作用的研究。

图1说明了该协议中的所有步骤：昆虫饲养，病毒获取，通过水稻喂养收集唾液蛋白以及蛋白质印迹。蛋白质印迹结果显示，在用Vg抗体孵育的膜上的喂养水稻和昆虫唾液腺的样品中观察到约220 kDa的特异性和预期条带。相比之下，在非饲料大米样品中未观察到条带。图2中的结果表明，Vg作为唾液蛋白释放到植物宿主中。在与RDV P8抗体?...

Watch this Scientific Journal Video about Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host at JoVE.com

Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host

该协议演示了如何使用植物宿主来检测叶蝉的唾液蛋白和叶蝉载体释放的植物病毒蛋白。

检测植物宿主中叶蝉载体的病毒和唾液蛋白

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran ...

该协议提供了一种检测叶蝉和植物宿主的唾液蛋白的技术，以进一步研究这些蛋白质在植物宿主中的功能。这种技术易于执行，系统稳定且可复制。该方法也有助于检测其他半翅目和植物宿主的唾液蛋白。首先，在40 x 35 x 20厘米的立方体笼子中饲养稻苗上的成年叶蝉。保持笼子的一侧盖上防虫网，以便通风。将带有叶蝉的笼子放在装有内置湿度控制器的培养箱中，温度为26摄氏度，相对湿度为60%至75%，照相时间为16小时，黑暗时间为8小时。每周一次，使用吸气器将所有成虫从笼子中轻轻转移到装有新鲜稻苗的新笼子中。允许200多个成年人在水稻中交配和产卵。保留老稻苗，让若虫出苗，并将这些新的非病毒若虫培育到第二龄期。使用抽吸器，小心地将第二龄非病毒若虫转移到玻璃培养管中一到两个小时以使其饥饿。然后将若虫释放到装有在盆中生长的水稻矮化病毒感染水稻植物的笼子中，让若虫以受感染的水稻植物为食两天。两天后，小心地将这些若虫转移到装有新鲜无病毒水稻幼苗的新笼子中，让若虫以无病毒水稻幼苗为食12天，以完成水稻矮化病毒的循环传播期。为了收集唾液蛋白，准备五个小管状喂食笼，一端覆盖着防虫网。在每个喂食笼中限制 15 到 20 个叶蝉后，用薄泡沫垫覆盖笼子的另一端。在笼子的末端和用胶带的泡沫垫之间固定一棵稻苗，确保饲养笼中的叶蝉可以以暴露在笼子内部的水稻幼苗为食。将幼苗根部浸入水中，使水稻植株保持活力，并让叶蝉以它们为食两天。两天后，将叶蝉从饲养笼中取出，收集叶蝉喂养的水稻幼苗。将幼苗切出网箱，恢复幼苗的摄食区。用800毫升无菌水稀释200毫升制备的5x缓冲液，制备1x Tris-甘氨酸缓冲液。然后准备10x TBS缓冲液，并在121摄氏度下高压灭菌溶液15分钟。接下来，准备4x蛋白质样品缓冲液。然后通过将800毫升Tris-甘氨酸缓冲液与200毫升甲醇混合来制备转印缓冲液。最后，通过加入 100 毫升 10x TBS 和 3 毫升吐温 20 至 900 毫升无菌水制备 TBST 溶液。为了检测唾液和病毒蛋白，用液氮研磨0.1克大米样品，直到组织变成粉末。然后将 200 微升 4x 蛋白质样品缓冲液加入样品中并煮沸 10 分钟。离心样品并将上清液转移到新小瓶中。将 10 μL 样品加载到 SDS-PAGE 凝胶上，并在 Tris-甘氨酸缓冲液中以 150 伏电压运行 45 至 60 分钟。在凝胶电泳时，将0.45微米硝酸纤维素膜和其他三明治用品置于转印缓冲液中30分钟。运行后，夹住凝胶并在转印缓冲液中以 100 伏电压转移 90 至 120 分钟。印迹完成后，将膜置于TBST中的7%脱脂奶粉封闭溶液中，孵育20分钟。之后，加入针对稻矮病毒P8或卵黄素的抗体，并将膜孵育两小时或过夜。孵育后，用TBST洗涤膜三次，每次洗涤之间孵育五分钟。然后加入山羊抗兔 IgG 作为二抗，并将膜孵育 60 至 90 分钟。使用ECL蛋白质免疫试剂盒在试剂盒中的混合检测试剂1和2中以1：1的比例进行化学发光检测。将膜放在混合试剂上，孵育印迹膜五分钟。排出多余的试剂，拍摄膜的化学发光和比色图像。将图片组合起来，看看后者与蛋白质条带。用于检测卵黄原蛋白的蛋白质印迹分析显示，在喂养水稻植物和昆虫的唾液腺中，特异性和预期条带约为220千道尔顿。相比之下，在非摄食植物中没有观察到条带，表明卵黄原蛋白作为唾液蛋白在植物宿主中释放。用于检测稻矮病毒P8蛋白的蛋白质印迹分析显示，在饲养植物和病毒昆虫体内，特异性和预期条带约为46千道尔顿。相比之下，在非摄食植物和非病毒性叶蝉体中未观察到条带，证明病毒蛋白也可以在摄食植物中检测到。在执行此协议时，应考虑叶蝉中的病毒载量，检测时间和重复。这种方法也可用于研究传播虫媒病毒的蚊子。

Research

JoVE Journal

Biology

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

从AE肠和唾液腺的解剖。埃及伊蚊蚊

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

科研

生物学

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 ...

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 ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

作为寄主植物挥发物的筛选工具electroantennographic生物活性

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

The MultiBac Protein Complex Production Platform at the EMBL

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞：隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

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 ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

肥胖的身体器官培养体系中蚊，一个向量 Zika 病毒

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...