这项工作是支持的BMBF Forschungsinitiative“BioEnergie 2021 - Forschung献给死去Nutzung冯Biomasse”创优“量身定制燃料生物质”，它是通过卓越计画经费由德国联邦和州政府，以促进科学的集群并在德国大学的研究。

1，生长野生型普通烟草植物<ol><li>成长N.烟草 L。 CV。对土壤佩蒂特哈瓦那SR1植物，放置在合适的花盆装满正常的盆栽土壤发芽烟草种子。 2周之后，苗转移至个别盆。孵育恒22°C（黑暗时期）或25℃（光周期）在温室中的植物和70％的相对空气湿度。在八分之一十六小时的光/暗周期提供照明（ 如飞利浦IP65 400瓦灯泡; 180微摩尔·秒-1·米-2，λ= 400〜700 nm）和水，每天。 </li></ol> TrCel5A和TrCel5A-mCherry的蛋白质2。瞬时表达<ol><li>在无菌条件下，挑Agarobacterium癌农杆菌含有两种pTRAkc-ER-TrCel5A或pTRAkc-ER-TrCel5A-的mCherry并转移到含有10ml酵母提取物锥形瓶中单菌落（GV3101 :: pMP90RK GMR，KMR，RIFR）肉汤（YEB，制备成每制造商的规格）介质和卡那霉素（50毫克/毫升），利福平（50毫克/毫升），和羧苄青霉素（100毫克/毫升）以供选择。孵育36-40小时，在26℃和180转。注意：适当的结构来表达设计是本文的范围，但作为指导，可遵循的结构设计麦克莱恩等[13]和芒罗和Pellham 14的方法。 </li><li>取200μl的各培养物接种到20毫升YEB与上面提到的抗生素浓度和以前一样在相同条件下孵育。 </li><li>经过36-40小时后，通过加入2微升acetosyringon（200μM的最终浓度），加入100μl的40％（重量/体积）的葡萄糖溶液和200微升的1M MES缓冲液pH 5.6。preinduce培养孵育过夜。 </li><li>制备100毫升2×缓冲液浸润（100克/升蔗糖，3.6克/升葡萄糖，8.6 g / L的Murashige和Skoog基础盐的用氢氧化钾调节pH值至5.6）。 </li><li>测量1:5稀释的培养物的OD 600，直到它达到的OD 600为5-6。计算出外径600需要30毫升渗透培养基培养体积。加入15毫升2X渗透缓冲，再加入去离子H 2 O至使缓冲高达〜30毫升。 </li><li>就拿植物温室和使用枪头缺口至5 日叶第三的远轴侧从上缓解渗透。 </li><li>用枪头缺口叶子的背面侧，以缓解渗透。 </li><li>填充注射器渗透介质，并把它（不带针）对先前作出的刻痕之一。支持注射器用手指在近轴叶边。仔细按介质进入脉间叶区的组织。注：浸润后，培养在温室中的植物如上文所述的相同的条件下进行。此外，保持untr同等比例eated，非瞬时表达植株（NTEPs）作为对照，在相同的条件下渗入植物下。 </li></ol>在植物叶片TrCel5A-mCherry的表达3。评估<ol><li>经过4-6天，采取植物渗透与甲农杆菌含有pTRAkc-ER-TrCel5A-的mCherry从温室，并将其放置在一个黑暗的房间荧光分析。 </li><li>可视化的荧光表达TrCel5A-的mCherry，光电厂使用的是便携式光源发射绿光在515 nm处有一个红色滤光片（620-750 nm）的叶节。注：在深度在植物叶片蛋白表达的鉴定可以通过薄切片用的叶子在光镜和荧光显微镜一vibratome和考试来实现。对于这种按照以下步骤操作： <ol><li>切约5×5毫米大小的小部分从叶渗透并嵌入在4％琼脂糖溶于50 mM磷酸盐缓冲液（pH 5.7）。 </li><li>切80-90微米大小使用一个vibratome嵌入式叶节。将它们放置在载玻片上，盖上缓冲或盖玻片，用显微镜在10倍放大倍率荧光检测器，使用相同的波长和过滤上述检查它们。 </li></ol></li></ol> 4。TrCel5A提取烟叶<ol><li>从感染A.植物从切烟叶片到1克的重量大约农杆菌含有pTRAkc-ER-TrCel5A并置于预冷用液氮在研钵。加液氮适量的样品。用预冷杵研磨叶粉。 </li><li>加入2毫升含有1mM苯甲磺酰氟的地面叶物质的磷酸盐缓冲盐水（PBS）中并混合，直到大部分的叶物质是在悬浮液中。离心提取液以15,000×g离心20分钟，在4℃，将含有蛋白的上清液。 </li><li>离心夫格提取物以15,000×g离心20分钟，在4℃，将含有蛋白的上清液，注意不要扰乱颗粒。弃沉淀。 </li><li>部分纯化TrCel5A孵育含蛋白质的上清液在55℃下10分钟。允许休息在室温下再保持10分钟，然后离心，15,000 xg下在RT下10分钟。重复部分纯化过程非渗透的植物来获得对照样品。使用这些样本为所有下面的实验。注：蛋白质溶液可以贮存于4℃下长达1周或在-20℃下长达3个月。 </li><li>重复该部分纯化过程非渗透的植物来获得对照样品。使用这些样本为所有下面的实验。注：蛋白质溶液可以贮存于4℃下长达1周或在-20℃下长达3个月。 </li></ol> 5，SDS-PAGE电泳，免疫印迹和TrCel5A-mCherry的&lt;中偶氮CMC酶谱/ P&gt; <ol><li>购买或准备12％（W / V）SDS-PAGE凝胶按照确定的方法15。 </li><li> CMC溶解在水中，得到1.5％（重量/体积）溶液。使CMC-SDS-PAGE凝胶，替换1毫升1.5％CMC的在10ml的SDS-PAGE凝胶的混合溶液中加入1ml水，造成了0.1％（重量/体积）CMC + 12％（w / v的）SDS-PAGE凝胶组合。通常倒入凝胶。注意：请确保管委会在原有的解决方案是完全由搅拌和加热的组合，以40℃溶解（重复制备凝胶溶液直接前）。 </li><li>通过在SDS存在下煮沸，按照已建立的方法12变性的样品。作为阳性对照，用1:500稀释从T.市售的纤维素酶混合物的里氏木霉 ，如T.里氏木霉 ATCC 26921。作为阴性对照，使用来自非渗透的叶（NTEPs）一个类似的方法制备的蛋白质溶液。 </li><li>进行电泳按正常协议15，例如：用200 V为〜50分钟，停止电泳一旦染料前沿到达凝胶的底部。 </li><li>用0.1％染色1的SDS-PAGE凝胶（重量/体积）考马斯亮蓝和脱色用甲醇/冰醋酸混合物。注意：快速染色和脱色可以通过轻轻加热染色和脱色的解决方案， 即通过使用微波对〜15秒预热（但不沸腾）第一染色，然后脱色溶液，改变在10分钟后的脱色液进行时，或者当冷却，并重复与新的脱色液。 </li><li>执行使用既定协议16,17 1 SDS-PAGE凝胶的印迹。使用下面的抗体：抗His-标签（主）和一个多克隆山羊抗兔抗体Fc区已被缀合至碱性磷酸酶（次要）的多克隆兔抗体。注：二级抗体，应选择使用硝基四唑来启用可视化鎓chloride/5-bromo-4-chloro-3&#39; - indolyphosphate对甲苯胺盐（NBT / BCIP），如所述18。 </li><li>进行凝胶内蛋白复性的0.15％（重量/体积），通过培养该凝胶在1.5％（重量/体积）的β-环糊精，20mM磷酸盐 - 柠檬酸盐缓冲液，pH 6.5，在室温下用CMC的SDS-PAGE凝胶轻轻摇动15分钟19。 </li><li>丢弃该β-环糊精溶液中，并简要用H 2 O洗涤该凝胶在50mM醋酸钠缓冲液（pH 4.8）进行15分钟在室温下孵育。使用复性0.15％CMC的SDS-PAGE凝胶在50℃下温育该凝胶在30mM乙酸钠缓冲液pH 4.8 20分钟进行CMC酶谱</li><li>使用复性为0.15％（重量/体积），通过20分钟在50℃下温育该凝胶在30mM的醋酸钠缓冲液（pH 4.8）的CMC的SDS-PAGE凝胶上进行的CMC酶谱</li><li>用0.1％的染色凝胶10分钟w / v的刚果红溶液和脱色30分钟或直到清晰的条带是使用1个月观察娜CL。用0.3％（V / V）乙酸为更清晰的成像洗凝胶。 </li></ol> TrCel5A 6 4-MUC活性测定<ol><li>制备10mM的4-MUC在50％（V / V）甲醇中的储备溶液，并且在黑暗中储存于RT </li><li>在测定之前立即，制备4-MUC 2个工作溶液，1mM的4-MUC组成和60mM的醋酸钠，pH值4.8。 </li><li>含100微升叶蛋白（带或不带TrCel5A可诱导表达），由T. 100微升市售的纤维素酶混合物样品进行这个测试里氏木霉 1:1,000，纯H 2 O的分别。一式三份地运行每个样品。 </li><li>移液管加入100μl的4-MUC工作液加入到96孔板的单独的孔中，再加入100微升各样品。 </li><li>确定0分钟时间点的4-MU荧光通过荧光光谱法，使用360nm的激发波长和465nm的发射波长。 </li><li>孵育板20分钟，在50℃下，覆盖有粘合剂的盖，以防止蒸发。使反应停止冻结（或通过组合加入100μl0.15M的甘氨酸，pH值为10.0，并加入100μl的样品在一个单独的板）。 </li><li>确定的4-MU荧光通过荧光光谱端点，采用360nm的激发波长和465nm的发射波长。减去从端点（20分钟）的时间点0分，获得荧光值的变化。 </li></ol> TrCel5A 7。偶氮羧甲基纤维素酶活性分析<ol><li>制备1.5％的股票（重量/体积）偶氮-CMC被存储在室温下，并含有18 mM的醋酸锌，0.5M醋酸钠，pH值为5和80％乙醇（V / V），在室温储存沉淀溶液温度。 </li><li>测定前夕，准备偶氮CMC 2X工作液，1％组成（重量/体积）偶氮-CMC（以H 2 O）和60 mM醋酸钠，pH值4.8。 </li><li>结合50μl的样品和50μl的工作ING液1.5 ml离心管。 </li><li>含50微升叶蛋白（带或不带TrCel5A瞬时表达）测试样品;将100μl来自T.市售的纤维素酶混合物的里氏木霉稀释1:1,000;纯H 2 O运行样品和标准，一式三份。 </li><li>孵育样品20分钟，在50℃下停止反应，加入250微升的沉淀溶液。涡大力为10秒每个样品，以确保溶液完全混合。 </li><li>再次孵育的样品在室温下10分钟，然后涡旋。离心样品以1,000×g离心10分钟。除去200μl的各样品上清液至96孔板中，注意不要扰乱颗粒。 </li><li>测定用吸收光谱在590nm的波长的光。 </li></ol> 8。PAHBAH TrCel5A的测定<ol><li>使用打孔切割的滤纸圆（〜5.5毫米直径）。加入100μl60 mM的醋酸钠，pH值4.8，和100微升的样品，无论是含叶蛋白（带或不带TrCel5A可诱导表达）或商业T的里氏木霉纤维素酶的混合物，稀释1:1,000，并孵育用滤纸圈20小时，在50℃与300 rpm振摇。对于培养用滤纸每个样品，也没有培养滤纸作为单独的采样控制重复的解决方案。 </li><li>通过在H 2 O的加入5毫升浓盐酸，总共为100毫升的溶液，并储存在室温下溶解PAHBAH制备330 mM的对羟基苯甲酸酰肼（PAHBAH）溶液A。注意：最好的结果，通过加入盐酸，以PAHBAH浆料， 即 30毫升，该温度下搅拌来帮助溶解使体积为100毫升H 2 O之前的一个较小的体积实现</li><li>制备PAHBAH溶液B，含有柠檬酸50mM的钠，10mM的氯化钙和500mM NaOH和存储在RT。 </li><li> IMMED测定iately之前，请准备1毫升PAHBAH试剂1.5倍的工作解决9毫升缓冲溶液，储存于冰上直至使用（须4小时内使用）。 </li><li>准备样品中耐热0.2或0.5 ml离心管。结合5μl的每种溶液的温育，用滤纸（步骤8.1）45微升H 2 O和100μl的PAHBAH工作溶液。样品对照（不育滤纸样品）应在相同浓度（5微升的样品，45微升H 2 O，加入100μlPAHBAH工作液）下运行。也考验5标准（5微升）介于0和葡萄糖0.2g / L的含，纯H 2 O运行样品和标准，一式三份。 </li><li>在100℃下保温样品进行准确的10分钟，然后冷至室温恰好10分钟。移液管溶液加入到96孔板中，并测定光谱在410纳米的波长。从样品中减去个别样品对照组（不带保温过滤纸），以获得最终值。 </li></ol>

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#8220;Western+Blotting&#8221;:+Electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate-polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">&#8220;Western Blotting&#8221;: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112, 195-203 (1981).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elelctrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets+-+procedure+and+some+applications.">Elelctrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets - procedure and some applications.</a> Proceedings of the National Academy of Sciences of the United States of America. 76, 4350-4354 (1979).</li><li>Blake, M. S., Johnston, K. H., Russell-Jones, G. J., Gotschlich, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+sensitive+method+for+detection+of+alkaline+phosphatase-conjugated+anti-antibody+on+Western+blots.">A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots.</a> Analytical Biochemistry. 136, 175-179 (1984).</li><li>Yamamoto, E., Yamaguchi, S., Nagamune, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+&#946;-cyclodextrin+on+the+renaturation+of+enzymes+after+sodium+dodecyl+sulfate&#8211;polyacrylamide+gel+electrophoresis.">Effect of &#946;-cyclodextrin on the renaturation of enzymes after sodium dodecyl sulfate&#8211;polyacrylamide gel electrophoresis.</a> Analytical Biochemistry. 381, 273-275 (2008).</li><li>Fischer, R., Vaquero-Martin, C., Sack, M., Drossard, J., Emans, N., Commandeur, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+molecular+farming+in+the+future:+transient+protein+expression+in+plants.">Towards molecular farming in the future: transient protein expression in plants.</a> Biotechnology and Applied Biochemistry. 30 (2), 113-116 (1999).</li><li>Sammond, D. W., 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=Cellulase+linkers+are+optimized+based+on+domain+type+and+function:+Insights+from+sequence+analysis,+biophysical+measurements,+and+molecular+simulation.">Cellulase linkers are optimized based on domain type and function: Insights from sequence analysis, biophysical measurements, and molecular simulation.</a> PLoS One. 7, (2012).</li><li>Boonvitthya, N., Bozonnet, S., Burapatana, V., O'Donohue, M. J., Chulalaksananukul, W. <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+the+heterologous+expression+of+Trichoderma+reesei+endoglucanase+II+and+cellobiohydrolase+II+in+the+yeasts+Pichia+pastoris+and+Yarrowia+lipolytica.">Comparison of the heterologous expression of Trichoderma reesei endoglucanase II and cellobiohydrolase II in the yeasts Pichia pastoris and Yarrowia lipolytica.</a> Molecular Biotechnology. 54, 158-169 (2013).</li></ol>

作者什么都没有透露。

纤维素酶的重组表达是非常关注的一个领域，因为更有效的生物燃料生产系统1的愿望。植物提供了许多可能性，成功的纤维素酶生产，具有的功能，如经济采伐技术和自动水解方法是非常理想的特性进行大规模的生物燃料生产。另外，使用不同的本地化的用于生产蛋白质允许（如果需要），翻译后修饰20。改变的植物，以使纤维素酶的组成型表达，同时提供明显的好处，是其可能...

<html>木质纤维素生物质，其转化为液体燃料的退化已经被设想为一个方法，以减少对化石燃料依赖。在建立经济上可行的生物处理系统中的一个显著障碍是在纤维素和半纤维素1的酶促降解。植物表达系统显示为酶在工业规模上生产的巨大潜力。农业系统收获植物经济和体积都已经建立起来，因为他们已经数千年之久。纤维素酶在植物中的生产是可取的，由于像易于自动水解2，以及用于最大限度地通过增加植物细胞壁1消化率使用较低的酶水平的潜在因素。最后，厂房系统允许重组蛋白的靶向植物细胞的特定区域，并能翻译后修饰酶在有需要时3。 ve_content“&gt; 烟草 （烟草）是很常用的植物模式生物的外源蛋白表达的研究，由于其快速增长和生物量的积累功能4。重组蛋白的瞬时表达是一种技术，它允许在短的时间周期5蛋白的生产，同时保持柔韧性，以定位和由此所选择的蛋白， 即纤维素酶的翻译后修饰。这使得生产的纤维素酶（S）的基本分析，同时也奠定了在植物中进一步表达策略。使用这样的瞬时表达的策略，从糖基水解酶家族5，Cel5A，从真菌宿主里氏木霉 （ 红褐肉座菌 ）衍生的6内切葡聚糖酶的产生（以下简称为TrCel5A）。 TrCel5A是一个42 kDa蛋白被糖化本身，是路政署高度活跃roylzing纤维素链7。 这里所描述的瞬时表达技术是基于一个较为常用的系统上，渗入植物叶与土壤杆菌携带目的基因在适当的表达载体中。以允许成功的在植物中表达的快速分析，TrCel5A也可以被表达为融合蛋白的mCherry，最初来源于Discosoma藻单体荧光蛋白。蛋白DsRed的8，具有一个额外的6组氨酸残基（His-标签）融合到在C-末端（TrCel5A-的mCherry）。异源融合蛋白，TrCel5A-的mCherry，表达因此可以生长的植物中通过使用绿色光检查的mCherry分散监控。如果需要，该植物材料的薄的部分可以根据绿色光被显微镜检查，以确定该蛋白的特异性定位。对于这项工作，信号和转录坐在肽在结构注册成立，本地化的异源蛋白出口到内质网9。 以分析植物中表达的纤维素酶，包括TrCel5A，一些纤维素酶活性测试可以被执行的活动。总可溶性植物蛋白的提取后，TrCel5A可以部分地用热温育技术纯化。蛋白质大小是使用SDS-PAGE随后通过Western印迹法确定。酶谱法可用于分析活性对底物， 例如纤维素这一直是羧基甲基化（使羧甲基纤维素：CMC）为溶解度10。纤维素酶和葡糖苷酶的活性可以通过用β-D-纤维二糖苷相关的荧光团4 - 甲基伞形酮（4-MU）（4-MUC组合）进行监测。评估切葡聚糖酶活性的另一种方法涉及到CMC的光谱测定分析已用的偶氮-D相关联的你们（Remazolbrilliant蓝R）11。此外，这两种内切葡聚糖酶和纤维素酶的范围中的蛋白质的活性可以通过使用糖分析测试，如对羟基苯甲酸酰肼（PAHBAH）测定12进行监测。这些技术可用于澄清和量化感兴趣的表达纤维素酶的活性。

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:308px;">4-methylumbelliferyl &#946;-D-cellobioside</td>
			<td style="width:127px;">SIGMA-ALDRICH</td>
			<td style="width:156px;">M6018</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>ROTH</td>
			<td>3738</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetosyringon (concentrated)</td>
			<td>ROTH</td>
			<td>6003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - kanamycin</td>
			<td>ROTH</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - rifampicin</td>
			<td>ROTH</td>
			<td>4163</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - carbenicillin</td>
			<td>ROTH</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibody - rabbit anti His(6) pAb</td>
			<td>ROCKLAND</td>
			<td>600-401-382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibody - goat anti rabbit AP, FC pAb</td>
			<td>DIANOVA</td>
			<td>111-055-008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Azo-carboxymethyl cellulose</td>
			<td>MEGAZYME</td>
			<td>S-ACMC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-cyclodextrin</td>
			<td>SIGMA-ALDRICH</td>
			<td>C4805</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carboxymethyl cellulose</td>
			<td>ROTH</td>
			<td>3333.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellulase from Trichoderma reesei ATCC 26921</td>
			<td>SIGMA-ALDRICH</td>
			<td>C2730</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Congo red</td>
			<td>SIGMA-ALDRICH</td>
			<td>75768</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coomasie brilliant blue G 250</td>
			<td>ROTH</td>
			<td>9598.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D(+)-glucose</td>
			<td>ROTH</td>
			<td>8337</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>ROTH</td>
			<td>K928</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter paper - Rotilabo blotting paper</td>
			<td>ROTH</td>
			<td>CL67</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NBT/BCIP</td>
			<td>SIGMA-ALDRICH</td>
			<td>72091</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES buffer</td>
			<td>ROTH</td>
			<td>4256</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>ROTH</td>
			<td>8388</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium citrate</td>
			<td>ROTH</td>
			<td>3580</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecylsulfate</td>
			<td>SERVA</td>
			<td>20765</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>ROTH</td>
			<td>6771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Soil, Einheitserde classic&#160;</td>
			<td>PATZER GMBH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectrometer - Infinite M200</td>
			<td>TECAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>SIGMA-ALDRICH</td>
			<td>S-5390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Hydroxy benzhydrazide&#160;</td>
			<td>SIGMA-ALDRICH</td>
			<td>H9882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>ROTH</td>
			<td>1058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV light source - BLAK RAY</td>
			<td>UVP</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vibratome - VT1000S</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

expression of recombinant cellulase cel5a from trichoderma reesei in tobacco plants

纤维素降解酶，纤维素酶，是研究和产业利益的目标。这些酶在难以培养微生物，如菌丝建设真菌和厌氧菌的优势，加快了在这一领域的使用重组技术。植物表达的方法是理想的系统，用于大规模生产的酶和其它工业上有用的蛋白质。本文中，对于真菌内切葡聚糖酶， 里氏木霉 Cel5A，在烟草中的瞬时表达方法被证实。成功的蛋白表达被示出，使用的mCherry-酶融合蛋白的荧光监测。此外，一组基本的测试是用来检查瞬时表达T的活动里氏木霉 Cel5A，包括SDS-PAGE，Western印迹，酶谱，以及荧光和基于染料的基质降解试验。这里所描述的系统可以被用来产生活性细胞ulase在很短的时间周期，从而通过组成型或诱导型表达系统，以评估植物中用于进一步的生产潜力。

TrCel5A和TrCel5A-的mCherry采用的瞬时表达系统（ 图1A和1B）中成功表达在烟草植物中。检查TrCel5A-的mCherry融合蛋白的表达模式的揭示健康的叶子（ 图1C），这下绿光（ 图1D）显示广泛的蛋白表达。 对烟草植物的叶子有包含被执行的总可溶性蛋白质的提取的瞬时表达TrCel5A蛋白和SDS-PAGE显示了大量的各种蛋白质的人出席（ 图2A...

Watch this Scientific Journal Video about Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants at JoVE.com

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

烟草植物被用于生产真菌纤维素酶，TrCel5A，通过一个瞬时表达系统。的表达可以用荧光融合蛋白进行监测，并且该蛋白质的活性进行了表征后表达。

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 ...

expression-recombinant-cellulase-cel5a-from-trichoderma-reesei

Research

JoVE Journal

Environment

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

12.7K 次观看.  RWTH Aachen University. 烟草植物被用于生产真菌纤维素酶，TrCel5A，通过一个瞬时表达系统。的表达可以用荧光融合蛋白进行监测，并且该蛋白质的活性进行了表征后表达。 

视频: Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

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 ...

科研

环境科学

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

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

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 ...

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

植物与放射性示踪剂测量矿质营养和毒物的通量

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

高分辨率红外热成像（HRIT）的冰核和冰传播的植物研究中的应用

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p&#8804;0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng.g-1 of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng.g-1. This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

We demonstrate a method for the analysis of aflatoxins and transgene expression in peanut seeds that contain RNA-interference signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. RNAi-mediated control of mycotoxins in plants has not been reported previously.

黄曲霉毒素在花生RNAi介导的控制：方法分析霉菌毒素的产生和转基因表达的花生/<em&gt;黑</em&gt;病害系统

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That ...

Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing seeds of Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum). We applied magnets under the culture dish. During the 4 days of application, we observed that the stem and root length increased. The group subjected to magnetic field treatment (n = 10) showed a 1.4 times faster rate of growth compared with the control group (n = 11) in a total of 8 days (p &#60;0.0005). This rate is 20% higher than that reported in previous studies. The tubulin complex lines did not have connecting points, but connecting points occur upon the application of magnets. This shows complete difference from the control, which means abnormal arrangements. However, the exact cause remains unclear. These results of growth enhancement of applying magnets suggest that it is possible to enhance the growth rate, increase productivity, or control the speed of germination of plants by applying static magnetic fields. Also, magnetic fields can cause physiological changes in plant cells and can induce growth. Therefore, stimulation with a magnetic field can have possible effects that are similar to those of chemical fertilizers, which means that the use of fertilizers can be avoided.

The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy.

采用静态磁场农业植物初始增长率提高

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based on the diffusion theory. A known volume of air overlaying a defined soil area is enclosed within a parallelepiped cover (named &#34;chamber&#34;), for a defined period of time. During this enclosure period, gases (methane (CH4) and nitrous oxide (N2O)) move from soil pore air near their microbial source (i.e., methanogens, nitrifiers, denitrifiers) to the chamber headspace, following a natural concentration gradient. Fluxes are then estimated from chamber headspace concentration variations sampled at regular intervals throughout the enclosure and then analyzed with gas chromatography. Among the techniques available for GHG measurement, the static closed chamber method is suitable for plot experiments, as it does not require large homogenously treated soil areas. Furthermore, it is manageable with limited resources and can identify relationships among ecosystem properties, processes, and fluxes, especially when combined with GHG driving force measurements. Nevertheless, with respect to the micrometeorological method, it causes a minimal but still unavoidable soil disturbance, and allows a minor temporal resolution. Several phases are key to the method implementation: i) chamber design and deployment, ii) sample handling and analyses, and iii) flux estimation. Technique implementation success in paddy fields demands adjustments for field flooding during much of the cropping cycle, and for rice plant maintenance within the chamber headspace during measurements. Therefore, the additional elements to be considered with respect to the usual application of non-flooded agricultural soils consist of devices for: i) avoiding any unintended water disturbance that could overestimate fluxes, and ii) including rice plants within the chamber headspace to fully consider gases emitted through aerenchyma transportation.

The overall goal of this protocol is to measure greenhouse gas emissions from paddy fields using the static closed chamber technique. The measurement system needs specific adjustments due to the presence of both a permanent water layer in the field and of the plants within the chamber headspace.

用静止封闭室维持顶空装置对稻田甲烷和一氧化二氮通量的评价

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast make 2-dimensions (2D) infrared images somewhat difficult to interpret. Viewing an IR image or the video of plants freezing in 3 dimensions (3D) would allow a more accurate identification of sites for ice nucleation as well as the progression of freezing. In this paper, we demonstrate a relatively simple means to produce a 3D infrared video of a strawberry plant freezing. Strawberry is an economically important crop that is subjected to unexpected spring freeze events in many areas of the world. An accurate understanding of the freezing in strawberry will provide both breeders and growers with more economical ways to prevent any damage to plants during freezing conditions.
The technique involves a positioning of two IR cameras at slightly different angles to film the strawberry freezing. The two video streams will be precisely synchronized using a screen capture software that records both cameras simultaneously. The recordings will then be imported into the imaging software and processed using an anaglyph technique. Using red-blue glasses, the 3D video will make it easier to determine the precise site of ice nucleation on leaf surfaces.

Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

植物冷冻三维红外视频制作协议

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

循环介导等温扩增 (灯管) 法快速识别烟粉虱

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

分离叶测定,以简化马铃薯中基因表达研究,在咀嚼昆虫曼杜卡性塔感染

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...