Name: two-layered membrane sandwich method for laodelphax striatellus saliva collection
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本研究由国家重点研发计划（编号：2019YFC1200503）、国家自然科学基金（编号：32072385）和中国科学院青年创新促进会（2021084）资助。

1. SBPH维护
<ol><li>在实验室的每个玻璃室中，将具有毒性和无RSV的SBPH个体置于玻璃培养箱（65 x 200 mm）中，每个玻璃室中含有5-6个大米（Oryza sativa cv. Nipponbare）幼苗。在25°C下在16小时光照/ 8小时黑暗光周期下种植水稻植物。 注：这些有毒和无RSV的SBPH个体最初是在中国江苏省捕获的。</li>
	<li>通过点酶联免疫吸附测定（dot-ELISA）检测SBPH中的RSV，该测定与针对RSV核糖核蛋白（RNPs）的兔RSV特...

<ol>
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1962年首次报道了人工日粮成功饲养昆虫，当时米特勒和爸爸描述了石蜡膜技术来保持人工日粮29，30。这种方法已经在昆虫生物学和行为的许多方面进行了探索，例如营养补充，dsRNA喂养和病毒获取。根据唾液分析的要求，本研究以5%蔗糖作为一般人工饮食，收集SBPH的唾液。为了成功收集唾液，这里有几个关键步骤值得注意。首先，在将实验昆虫引入腔?...

水稻条纹病毒（RSV）是Tenuivirus属中的一种阴性链RNA病毒，在东亚的水稻生产中引起严重疾病1，2，3。RSV从受感染的水稻植物传播到健康的水稻植物取决于昆虫载体，主要是Laodelphax striatellus，它以持久繁殖的方式传播RSV。SBPH在以RSV感染的植物为食后获得病毒。一旦进入昆虫体内，RSV在进食后一天感染中肠上皮细胞，然后穿过中肠屏障穿透血淋巴。随后，RSV通过血淋巴扩散到不同的组织中，然后传播。在获得后约10-14天的潜伏期后，唾液腺内的病毒可以通过分泌的唾液传播给健康的寄主植物，而SBPH则从韧皮部4，5，6，7，8，9，10中吸收汁液.有效的摄食过程和唾液中的各种因素对于RSV从昆虫传播到寄主植物至关重要。
唾液腺分泌的昆虫唾液被认为介导昆虫，病毒和寄主植物。半翅目昆虫通常产生两种类型的唾液：胶状唾液和水性唾液11，12，13。胶凝唾液主要分泌到脑浆中，以维持寄主细胞间的蠕动，也与克服植物抗性和免疫反应有关14、15、16、17。在进食的探测阶段，昆虫间歇性地分泌凝胶状唾液，立即被氧化形成表面法兰。然后，单层或分支鞘包裹样式以保留管状通道18，19，20。表皮上的表面法兰被认为通过充当锚点来促进样式的穿透，而样式周围的护套可以提供机械稳定性和润滑16，21，22，23。Nlshp被确定为唾液鞘形成和成功喂养棕色飞虱（Nilaparvata lugens，BPH）的必需蛋白质。抑制由蚜虫Acyrthosiphon pisum分泌的结构鞘蛋白（SHP）的表达通过破坏宿主筛管24的进食来减少其繁殖。此外，在一些昆虫物种中，凝胶唾液因子应该通过形成所谓的食草动物相关分子模式（HAMPs）来触发植物的免疫反应。在N. lugens中，NlMLP，一种与鞘形成相关的粘蛋白样蛋白质，诱导植物防御进食，包括细胞死亡，防御相关基因的表达和胼脂沉积25，26。 此外，蚜虫中的一些凝胶唾液因子已被证明可以通过类似于病原体相关分子模式的基因间相互作用触发植物防御反应12，15，27。
为了研究昆虫喂养和/或病原体传播所必需的唾液因子，有必要分析分泌的唾液。这里描述了人工喂养和收集方法以获得足够量的唾液，以便进一步分析。使用仅含有单一营养元素的培养基，通过银染和蛋白质印迹收集和分析许多唾液蛋白。该方法将有助于进一步研究唾液中对SBPH传播RSV至关重要的因素。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10-KD centrifugal filter</td>
			<td>Merck Millipore</td>
			<td>R5PA83496</td>
			<td>For concentration</td>
		</tr>
		<tr>
			<td>10x Protein Transfer Buffer(wet)</td>
			<td>macGENE</td>
			<td>MP008</td>
			<td>Transfer buffer for western blotting</td>
		</tr>
		<tr>
			<td>10x TBST buffer</td>
			<td>Coolaber</td>
			<td>SL1328-500mL&#215;10</td>
			<td>Wash buffer for western blotting</td>
		</tr>
		<tr>
			<td>Azure c600 biosystems</td>
			<td>Azure Biosystems</td>
			<td>Azure c600</td>
			<td>Imaging system for western blotting and silver staining</td>
		</tr>
		<tr>
			<td>Color Prestained protein ladder</td>
			<td>GenStar</td>
			<td>M221-01</td>
			<td>Protein marker for western blotting</td>
		</tr>
		<tr>
			<td>ECL&#160;western blotting detection reagents</td>
			<td>GE Healthcare</td>
			<td>RPN2209</td>
			<td>Western blotting detection</td>
		</tr>
		<tr>
			<td>Enchanced HRP-DAB Chromogenic Kit</td>
			<td>TIANGEN</td>
			<td>#PA110</td>
			<td>Chromogenic reaction</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase-conjugated goat anti-rabbit antibodies</td>
			<td>Sigma</td>
			<td>401393-2ML</td>
			<td>Polyclonal secondary antibody for western blotting</td>
		</tr>
		<tr>
			<td>Immobilon(R)-P Polyvinylidene difluoride membrane</td>
			<td>Merck Millipore</td>
			<td>IPVH00010</td>
			<td>Transfer membrane for western blotting</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>Bio-Rad</td>
			<td>1725125</td>
			<td>For quantitative real-time PCR (qRT-PCR)</td>
		</tr>
		<tr>
			<td>KIT,iSCRIPT cDNA SYNTHES</td>
			<td>Bio-Rad</td>
			<td>1708891</td>
			<td>For Reverse-transcriptional PCR (RT-PCR)</td>
		</tr>
		<tr>
			<td>Millex-GP Filter, 0.22 &#181;m</td>
			<td>Merck Millipore</td>
			<td>SLGP033RB</td>
			<td>For filtration</td>
		</tr>
		<tr>
			<td>Mini-PROTEAB TGX Gels</td>
			<td>Bio-Rad</td>
			<td>4561043</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>Thermo Scientific</td>
			<td>ND-ONEC-W</td>
			<td>Detection of protein concentration</td>
		</tr>
		<tr>
			<td>Nylon membrane</td>
			<td>PALL</td>
			<td>T42754</td>
			<td>Membrane for dot-ELISA</td>
		</tr>
		<tr>
			<td>Parafilm M Membrane</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td>Making artifical diet sandwichs</td>
		</tr>
		<tr>
			<td>Rabbit anti-LssgMP polyclonal antibody against LssgMP peptides</td>
			<td>Genstript</td>
			<td></td>
			<td>Rabbit primary anti-LssgMP polyclonal antibody for western blotting</td>
		</tr>
		<tr>
			<td>Rabbit anti-RSV polyclonal antibody</td>
			<td>Genstript</td>
			<td></td>
			<td>Rabbit primary anti-RSV polyclonal antibody for western blotting and dot-ELISA</td>
		</tr>
		<tr>
			<td>RNAprep pure Micro Kit</td>
			<td>TIANGEN</td>
			<td>DP420</td>
			<td>For RNA Extraction</td>
		</tr>
	</tbody>
</table>

two-layered membrane sandwich method for laodelphax striatellus saliva collection

稻条病毒（RSV）在东亚造成农业的重大经济损失，其在宿主水稻之间的有效传播完全依赖于昆虫媒介。 Laodelphax striatellus（ 小型棕色飞虱，SBPH）是主要昆虫媒介，在从韧皮部吸食汁液时水平传播RSV。唾液在昆虫的进食行为中起着重要作用。这里描述了一种方便的方法，可用于研究具有穿刺 - 吸食进食行为的昆虫唾液。在这种方法中，允许昆虫以夹在两个拉伸的石蜡膜层之间的人造饮食为食。每天收集含有唾液的饮食，过滤并浓缩以进行进一步分析。最后，通过蛋白质染色和免疫印迹检查收集的唾液的质量。该方法通过检测SBPH唾液中RSV和粘蛋白样蛋白的存在来举例说明。这些人工摄食和唾液收集方法将为进一步研究昆虫唾液中与摄食行为和病毒传播相关的因素奠定基础。

人工饲喂装置及唾液采集示意图 图1A 描绘了用作收集唾液的进食室的玻璃圆筒（15厘米×2.5厘米）。首先，将SBPH幼虫饥饿数小时以提高收集效率，然后通过冷却5 min固定。将昆虫转移到玻璃圆筒中后，腔室的两个开口端都覆盖着拉伸的石蜡膜。在一端，将200μL5%蔗糖夹在两层石蜡膜之间，延伸至其原始面积的约两倍（图1B）。腔室被铝箔?...

Watch this Scientific Journal Video about Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection at JoVE.com

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

本方案描述了一种使用人工介质从穿刺-吸吮昆虫中收集足够唾液的方法。这是一种收集昆虫唾液和研究唾液对昆虫进食行为和媒介传播病毒传播的有用方法。

用于 老德法纹 状体唾液采集的双层膜夹层法

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective ...

人工喂养和唾液收集为检测昆虫唾液中的效应物提供了一种直接的方法。但是，这在以前没有详细描述过。该协议对从腐生昆虫收集唾液有效。此外，这是一种简单的技术，因为我们使用仅含有蔗糖的培养基作为人造饮食。腐生昆虫的唾液可能会影响宿主的免疫系统，以利于喂养行为并传播病原体。这种药物可用于昆虫行为和昆虫骨路的研究。这是一种简单的执行方法。然而，这表明正在收集唾液以进行进一步研究，因此需要安装清洁的实验环境。首先为人工饮食制备5%蔗糖溶液，并通过0.22微米过滤器过滤溶液以除去细菌污染和杂质。将大约200个小的棕色植物料斗幼虫饿死三到五个小时，然后再将它们引入喂食室。接下来，准备玻璃圆筒作为进料室，在引入实验昆虫之前用石蜡膜覆盖腔室的一个开口端，然后将昆虫转移到玻璃圆筒中，并用拉伸的石蜡膜覆盖腔室的另一端。在膜上加入200微升人造饮食，然后用另一层拉伸的石蜡膜覆盖液体。最后，用铝箔覆盖腔室，使末端的人造饮食装置暴露在光线下。24小时后，通过将圆筒冷却在4摄氏度下以固定昆虫来收集SBPH唾液，然后揭开外膜并使用无菌移液管将人造饮食收集到1.5毫升无菌管中。将收集的唾液保持在零下80摄氏度，直到分析。接下来，用50微升新鲜人造饮食轻轻移液三次冲洗内膜，并将人工洗涤饮食收集到1.5毫升无菌管中。冲洗三次后，在内膜上加入新鲜的人造饮食，并在顶部放置新鲜拉伸的石蜡膜。最后，通过0.22微米过滤装置过滤收集的样品，以去除微生物和其他污染物。将集体唾液样品转移到500微升，10千道尔顿离心过滤器中。离心样品。收集上清液并将最终体积提高到100微升。要测量收集的唾液的浓度，请打开紫外可见分光光度计，并用双蒸馏水冲洗基座三次。按以下顺序在屏幕上选择选项，蛋白质，蛋白质A280。选择类型和一个吸光度单位，等于每毫升一毫克。然后选中基线校正框，340纳米。接下来，加载两微升5%蔗糖水溶液作为空白，然后触摸屏幕底部的空白。最后，在设定标准后，加载收集的唾液的两微升并记录蛋白质浓度。当以5%蔗糖为食时，超过80%的SBPH在前四天存活下来。然而，从第五天开始，死亡率迅速上升到40%，在第七天，只有不到一半的SBPH存活下来。SDS页面分析显示，来自RSV感染的SBPH唾液的浓缩唾液样本比5%蔗糖阴性对照含有更多的蛋白质。在用于检测RSV衣壳蛋白的蛋白质印迹分析中，一种未感染和有毒的昆虫。在毒力样品中成功检测到RSV衣壳蛋白，一种针对LssgMP基因产物设计的抗体在未感染和有毒样品中检测到78千道尔顿蛋白，证明LssgMP是一种唾液蛋白。对不同组织中LssgMP转录本水平的QRT PCR分析表明，唾液腺中的LSSG转录本水平比全身和肠道高20倍，证实了LssgMP在唾液腺中的特异性表达。在将实验昆虫引入腔室之前，必须使它们挨饿，以确保我们唾液收集的效率。此外，应每24小时收集和更换一次人工培养基，以减少收集样品中的微生物污染。该技术为研究唾液在传播病原体中的功能提供了直接方法。此外，它还可以帮助研究昆虫病原体宿主的相互作用。

Research

JoVE Journal

Biology

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

科研

生物学

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

在成年斑马鱼的生化分析采血车

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

一种实用的新的方法来提取基因组DNA保存血浆蛋白从血液采集套件

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

收集和分析<em&gt;拟南芥</em&gt;韧皮部分泌物使用EDTA促进方法

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

重复采血车在成年斑马鱼验血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

用于 老德法纹 状体唾液采集的双层膜夹层法

用于老德法纹状体唾液采集的双层膜夹层法