MultiBioPro要感谢下面的人谁也有助于项目：多米尼克·斯温顿（绿色燃料），托马斯·洛厄里（绿色燃料），山姆Buekenhout（CAPAX）和西尔维亚Drouven（CAPAX）。

1，植物材料成长粉蓝烟草植物中含30 M2的专业成长中公分直径的花盆。 生长的植物在温室中，用20-25℃的日间温度和15℃。夜间温度使用16小时光照和8小时黑暗周期为补充光照制度。 2，样品制备<ol><li> 对于初级代谢产物： <ol><li>收获的植物材料（叶和茎北路栎 ），并立即冷冻材料。 </li><li>通过冷冻干燥冻干冷冻的植物材料为三天。 </li><li>通过搅拌机磨与金属球研磨冻干样品。 </li><li>等分细地干物料到2ml离心管或玻璃小瓶中。 </li></ol></li><li> 对于次级代谢产物： <ol><li>收获植物组织，并立即冷冻材料。 </li><li>用m冷冻研磨植物组织ixer磨不锈钢球或研钵和杵。 </li><li>分装精细地组织分为2 ml离心管（约20毫克湿重）。 </li></ol></li></ol> 3，萃取气相色谱 - 质谱协议的代谢物谱的初级代谢产物<ol><li>等分部分地干燥的植物材料（10毫克叶和20mg的干物料）在2毫升，螺旋盖圆底试管中。 </li><li>新增1,400微升100％甲醇和漩涡，持续10秒。 </li><li>加入60微升核糖醇（0.2 mg / ml的股票在H 2 O）作为内部量化的标准，涡旋10秒。 </li><li>新增一个不锈钢（或氧化锆）球和均质材料用搅拌机磨在25赫兹2分钟。 </li><li>离心混合10分钟，11,000×g的。 </li><li>将上清液转移至玻璃小瓶中。 </li><li>加入750μL氯仿。 </li><li>新增1,500微升H 2 O和旋涡振荡混合10秒。 </li><li>从转移150微升上层相（极性相）到一个新的1.5毫升离心管中。 </li><li>用快速真空干燥样品。至关重要的是，将样品浓缩至干24之间3和小时，直到没有残留液体存在。 </li><li>加入40微升甲氧胺盐酸盐（20毫克/毫升吡啶）的。 </li><li>在37°C摇混在2小时水平散热块振动筛</li><li>加入70微升的N-甲基-N - （三甲基硅基）三氟乙酰胺三氟乙酰胺混合。 </li><li>摇动混合2小时，在37°C。 </li><li>通过短暂离心〜1分钟，11,000×g的降速封面上的下降。 </li><li>液转移到合适的用于GC-MS分析的玻璃小瓶中。 </li></ol> 4，提取的代谢物谱的次生代谢产物的LC-MS <ol><li>加入500μl甲醇的冻土样品。 </li><li>具有恒温15分钟摇动，在70℃（1,000秒-1）。 </li><li>离心样品（5英里N，20000×g离心），并转移到1.5ml的离心管中的液体相。 </li><li>冻干液相用离心蒸发器（Speed​​Vac中）。 </li><li>加入100μl环己烷和100μL超纯水的干颗粒。 </li><li>摇晃样品5分钟，在室温下（利用涡流）。 </li><li>离心样品（5分钟，20,000×g离心）。 </li><li>转移80μL水相（下层相），以LC-MS注射瓶带有锥形底部的LC-MS分析。 </li></ol> 5。数据分析<ol><li>配置Xcalibur，MarkerLynx，Metalign 7或XCMS 8选择数据分析进行处理。 </li><li>准备你按照表1复方阶级利益的峰值检测表。 </li><li>通过标准的化合物共流出识别峰。 </li><li>使用MSN分析，结构表征算法9,10，文学s随后注释检测到的峰urvey和代谢物数据库检索11,12。 </li></ol> 6，油气开采13 <ol><li>浸入新鲜收获N.青冈叶（〜200mg）的溶剂（甲醇，乙醇，氯仿，己烷或石油醚（沸点40-60℃或60-80℃）（5毫升; HPLC级）为2至20分钟。 </li><li>除去叶子，彻底擦干溶剂用旋转蒸发器。 </li><li>在己烷中重悬的样品（1毫升; HPLC级）。 </li><li>用气相色谱法和连字符到一个5973MSD进行GC-MS分析。工作条件应如下;载气，氦气为0.9毫升·分-1 / 11磅的流率。注入样品（1微升）用不分流进样器在280℃下升温，在4℃/分钟至320℃之前，容纳了气相色谱烘箱中在70℃下进行5分钟保持最后的温度下再10分钟后，使67.5分钟的总时间。与MS设置接口在280℃和perform MS在使用70伏特EI +全扫描模式和从10到800 D.扫描</li><li>最初处理的色谱部件通过自动去卷积MS和识别系统，和那些正在使用NIST 98 MS谱库鉴定。 </li><li>通过比较保留时间和经典的碎片模式，以已知的真实标准确认饱和的长链烷烃（hexacosenol，二十九烷，三十烷，octacosenol，nonacosonal，hentriacontane，三十二烷和tritriacontane）的标识。 </li><li>通过比较剂量反应曲线（0.07至2.5毫克），从真实标准构建集成峰面积定量确定烃。 </li><li>计算方法和使用Excel软件的方法（SEM）的标准误差。 </li></ol>类异戊二烯14 7。分析<ol><li>在第2节中所述进行均质。称取10毫克匀浆冻干粉到离心管中。</li><li>依次加入250ml甲醇上粉，拌匀，然后加入500氯仿溶液（实验室试剂级），并通过涡旋混合。 </li><li>留在冰上的样品在黑暗中20分钟。 </li><li>添加250毫升的Tris-HCl缓冲液（100毫米，pH值7.5）或水（HPLC级）悬浮液中并涡旋混合。 </li><li>以12,000 rpm（13,523×g离心）离心混合物5分钟，以非极性从水相分离。含有异戊二烯的萃取非极性氯仿相是在底部。 </li><li>相转移至新的离心管中。 </li><li>增加一个额外的500毫升氯仿至水相，并进行第二次提取由涡流并如上所述离心。 </li><li>合并氯仿萃取液，并把样品用蒸发器，并储存于-20℃直至分析至干。 </li><li>加入50毫升的乙酸乙​​酯（HPLC级），以干燥后的类异戊二烯萃取再溶解的物质。 </li><li>注入3毫升到C18使用的Acquity UPLC分离模块列。用于分离色素的梯度应包括;答：甲醇/ H 2 O（50:50）和B：乙腈/乙酸乙酯（75:25），和初始条件应该是A的30％（体积/体积）和B 70％（V / V）将100 ％B，历经6分钟。用0.6毫升/分钟的流速。 </li><li>与光电二极管阵列（PDA）检测器在250〜600 nm的连续监测洗脱液。 </li><li>通过共同色谱和正宗的标准，β-胡萝卜素（维生素原A），八氢番茄红素，番茄红素，叶黄素和玉米黄质的光谱进行比较识别组件。 </li><li>从剂量反应曲线进行定量。 </li><li>用LC-MS确认的化合物;使用类似的色谱条件。应使用APCI电离正离子模式与Q-TOF仪器上完成检测。 </li><li>做的修改对于生育酚判定与已提取和皂化在6％的KOH在50℃下1.5小时25毫克冻干材料。 </li></ol><p类=“jove_title”&gt; 8。 RNA提取的RNA序列该协议结合了Trizol法提取RNA用RNeasy试剂​​盒获得高质量的RNA。 <ol><li>通过混合器研磨机研磨100毫克冷冻植物材料在2毫升的离心管中与金属球。 </li><li>加入1 ml Trizol法。 </li><li>离心10分钟，该材料在12,000×g离心在4℃下</li><li>将上清液转移到一个新的反应管中。 </li><li>添加200μl的氯仿，颠倒试管几次，孵育3分钟，在室温下进行。 </li><li>离心反应混合物15分钟，以12,000×g离心在4℃下</li><li>转移上层水相（约700微升）到一个新的反应管中。 </li><li>加约2.5倍体积的乙醇​​（100％），颠倒试管几次，并孵育30分钟，于-80℃。 </li><li>移动样品包括任何沉淀物，以从试剂盒中的旋转柱。 </li><li>离心柱为15秒，8,000×g离心，弃了F低通。 </li><li>加入700μl缓冲液RW1到离心柱，离心15秒，8,000×g的。通过丢弃的流量。 </li><li>加入500μl缓冲液RPE到离心柱，离心15秒列在8000×g的。通过丢弃的流量。 </li><li>加入500μl缓冲液RPE到离心柱和离心2分钟，8,000×g的。 </li><li>将离心柱到一个新的1.5 ml反应管中，加入50μl的不含RNA酶的水。 </li><li>离心1分钟的列在8000×g离心来洗脱RNA。 </li><li>使用Ambion公司的DNA-free试剂盒根据制造商的说明去除残留的DNA。 </li><li>确定RNA的质量。 RNA的应该有超过8 RNA的完整性号码（RIN）。 </li><li>送行的RNA为下一代测序。 </li></ol>

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这项研究导致这些结果根据赠款协议号311804已收到的资金从欧洲第七框架计划（[FP7/2007-2013]）。

这里介绍的协议提供了一个全面的框架来分析烟草树叶的代谢物和成绩单。根据设想，这些联合的努力应该为我们提供了新的见解的碳氢化合物和目前在这个组织中的高价值化合物的合成和挤出相关的进程。因此，这些方法应该给我们怎样的化合物被合成一个更好的理解。除了烟草树方面的工作，目的也是为了提高杨树生物量，特别是针对次生壁结构的木质化，更要研究我们是否可以使用树皮提取有价值的化合物。 在本文提出的方法是标准化的方法代谢物谱稍作修改。这些方法当然不限于已知的代谢概况，并且有可能是几个新的代谢峰可能为瓦特以下方式获得HICH无化合物是已知的。我们希望通过合并代谢产物和成绩单在发育的时间序列行为把这些化合物在上下文中的其他代谢产物。 没有这里介绍的方法，从方法通常用于植物材料的显著改变。有趣的方面在于相结合的方法来理解主要是长链烃的生产和改装烟草树叶的底层框架。一种用于获得该信息的关键步骤是在不同的数据类型的随后的结合。我们设想，将数据作为第一评价的基础上，代谢/转录在发展，而这些数据将被用于推断的转录与代谢物的行为，并且还可能分配某些代谢产物途径的行为将被划分为不同的簇。另外，更复杂的基于网络的分析，然后设想吨Ø利用因果关系。 这里提出的分析方案也将提供现场试验和工业开发的生物质的基础。要做到这一点，MultiBioPro财团包含几个工业合作伙伴有能力进一步开拓生物质，目的是提供生物柴油，生物乙醇和其他高价值的化合物。这些类型的生物质利用，将根据评估; （1）测试生产生物产品的稳定性和质量（典型的工业标准测试将进行，以确保产生的产品具有良好的市场价值），（2），这些技术的经济，社会和环境评估将被执行采用文献资料，访谈，并且是在现场试验和试点生物精炼厂产​​生的评估材料。这些活动将包括成本效益和生命周期分析，环境卷宗和市场以及商务的产生SS策略。我们认为，这条管道将成为学​​术界的一个有用的融合，应用科学和工业开发，进一步杨树和烟草树生物质能消费的终端产品。

A correction was made to: <a href="http://www.jove.com/video/51393/transcript-metabolite-profiling-for-evaluation-tobacco-tree-poplar-as">Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry</a>.
There was a spelling error in one of the authors' surname. The author's name was corrected from: 
Juan Pedro Navarro
to:
Juan Navarro-Avi&#241;&#243;

A correction was made to: Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry.

Erratum: Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

人口和经济增长造成了对食物，水和燃料的需求不断增加。许多这些用品的生产，加工和利用有限的矿物化手段，如石油输送。它是，但是，显然，这种做法是不可持续的，因此可替代资源的发展将具有十分重要的意义1。许多可再生资源，在不同程度上，当前正被利用，包括风，水运动，太阳能，地热能和波浪为主的能源来源。另一种可持续的，主要是未开发的资源是来自植物的生物量。这个资源还提供了一个非常符合成本效益的方法来转换太阳能所产生的能量转化成燃料2。除了提供生物基燃料，植物生物量还提供了替代产品，包括塑料，洗涤剂和有价值化学品的独特机会。 植物细胞壁，这在很大程度上是由基于糖的聚合物，麦的es了植物的生物量的主要大宗，目前正在投入了大量精力在其高效转化为生物乙醇。余下的生物质可随后被加工成沼气和石油相关的产品3。大部分的多年生植物，包括各种草和树木，产生大量的纤维素生物质的一般生长最好在温带地区。然而，土地面积约20％，属半干旱，因此也容易发生旱灾4。显然，这将是感兴趣的也培养这些干旱地区的植物，可以有效地促进可持续能源生产和材料。这些植物需要有一个最佳的水分利用效率和抗旱性，并会包括烟草树（ 粉蓝烟草 ），从龙舌兰属的物种。 该MultiBioPro财团的目标是实现一个集成的生物精炼厂的管道，使用两个重要的CR运种，杨树和烟草树。杨树已成为一个有前途的生物燃料作物，因为它是生长速度快，容易无性繁殖和高度适应广泛的气候和土壤条件。它还提供了一个广泛的木材，纤维，燃料木材和其他林产品5。烟草树也成为一个合适的植物的生物燃料和生物炼制的目的。它通常会产生相当大量的生物质，含有大量的非结构性碳水化合物6的，也有积累大量的易于提取的非食用油脂（包括长链C 29-C 31饱和烃和三萜类），适合在生物柴油罕见的能力生产。烟草树，而且，服从遗传改良，具有较高的发芽能力，并愉快地在不用于食品生产半干旱地区土壤中生长。因此，似乎这两个杨树和烟草树具有内在潜力MULTIPurpose作物， 即作为新的高价值原料为一体的生物基工业。在本文中，我们着重于方法多样化辨别烟草树存款有多长链烃。 在试图确定负责对烟叶的饱和长链烃的生产和分泌潜在的分子机制，我们应用基础的技术现代的“组学”。这包括发育叶系列（10级）的RNA序列，以及多平台代谢物谱方法，使用LC-和GC-MS（用于极性和非极性代谢物和脂质组学）。这些数据将用于挖掘的基因表达，与相关联，或者先于，上文所述的分子的生物合成的起始。出现这些努力有希望的基因和途径，将用于在模式植物拟南芥功能测试，并可能最终将服从于TOB生物技术工程ACCO树。

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			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:124px;">Company&#160;</td>
			<td style="width:144px;">Catalog number&#160;</td>
			<td style="width:64px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol reagent</td>
			<td>Invitrogen&#160;</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>Merck</td>
			<td>102445</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Merck</td>
			<td align="right" style="width:144px;">101986</td>
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			<td height="21" style="height:21px;">Rneasy Mini Kit</td>
			<td>Qiagen</td>
			<td align="right">74104</td>
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			<td height="21" style="height:21px;">TURBO Dnase</td>
			<td>Invitrogen&#160;</td>
			<td>AM2238</td>
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			<td height="21" style="height:21px;">RNA 6000 Nano Kit</td>
			<td>Agilent</td>
			<td>G2938-90034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2100 Electrophoresis Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 ml and 2 ml safe-lock tubes</td>
			<td>Eppendorf</td>
			<td>&#160; 0030 120.086, 0030 120.094</td>
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			<td height="21" style="height:21px;">Steel balls</td>
			<td>Geyer Berlin GmbH</td>
			<td>VA2mm</td>
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			<td height="21" style="height:21px;">Mixer mill MM 300</td>
			<td>Retsch</td>
			<td>YO-04182-09</td>
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			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>Eppendorf</td>
			<td align="right">5424</td>
			<td></td>
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transcript and metabolite profiling for the evaluation of tobacco tree and poplar as feedstock for the bio-based industry

用于食品，饲料，能源和水资源的全球需求带来的后代寻常的挑战。显而易见的是，健壮平台为可再生资源的勘探是必要的，以克服这些挑战。在多国框架MultiBioPro我们正在开发的生物精炼厂的管道，以最大限度地利用植物生物量。具体而言，我们使用杨树和烟草树（ 粉蓝烟草 ）作为目标作物品种改善糖化，异戊二烯，长链烃含量，纤维品质和木栓质和木质素含量。用于获得这些输出的方法包括GC-MS，LC-MS和RNA测序平台。代谢物管线完善的工具来生成这些类型的数据，但也有只有充分表征代谢物的限制都可以使用。深测序将使我们能够包括所有笔录在烟草树的叶子发育阶段存在，但ħ以被映射回烟草的序列。有了这些设置窗口，我们的目标是一个基本的了解的基本流程，并在建立工业框架，利用这个结果。在一个更长远的角度来看，我们认为，这里所产生的数据提供装置，用于利用杨树和烟草树为原料可持续生物炼制过程。迄今为止代谢物的基础水平的样本中进行了分析，并利用该协议本文中提供。

图1中的HPLC图谱显示了N的类异戊二烯分析的代表性结果青冈叶提取物。采用光电二极管阵列（PDA）检测器进行检测C40的不同的类异戊二烯及以上。基于共同色谱和正宗的标准，β-胡萝卜素（维生素原A），八氢番茄红素，番茄红素，叶黄素和玉米黄质的光谱比较峰注释。 图2中的2的MS色谱图显示从N.初级代谢产物的分析结果青冈叶和茎的材料，分别。对应于丝氨酸（由箭头指示）的峰的质谱图，也给出一个例子。 图3显示了生物分析仪用于测定RNA的质量和从设备输出一个代表。在色谱图中的两个主峰对应于18 S和25秒的核糖体RNA，表明完整的RNA样品中。零散的肋骨附加峰osomal RNA会出现的情况下部分或严重降解的RNA。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51393/51393fig1highres.jpg" src="/files/ftp_upload/51393/51393fig1.jpg" /> 图1显示存在于全叶提取物类异戊二烯HPLC图谱青冈。C40及以上的线路异戊二烯不适合于GC-MS分析。因此，我们用HPLC分离光电二极管阵列检测。显示记录在450 nm的典型色谱图。类胡萝卜素颜料是典型的叶黄素光合一边倒组织。同时出席的玉米黄质，这是很少的叶组织中发现，除非放置在高光的压力。玉米黄质的含量使其成为这个高价值的化合物的良好来源。 <a href="https://www.jove.com/files/ftp_upload/51192/51192fig1highres.jpg" target="_blank">请点击这里查看更高的V</a>版为这个数字的。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51393/51393fig2highres.jpg" src="/files/ftp_upload/51393/51393fig2.jpg" /> 图2：MS色谱和N的频谱叶木莲组织提取物，总离子质谱色谱图（TIC）和茎用GC-MS测定提取物呈现（70-600 M / Z）。如在15前面的描述进行GC-MS分析。检测到的峰均采用质谱标记库的注解。丝氨酸（2TMS）的MS光谱示作为一个例子。 MS色谱：X轴和Y轴表示保留时间（分钟），并将该信号的强度（丰度），质谱：X轴和Y轴表示的M / Z比率和该信号的强度（丰度），分别为。 <a href="https://www.jove.com/files/ftp_upload/51393/51393fig2highres.jpg" target="_blank">点击这里查看大</a>图像。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51393/51393fig3highres.jpg" src="/files/ftp_upload/51393/51393fig3.jpg" /> 图3为N的序列的RNA制备的RNA的生物分析仪测量青冈叶的材料。为了获得所需要的，我们提取的RNA用Trizol试剂，其后使用的列从RNeasy Mini试剂盒（Qiagen公司，希尔登，德国）纯化的RNA的RNA起高纯度的RNA。我们决定使用生物分析仪（安捷伦，德国Waldbronn）显示在左侧的RNA的质量。一个生物分析仪的输出的一个例子给出在右侧。样品中的两个主要的峰代表18 S和25秒的核糖体RNA。我们的样品显示出RNA的完整性数9.2（RIN），这是远高于8所需的值。 <a href="https://www.jove.com/files/ftp_upload/51393/51393fig3highres.jpg" target="_blank">点击她</a>E要查看大图。

Watch this Scientific Journal Video about Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry at JoVE.com

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

植物生物质，提供对多个产品，包括燃料，饲料，食品和各种材料的可再生资源。在本文中，我们探讨烟草树（ 粉蓝烟草 ）和杨树为适合来源的生物炼制管线的属性。

成绩单及代谢物谱烟草树和杨树的评价作为原料的生物基工业

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

transcript-metabolite-profiling-for-evaluation-tobacco-tree-poplar-as

Research

JoVE Journal

Environment

15.0K 次观看.  Max Planck Institute for Molecular Plant Physiology. 植物生物质，提供对多个产品，包括燃料，饲料，食品和各种材料的可再生资源。在本文中，我们探讨烟草树（ 粉蓝烟草 ）和杨树为适合来源的生物炼制管线的属性。 

视频: Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

技术树内组织学习节肢动物和微生物群落

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

科研

环境科学

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

重组纤维素酶Cel5A从表达<em&gt;里氏木霉</em&gt;在烟草

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal carbonization (HTC) are promising technologies for bioenergy production from biomass in terms of biogas and HTC biochar, respectively. In this study, the combination of AD and HTC is proposed to increase overall bioenergy production. Wheat straw was anaerobically digested in a novel upflow anaerobic solid state reactor (UASS) in both mesophilic (37 &#176;C) and thermophilic (55 &#176;C) conditions. Wet digested from thermophilic AD was hydrothermally carbonized at 230 &#176;C for 6 hr for HTC biochar production. At thermophilic temperature, the UASS system yields an average of 165 LCH4/kgVS&#160;(VS: volatile solids) and 121 L CH4/kgVS&#160;at mesophilic AD over the continuous operation of 200 days. Meanwhile, 43.4 g of HTC biochar with 29.6 MJ/kgdry_biochar was obtained from HTC of 1 kg digestate (dry basis) from mesophilic AD. The combination of AD and HTC, in this particular set of experiment yield 13.2 MJ of energy per 1 kg of dry wheat straw, which is at least 20% higher than HTC alone and 60.2% higher than AD only.

A novel Upflow Anaerobic Solid State (UASS) reactor was used for biogas production from fibrous feedstock. Digestate from UASS reactor was hydrothermally carbonized into HTC biochar in a pressurized batch reactor. The integration of the two bioenergy concepts was applied in this study to increase overall bioenergy production.

评定一体化厌氧消化和水热碳化对生物能源生产

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

协议塑料微粒采样的海面和样品分析

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

树叶颗粒物去除能力的评价

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

一种用自动表型分析预测线虫化学毒性的高通量测定

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, phloem transport and vascular patterning. This protocol shows bottom-to-top carboxyfluorescein (CF) movement in the Arabidopsis by using the root-cutting and the hypocotyl-pinching procedure respectively. These two different procedures result in different efficiencies of CF movement: about 91% appearance of CF in the shoots with the hypocotyl-pinching procedure, whereas only about 70% appearance of CF with the root-cutting procedure. The simple change of loading sites, resulting in significant changes in the mobile efficiency of this symplastic dye, suggests CF movement might be subject to the symplastic regulation, most probably by the root-hypocotyl junction.

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The goal of this protocol is to show how to load the CFDA into different sites of the bottom parts of Arabidopsis. We then present the resulting distribution pattern of CF in the shoots.

美国食品药品监督管理局追踪者在拟南芥底部水槽组织中的射击运动

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, ...