根据美国农业部CRIS软件项目5325-42000-037-00D与CRADA 58-3K95-7-1198和TFCA 58-5325-8-419的结果进行了这项研究。作者感谢Suterra的礼物（z，z）-11,13-hexadecadienal，希格比B.富有成效的讨论，提供技术援助和J.贝克。

1。东亚运动会筛选检测寄主植物挥发物的制备<ol><li>适当的标识和认证通过的GC-MS挥发后，执行训练班粉扑每个可用挥发性分析。初步筛选可以是低复制数量为每个性别的触角反应（n = 3-5），以实现在很短的时间量（ 见表1）相对chemoreceptivity的指示。 </li><li>准备在5毫克/毫升戊烷浓度的每一个波动的解决方案。紧紧密封和冷藏直到准备立即使用（例如，苯乙酮，密度= 1.03克/毫升，体积=（0.005 g/1.03克/毫升）×1,000 = 4.85μL到1.0 mL容量瓶苯乙酮和稀释样品用戊烷1.0毫升）。 </li><li>只是之前东亚运动会的分析，取出小瓶含5毫克/毫升的浓度进行测试的挥发，使预热到室温。同时，标签的巴斯德移液器的正确数量相关的挥发性每次进行测试，并超过枪头包装的封口膜小片（约15×15毫米）。对初步筛选的10个刺激移液器（N = 4时为每个男性和女性）都装在一个坏的准备或其他不可预见的障碍事件。 </li><li>使用镊子，轻轻折了一半的生物活性光盘，以便将吸管的位置，然后部分放入大底的吸管折叠，使CA光盘。 2-3毫米的光盘被曝光。在机架支架对齐标记和prepped移液器。 </li><li>使用移液器或注射器，加载到每个盘的挥发性溶液10μL（50微克），并允许2分钟前通过完全插入吸管光碟。一旦加载，立即查封移液器年底，封口膜（约15×30毫米）。要加载和自高自大的挥发性刺激量将最有可能随种昆虫。 </li><li>遵循相同的协议，上面提到的准备控制到包括在每一个分析。负控制负载10μL刚戊烷，到每张光盘，等待2分钟，装入标记移液器，密封。为阳性对照，10μL（50微克）加载到光盘上，等待2分钟，将标记吸管的负载，并用封口膜密封。 </li><li>昆虫触角准备的可行性，将最有可能与物种而有所不同，但我们发现，Amyelois transitella天线通常保持活跃切除后30分钟使用所描述的协议和一致。这段时间内，一般会允许长达4-10挥发性样品进行评估。 表2提供了时间和序列的例子。 </li></ol> 2。为筹备东亚运动会的生物活性昆虫触​​角<ol><li>这个实验的重点是分析东亚运动会，因此，假设每个研究者将有机会获得适当的饲养昆虫。 </li><li>在这个实验中，我们将学习3-4天OLD，交配的雄性和雌性飞蛾。个别飞蛾被转移到一个小的，有盖，塑料容器分析的日子。前夕的生物活性，被测试的蛾被转移到第一头控股仪器（即，从各种塑料移液器提示），从背后担保。昆虫可以被视为低功耗立体声方便切除显微镜下操作。 </li><li>触须梳理出使用电线尖的工具。叉架，用电极胶的小电影，被放置在靠近天线的快速传输。第一天线采用微型剪刀切除，放在叉，叉（漠不关心的接地电极）非红色部分放在确保天线的基础上。一个10分钟的计时器集开始，第二个天线被切除，放在旁边的第一天线上叉同方的两个基地。 </li><li>另外，天线可以离开担保范围内的牵着Ğ管，切除，然后轻轻地从管和昆虫，用导线尖端的工具，民建联上月底的凝胶。 </li><li>触角检查，以确保基地和尖端沉浸在电极胶，无气泡凝胶。可以修剪完/提示，以保证充分暴露了电极凝胶。应谨慎使用，以确保每个天线的其余部分不与凝胶覆盖，从而保证最大触角表面积暴露粉扑。 </li><li> prepped叉，然后插入探头前置放大器和下恒定加湿空气流保持在200毫升/分钟的轮候时间为10分钟的剩余时间。指出的消费时间和东亚运动会的起始时间（ 见表2）和30秒之前，首先刺激粉扑，吸管含有阳性对照启封，并放置在持有人调整直接空气/粉扑流2-3毫米prepped触角。 </li><li>经过各experiment，测试飞蛾安乐死在干冰环境，并妥善处理。 </li><li>解剖检查雌蛾交配状态。同居的男性被认为已交配。 </li></ol> 3。东亚运动会为单独的组件的议定书<ol><li>触角反应记录通过用包含的Syntech东亚运动会仪器的AutoSpike软件。在与东亚运动会的探头通道的AutoSpike属性"选项卡的配置设置的106.7采样率，无以纠正，0-42赫兹的过滤器。对于"筛选"选项卡，东亚运动会的过滤器是ON和低截止到0.1 Hz。 </li><li>刺激喷1-2秒。 1分钟设置一个定时器启动后，每个噗。 </li><li>接下来的测试挥发性移液器启封，并放置在持有人。测试挥发随机之间的顺序运行，以确保没有测试化合物一贯遵循另一个测试挥发性（例如， 表2），随后仔细allowi纳克之间泡芙1分钟。 </li><li>为了方便易于阅读EAG反应，统治者的部分放置在电脑屏幕上，顶点向下偏转信号的基线水平，以毫米测量和记录数据表（即33毫米为5 mV的设置， = 2.5毫伏）。该软件具有播放保存的录音，供日后分析的能力。 mV或μV的振幅毫米的转换进行分析后的数据。 </li><li>决赛后，积极控制粉扑（例如，记录＃12）管理，被删除的触角，叉饱和乙醇擦拭清洗，并允许晾干后方可后续使用。 </li><li> ，以增加检测的吞吐量，可以切除触角下一组开始了第二次的实验室工作人员，目前的实验结束前约10分钟-之后的第四个粉扑;第二次测试，目前的实验中挥发，按表2。这将允许FOr为目前的实验结束后直接下一个实验的开始。 </li><li>可以执行较高的重复数（在不同的天线），感兴趣的化合物，以提供进一步的统计验证。 </li></ol> 4。东亚运动会的混合物或其他矩阵的分析为例（见表3） <ol><li>接下来，两个大专混合物（3组分混合物）的比率和数量计算提供了一个例子引出的EAG反应（ 表4）相结合的挥发。计算一些基本比率，α-蛇麻烯摩尔比为1:1:1:2 - 十一酮:2 - 苯乙醇，然后比较第二次混合比例是1:2:4。 </li><li>使用类似的协议中所述的混合物准备和标记巴斯德移液器加载必要的阳性和阴性对照，沿。 </li><li>挥发性样品膨化跨越男性和女性的触角和响应测量和记录数据表（ 表3）。 </li></ol> 5。代表结果对于女性肚脐orangeworm的以下设置:2秒喷，10秒录音时间，10秒的窗口，和5 mV的规模。负偏差是典型的反应，但绝对值记录（如-3,400μV的偏转被记录为3400μV）。阳性对照的一个相对薄弱的准备响应将被丢弃。 图1提供了一个图形表示阳性对照由肚脐orangeworm的反应不佳。 例如，对于一个贫穷的控制效果，平均女性的触角，以苯乙酮的反应通常是CA。 2600μV（ 图2），如果只准备了CA的响应。 1300μV的，它会被丢弃，另一对触角prepped。同样，男性平均响应（z Z）-11,13-hexadecadienal是典型地Ÿ3000μV的;任何回应，因此，低于1500μV的，通常被丢弃。 在每个实验的开始和结束时的阳性对照，也提供了信息触角条件。我们快速筛查遵循一个法则是，如果触角反应后控制（记录＃12， 表2）粉扑或者低于75％的预控第一粉扑（记录＃1 ， 表2），或超过预控（纪录＃2， 表2）实验不使用的数据分析，由于可能退化的准备（ 图3） 第二粉扑。拇指的第一条规则的一个例子将记录＃1 = 2,730μV和记录＃12 = 1,680μV;和拇指的第二条规则，如果记录＃2 = 2350μV和记录＃12 = 1,680μV，在这些案件中的每个，准备和实验的结果将被丢弃。 一纠正EAG反应值作为衡量阳性对照的有代表性的例子如下。 <table border="1"><tbody><tr><td> 运行＃ </td><td> 东亚运动会（μV峰） </td><td> 运行＃2 </td><td> 东亚运动会（μV峰） </td><td> 运行＃3 </td><td> 东亚运动会（μV峰） </td></tr><tr><td> （）CTRL </td><td> 2800 </td><td> （）CTRL </td><td> 2420 </td><td> （）CTRL </td><td> 3030 </td></tr><tr><td> CMPND一个</td><td> 3000 </td><td> CMPND一个</td><td> 2500 </td><td> CMPND一个</td><td> 3440 </td></tr><tr><td> （ - ）CTRL </td><td> 530 </td><td> （ - ）CTRL </td><td> 755 </td><td> （ - ）CTRL </td><td> 910 </td></tr><tr><td> CMPND乙</td><td> 2400 </td><td> CMPND乙</td><td> 2000 </td><td> CMPND乙</td><td> 2560 </td></tr><tr><td> （）CTRL </td><td> 2770 </td><td> （）CTRL </td><td> 2400 </td><td> （）中心升</td><td> 3020 </td></tr></tbody></table>对于N = 3的实验上面的值，阴性对照反应在每个实验中减去阴性对照基线触角机械粉扑和溶剂残留的假设下，从每个值。 <table border="1"><tbody><tr><td> 运行＃1 </td><td> 东亚运动会（μV峰） </td><td> 运行＃2 </td><td> 东亚运动会（μV峰） </td><td> 运行＃3 </td><td> 东亚运动会（μV峰） </td></tr><tr><td> （）CTRL </td><td> 2270 </td><td> （）CTRL </td><td> 1665 </td><td> （）CTRL </td><td> 2120 </td></tr><tr><td> CMPND一个</td><td> 2470 </td><td> CMPND一个</td><td> 1745 </td><td> CMPND一个</td><td> 2530 </td></tr><tr><td> （ - ）CTRL </td><td> 0 </td><td> （ - ）CTRL </td><td> 0 </td><td> （ - ）CTRL </td><td> 0 </td></tr><tr><td> CMPND乙</td><td> 1870 </td><td> CMPND乙</td><td> 1245 </td><td> CMPND乙</td><td> 1650 </td></tr><tr><td> （）CTRL </td><td> 2240 </td><td> （）CTRL </td><td> 1645 </td><td> （）CTRL </td><td> 2110 </td></tr></tbody></table>每个实验的阳性对照，然后将平均和纠正1000μV的，并为修正至1000μV的比例。数据表（如Excel）可以很容易地操纵转换成可用的数据。 <table border="1"><tbody><tr><td> 运行＃1 </td><td> AVG（μV峰） </td><td> 运行＃2 </td><td> AVG（μV峰） </td><td> 运行＃3 </td><td> AVG（μV峰） </td></tr><tr><td> （）CTRL </td><td> 2255 </td><td> （）CTRL </td><td> 1655 </td><td> （）CTRL </td><td> 2115 </td></tr><tr><td> （+）CTRL形容词。 </td><td> 1000（0.443） </td><td> （+）CTRL形容词。 </td><td> 1000（0.604） </td><td> （+）CTRL形容词。 </td><td> 1000（0.473） </td></tr></tbody></table>校正比率乘以在每个实验中，化合物A和化合物B的值，然后调整。 <table border="1"><tbody><tr><td> 运行＃1 </td><td> ADJ。东亚运动会（μV峰） </td><td> 运行＃2 </td><td> ADJ。东亚运动会（μV峰） </td><td> 运行＃3 </td><td> ADJ。东亚运动会（μV峰） </td></tr><tr><td> CMPND一个</td><td> 1094 </td><td> CMPND一个</td><td> 1054 </td><td> CMPND一个</td><td> 1197 </td></tr><tr><td> CMPND乙</td><td> 828 </td><td> CMPND乙</td><td> 752 </td><td> CMPND乙</td><td> 780 </td></tr></tbody></table>平均每个化合物（手段），然后确定，然后进行评估与其他相关统计数据和测试化合物的EAG反应候选人作进一步调查。 <table border="1"><tbody><tr><td> 复合 </td><td> 东亚运动会（μV峰） </td><td> 号运行N = </td></tr><tr><td>一</td><td> 1115 </td><td> 3 </td></tr><tr><td>乙</td><td> 787 </td><td> 3 </td></tr></tbody></table><img alt="图1" src="/files/ftp_upload/3931/3931fig1.jpg" /> 图1代表男性的触角反应（1,180μV），预先控制（1 ST阳性对照噗）将被丢弃后，确保触角的由于贫困触角响应东亚运动会与凝胶有良好的接触。在底部窗口的蓝色条代表的挥发性的第二粉扑。 <a href="http://www.jove.com/files/ftp_upload/3931/3931fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3931/3931fig2.jpg" /> <s阮富仲&gt;图2代表女性的触角反应（3,400μV），东亚运动会第一噗会被认为是适当的预控。 <a href="http://www.jove.com/files/ftp_upload/3931/3931fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/3931/3931fig3.jpg" /> （1,680μV），女性的触角反应的控制后（去年为每个实验的阳性对照），将被视为穷人，触角退化（&lt;75％1 日预控或暗示的代表东亚运动会&lt; 图3。 第二预控粉扑值）。对于这个例子中，1 日预控粉扑是2,730μV至2 次，预控粉扑2350μV的。 <a href="http://www.jove.com/files/ftp_upload/3931/3931fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <spa一类n ="pdflinebreak"&gt; <img alt="图4" src="/files/ftp_upload/3931/3931fig4.jpg" /> 图4。女性的触角反应（3800μV），粉扑候选人挥发性混合，随后测量最大的初始挠度（3800μV），在粉扑时间初步斜率（代表东亚运动会0.3 s/1.2 MV = 0.25 / MV），其余粉扑时间的斜率（1.6 s/1.9 MV = 0.84 S / MV）。 <a href="http://www.jove.com/files/ftp_upload/3931/3931fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" src="/files/ftp_upload/3931/3931fig5.jpg" /> 图5。小修改含有样品基质及相关挥发的船只，以张狂跨答transitella触角。 <img alt="表1" src="/files/ftp_upload/3931/3931table1.jpg" /> 表1。 在S国际电联的挥发排放无敌的杏仁（2007年）和在不同的Autospike方案和较不敏感的配置决定EAG反应。 <img alt="表2" src="/files/ftp_upload/3931/3931table2.jpg" /> 表2。形式记录个别挥发性成分的男性和女性的触角反应的例子。 <img alt="表3" src="/files/ftp_upload/3931/3931table3.jpg" /> 表3。例如一个记录男性和女性的触角反应挥发性混合物和/或花束的形式。 <img alt="表4" src="/files/ftp_upload/3931/3931table4.jpg" /> 表4。准备10毫升5毫克/毫升溶液两种不同比例的混纺的例子。 <img alt="表5" src="/files/ftp_upload/3931/3931table5.jpg" /> 举例记录2 conse的形式表5。男性和女性的触角跨单一的挥发性成分cutive泡芙。

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Ecol. 36, 584-591 (2010).</li><li>Takacs, S., Gries, G., Gries, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semiochemical-mediated+location+of+host+habitat+by+Apanteles+carpatus+(Say)+(Hymenoptera:+Braconidae),+a+parasitoid+of+cloths+moth+larvae.">Semiochemical-mediated location of host habitat by Apanteles carpatus (Say) (Hymenoptera: Braconidae), a parasitoid of cloths moth larvae.</a> J. Chem. Ecol. 23, 459-472 (1997).</li><li>Karimifar, N., Gries, R., Khaskin, G., Gries, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+food+semiochemicals+attract+omnivorous+German+cockroaches,+Blattella+germanica.">General food semiochemicals attract omnivorous German cockroaches, Blattella germanica.</a> J. Agric. Food Chem. 59, 1330-1337 (2011).</li><li>Molyneux, R. J., Schieberle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound+identification:+a+Journal+of+Agricultural+and+Food+Chemistry+perspective.">Compound identification: a Journal of Agricultural and Food Chemistry perspective.</a> J. Agric. Food Chem. 55, 4625-4629 (2007).</li><li>Marion-Poll, F., Thiery, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+EAG+responses+to+host-plant+volatiles+delivered+by+a+gas+chromatograph.">Dynamics of EAG responses to host-plant volatiles delivered by a gas chromatograph.</a> Entomol. Exp. Appl. 80, 120-123 (1996).</li><li>Beck, J. J., Higbee, B. S., Merrill, G. B., Roitman, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+volatile+emissions+from+undamaged+and+mechanically+damaged+almonds.">Comparison of volatile emissions from undamaged and mechanically damaged almonds.</a> J. Sci. Food Agric. 88, 1363-1368 (2008).</li><li>Lucas, P., Renou, M., Tellier, F., Hammoud, A., Audemard, H., Descoins, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+and+field+activity+of+halogenated+analogs+of+(E,E)-8-10-dodecadien-1-ol,+the+main+pheromone+component+in+codling+moth+(Cydia+pomonella+L.).">Electrophysiology and field activity of halogenated analogs of (E,E)-8-10-dodecadien-1-ol, the main pheromone component in codling moth (Cydia pomonella L.).</a> J. Chem. Ecol. 20, 489-503 (1994).</li><li>Rodriguez-Saona, C., Poland, T. M., Miller, J. R., Stelinski, L. L., Grant, G. G., de Groot, P., Buchan, L., MacDonald, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+electrophysiological+responses+of+the+emerald+ash+borer,+Agrilus+planipennis,+to+induced+volatiles+of+Manchurian+ash,+Fraxinus+mandshurica.">Behavioral and electrophysiological responses of the emerald ash borer, Agrilus planipennis, to induced volatiles of Manchurian ash, Fraxinus mandshurica.</a> Chemoecology. 16, 75-86 (2006).</li><li>Burks, C. S., Brandl, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+abundance+of+navel+orangeworm+(Leipidoptera:+Pyralidae)+in+figs+and+effect+of+peripheral+aerosol+dispensers+on+sexual+communication.">Seasonal abundance of navel orangeworm (Leipidoptera: Pyralidae) in figs and effect of peripheral aerosol dispensers on sexual communication.</a> J. Insect Sci. 4, 1-8 (2004).</li></ol>

笔者派拉蒙养殖公司，Suterra有限责任公司的关系公司与现有的合作研究和开发协议。

作为一种生物活性，以确定目标昆虫的化学感受反应电位录音使用相当普遍，许多可以利用探测器从气相色谱（GC-EAD）的污水作为东亚运动会的研究在文献中发现。9,10的方法证明将提供等量的挥发性成分与自信分配的相对响应的高重复的快速筛选。 AutoSpike在Syntech软件程序是为筛选挥发好的方案，因为它是能够提供最大挠度从天线的幅度信号（ 图1-3），这是我们这里提出的"?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>苯乙</td><td>阿尔法（天津）化学有限公司</td><td> A12727 </td><td>女性阳性对照</td></tr><tr><td> （Z，Z）-11,13-Hexadecadienal </td><td> suterra </td><td></td><td>男阳性对照</td></tr><tr><td> α-蛇麻烯</td><td>奥尔德里奇</td><td> 53675 </td><td>倍半萜</td></tr><tr><td> 2 - 十一酮</td><td>奥尔德里奇</td><td> U1303 </td><td>脂肪酸衍生物</td></tr><tr><td> 2 - 苯乙醇</td><td>奥尔德里奇</td><td> 77861 </td><td>苯</td></tr><tr><td>戊烷</td><td>电解二氧化锰</td><td> PX0167-1 </td><td>溶剂</td></tr><tr><td>收购4通道控制器</td><td> syntech </td><td>我DAC-4 </td><td></td></tr><tr><td>东亚运动会探头，前置放大器</td><td> syntech </td><td> PRG-2型</td><td></td></tr><tr><td>天线支架</td><td> syntech </td><td> PRG-2 </td><td>叉电极</td></tr><tr><td>刺激控制器</td><td> syntech </td><td> CS-55 </td><td>空气流和喷</td></tr><tr><td>光谱电极凝胶</td><td>帕克</td><td> 12-02 </td><td></td></tr><tr><td>生物活性光盘</td><td>瓦特曼</td><td> 2017-006 </td><td> 6毫米</td></tr><tr><td>巴斯德滴管</td><td>厂商VWR </td><td> 14673-010 </td><td> 5¾英寸（14.6厘米） </td></tr><tr><td>封口膜中号</td><td>比米斯</td><td> PM-992 </td><td></td></tr></tbody></table>

electroantennographic bioassay as a screening tool for host plant volatiles

植物挥发物发挥重要作用，在植物，昆虫相互作用。食草昆虫利用植物挥发物，如米蛾利他素，找到他们的寄主植物。1,2当寄主植物是一个重要农艺商品饲养害虫的损害，可以给种植者造成严重的经济损失。因此，米蛾利他素可以被用来作为这些昆虫引诱或混淆，因此，提供一个环境友好的替代杀虫剂控制昆虫的引诱。不幸的是，植物能发出不同的成分和比例的排放量取决于物候的一个巨大的数量挥发商品或一天的时间。这使得生物活性成分的鉴定或一个艰难的过程挥发性成分的混合物。为了帮助确定我们聘请的实验室为基础的筛选生物活性electroantennography（东亚运动会）的寄主植物挥发物的活性成分。东亚运动会是一个有效的工具来评估和recorð电生理通过他们的触角感受器的昆虫的嗅觉反应。东亚运动会的筛选过程，可以帮助减少一些测试，以确定有前途的生物活性成分的挥发。然而，东亚运动会的生物测定只提供有关受体激活的信息。它不提供有关昆虫行为的化合物引起的类型的信息，这可能是作为引诱剂，驱虫剂或其他类型的行为反应。挥发引出由东亚运动会上，相对适当的阳性对照显着的反应，通常采取进一步虫害的行为反应测试。实验设计将详细的方法来筛选基于杏仁寄主植物挥发物的触角电生理反应测量成人虫害肚脐orangeworm（Amyelois transitella）单部件和组件的简单混合物通过东亚运动会的生物活性4,5。该方法利用两个前cised天线置于整个一个"叉"的电极支架。这里展示的协议提出了一种快速，高通量筛选挥发物的标准化方法。每个波动是在一组，固定金额，以规范的刺激水平，从而使触角反应相对chemoreceptivity的指示。阴性对照，有助于消除粉扑残留溶剂和机械力的电生理反应。阳性对照组（在这种情况下苯乙酮）是一个单一的化合物，已引起了男性和女性的肚脐orangeworm（NOW）蛾的一致响应。一个的额外semiochemical标准，提供一致的响应，并用于生物活性的研究与男性现在蛾是（Z，Z）-11,13-hexdecadienal，从女性的生产性信息素醛组件。6-8

Watch this Scientific Journal Video about Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles at JoVE.com

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

一个迅速屏幕寄主植物挥发的成人肚脐orangeworm的电生理反应（测量方法<em&gt; Amyelois transitella</em&gt;）天线，单一组件和通过electroantennographic分析混纺证明。

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 ...

electroantennographic-bioassay-as-screening-tool-for-host-plant

Research

JoVE Journal

Biology

13.6K 次观看.  Agricultural Research Service. 一个迅速屏幕寄主植物挥发的成人肚脐orangeworm的电生理反应（测量方法 Amyelois transitella）天线，单一组件和通过electroantennographic分析混纺证明。

视频: Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

一个β-葡萄糖醛酸酶（GUS）的基细胞死亡含量

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

科研

生物学

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

蓝莓虫害诱导挥发物和植物内信令

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

荧光显微镜筛选和新一代测序:参与细胞器完整性的基因鉴定的有用工具

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

RNA催化剂作为记者为筛选药物对RNA编辑在锥虫

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

一个简单的生物测定血管内皮生长因子的评价

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

一种快速的疾病抑制初步评估的板块竞争分析法

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify&#160;molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts.
Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is&#160;carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.