我们感谢在RIKEN的PSC和比约恩·Usadel博士和树齐藤教授有用讨论MPIMP的。 TT是从亚历山大·冯·洪堡基金会奖学金支持。

1。样品制备<ol><li>植物材料的收获，并立即冻结。 </li><li>冷冻材料厂搅拌机磨制成粉末（或砂浆）和猎鹰管或Eppendorf管中于-80°C。 </li></ol> 2。提取代谢物谱<ol><li>植物材料，在2毫升的Eppendorf管分装冷冻。 </li><li>添加5μL提取每毫克冷冻厂材料的鲜重的缓冲区。 </li><li>新增一个金属（或gilconia）的球，并融汇冻粉与调音台轧机在25 LS -1 2分钟。 </li><li>在12000转离心10分钟。 </li><li>转移上清NANOSEP离心式过滤器。 </li><li>在4000转速离心2分钟。 </li><li>将上清转移至新的Eppendorf管中，并储存在-20°C，直到使用。 </li></ol> 3。代谢物谱的LC-MS <ol><li>建立高效液相色谱法和检查柱箱温度和样品盘。 </li><li>成立MS条件和检查真空和加热毛细管的状态。 </li><li>执行的m / z MS检测校准。 </li><li> 50μL提取物转移到玻璃小瓶的LC-MS。 </li><li>注入5μL提取物的LC-MS。 </li></ol> 4。数据分析<ol><li>配置神剑传奇或Metalign 4，选择要处理的数据进行分析。 </li><li>准备检测到您的利益峰表，按照表I类化合物。 </li><li>识别标准化合物的共流出峰。 </li><li>注释使用MS 分析 ，文献调查，代谢物数据库搜索（ 图2），12,13检测峰。 </li></ol> 5。代谢途径的预测<ol><li>构建与检测的化合物可能的途径。预测使用高峰注解的途径，应根据检测到的化合物的化学结构S的 ​​预测连接酶的功能，代谢途径13。构建合成步骤应进行精确的峰值注释。但详细的化学结构的测定，例如糖基，是不可缺少的这一步，因为糖基和内收位置的预测是非常困难的，以确定由MS分析。酶法测定糖捐助的测定糖如hexoside和pentoside的类型将确定，在项目的最后一步。主要建设途径的预测，应为小分子较大分子的中间，除了在某些情况下，如脱水反应。原子分子量的名单，例如16米/ Z - H之间的差异和OH基（氧化），14米/ Z（碳原子）之间的-OH和-OMe的（甲基）差价和162 m / z为 （兆瓦H 2 O的己糖（糖基化）），是有用的预测。测定修改相关组织的具体分析的类型，有助于代谢途径的预测。一般代谢途径，如KEGG数据库（数据库<a href="http://www.genome.jp/kegg/" target="_blank">http://www.genome.jp/kegg/</a> ）PlantCyc（ <a href="http://plantcyc.org/" target="_blank">http://plantcyc.org/~~V</a> ），为您感兴趣的代谢途径的预测是非常有效的。 </li></ol> 6。基因名单的制备与拟南芥同源基因ID <ol><li>下载从基因组数据库，基因ID列表。 </li><li>添加同源基因的拟南芥基因ID，在你的目标工厂的情况下是不拟南芥。 </li><li>准备一个利益的途径中的基因名单。在TAIR网页（ <a href="http://www.arabidopsis.org/" target="_blank">http://www.arabidopsis.org/~~V</a> ），拟南芥的通路数据和基因家族的数据诠释。如果你准备拟南芥同源基因的清单，你可以随后组合NE他们。 </li></ol> 7。共表达基因分析<ol><li>测试使用搜索最佳通路的数据库，使用众所周知的基因对您的利益途径（ 表二 ）检查相关的准备基因ID列表。如果数据库或共同表达的基因表达数据库是不是你的兴趣在植物，植物共表达数据库，应使用与拟南芥的同源基因的清单。在大麦的情况下，可用于水稻，杨树，小麦，苜蓿，大豆，植物物种的共同表达分析（ 见表二 ）。 </li><li>基于利益的途径著名的基因连接，构建框架为目标的共同表达网络。 </li><li>新增相关的候选基因（R &lt;0.4〜0.90系数的途径利益知名的基因之间的连接价值的近似值，内），并检查他们在你的PR基因注释edicted家庭网络连接，寻找最佳的候选基因（ 图3）。系数阈值应根据网络结构和密度相关基因的协调。 </li><li>使您可以作为专门为你的目标的途径缩小的基因列表。 </li><li>检查你的候选基因的器官特异性和应激反应的基因表达。 </li></ol> 8。的所有信息整合到预测的新途径<ol><li>新增查询共表达分析已用于预测代谢途径的基因特征。 </li><li>检查这个途径在未知的部分，未知酶的步骤，例如运输蛋白和转录因子。 </li><li>预测这些失踪未知的步骤最合适的基因注释。 </li><li>结合GE 在硅片的代谢产物分析和候选基因的结果NE表达的基础上预测的途径。 </li><li>安排您的候选基因，根据基因功能的预测途径，例如，乙酰转移酶的代谢产物乙酰化，糖基苷，P450的氧化复合。氨基酸序列的系统发育树分析是有用的宽一些，例如P450和糖基转移酶的基因家族。 </li><li>检查的组织特异性应激反应或代谢产物的积累和候选基因的基因表达水平之间的一致性。 </li><li>检查提供基板和应激反应的基因连接到其他代谢。 </li></ol> 9。实验中使用的生物资源的基因鉴定<ol><li>检查的生物资源提供便利为候选基因鉴定实验。 </li><li>执行实验利用生物资源，如高突变体库和全长cDNA文库，基因功能鉴定。 ţ他的实验与编制过度植物及敲除突变体，酶的检测和启动子结合试验，基因功能鉴定，必须在你的预测最好的候选基因进行。蛋白质性质的表征和过度植物准备好要进行确认后代谢产物的文件，使用正突变体，因为它需要相当长的时间准备改造重组蛋白和基因的克隆，重组蛋白检测。 </li></ol> 10。代表结果在本协议中所述的综合分析的过程有很多可能性，取决于指定的实验目的和选择生物和分析组合。程序和实验设计的选择，应进行正确目标的途径，化合物和植物物种的基础上。在本协议中所述的一体化战略是FOC<html>用于植物基因功能和新基因功能的发现与一些生物和数据资源的有效使用注释。预期的结果是承诺提供与确凿预测的唯一情况。这一事实表明，如果有足够的证据不能组合型材，实验不应该被启动。出于这个原因，在任何情况下，可以支持额外的初步实验，RT-PCR技术，如针对性的基因表达谱，基因功能的预测。预测的准确性和正确性的相关性更高的质的区别和数量组合的变化而定。此外，良好的候选人和有效的结果，只能来自准确预测的途径。高峰应进行注释的几种方法的组合，例如文献调查，参考植物提取物，MS n分析，器官特异性和突变分析13。 1“SRC =”/ files/ftp_upload/3487/3487fig1.jpg“/&gt; 图1。实验相结合的方法，通过基因注释流程的概述。在某些情况下，项目开始发现这是在特殊条件下或组织发现一个新的高峰，和渴望了解在其新陈代谢作用。在其他情况下，该项目的目的是关键调控因子，如转录因子的基因鉴定或发现。实验设计应刨用一组数据显示了明显的差异，在你的目标途径的代谢水平，从不同器官的组织样本的广泛使用，接触应力条件差异生长的植物或植物，并受到材料代谢产物分析。突变和转基因植物以及QTL窝藏育种材料，也代表了适合这些研究的遗传材料。应认真进行准确预测新途径如metabolotype机关根据证券及应激反应的基因表达数据的途径利益的不同类型的峰值注释和相结合的办法。在最后一步，代谢物和成绩单分析应执行最终，当网络资源，在硅片分析和基因表达的特征， 通过异源表达在体外结合，导致确认的候选基因，并阐明其功能内的代谢途径中的地位。缩写：QTL定位，QTL定位。 <img alt="图2" src="/files/ftp_upload/3487/3487fig2.jpg" /> 图2。工作流高峰注释的组合方式。标准化合物的峰识别和注释，比较野生型和敲除突变体的过程，多维目标峰的质谱质谱纯COM从数据库12磅。缩写：DB，数据库;敲，敲了一维，一维，二维，二维核磁共振，核磁共振，红外光谱，红外多级质谱 ，质量质量spectrometries。 <img alt="图3" src="/files/ftp_upload/3487/3487fig3.jpg" /> 图3。例如合作的花青素途径调控网络分析的共表达分析进行了使用与总理（ <a href="http://prime.psc.riken.jp/?action=coexpression_index" target="_blank">http://prime.psc.riken.jp/?action=coexpression_index</a> ）设置ATTEDII版本的数据3 8,2的pajek计划（ <a href="http://vlado.fmf.uni-lj.si/pub/networks/pajek/" target="_blank">http://vlado.fmf.uni-lj.si/pub/networks/pajek/</a> ）。呈正相关（R &lt;0.5）被用来进行网络连接。红色节点：12花色苷酶基因（At5g13930，社区卫生服务，TT4，查尔酮合酶; At3g55120，C您好，TT5，查耳酮isomerise; At3g51240，F3H，TT6，黄烷酮3 -羟化酶; At5g07990，甘蓝型油菜F3&#39;H，TT7，类黄酮3&#39;-羟化酶; At5g17050，Fd3GT，UGT78D2，类黄酮3 - O型葡萄糖; At5g17220，AtGSTF12，TT19 ; At5g42800，DFR的二氢叶酸还原酶，TT3 At4g22880时，ANS / LDOX，TT18，花青素的合成; At4g14090 A5GT，花青素5 - O型葡萄糖; At5g54060，A3G2“XT的，公认的花青素3 - O-葡萄糖苷2” - O - xylosyltransferase; At3g29590，A5GMaT，花青素5 - O-葡萄糖苷6&#39;&#39;&#39; - O型 malonyltransferase; At1g03940，A3GCouT，花青素3 - O-葡萄糖苷6“ - O - P-coumaroyltransferase）和两个花青素生产的转录因子（At1g56650， PAP1; At1g66390，PAP2）用于寻找候选基因的候选基因被发现一个“集合的交集”搜索与阈值的系数 r &lt;。/ EM &gt;&gt;质疑0.50套所有质疑的基因（十四花青素合成基因）的交点。共同表达网络，包括相关的候选基因（68个基因）和查询基因（14个基因），是由一个“套联网”重新构建与R&gt; 0.50总理数据库搜索。与“网”从总理的数据库和网络的文件格式的输出文件使用Pajek软件绘制。蓝色节点代表候选人花青素基因相关的基因。 <table border="1"><tbody><tr><td> 种类 </td><td> 主要次生代谢产物 </td></tr><tr><td> 拟南芥 </td><td>硫代葡萄糖甙，黄酮，花青素，sinapoyl的衍生</td></tr><tr><td> 杨毛果 </td><td>黄酮，花青素，水杨酸衍生物</td></tr><tr><td> 葡萄 </td><td>黄酮，花青素，单宁酸，苯乙烯</td></tr><tr><td> 马铃薯lycopersicum </td><td>黄酮，花青素，glycoalkaloid，chrologenate相关， </td></tr><tr><td> 烟草 </td><td>黄酮，花青素，nicotianamide，chrologenate相关acylsugar </td></tr><tr><td> 水稻 </td><td> glycoflavone，花色苷，甾醇衍生物</td></tr><tr><td> 玉米5月 </td><td> glycoflavone，花青素，benzoxazinone，甾醇衍生物</td></tr><tr><td> 苜蓿 </td><td>花青素，异黄酮，皂甙， </td></tr><tr><td> 莲花粳稻 </td><td>大豆异黄酮，花青素，黄酮，皂甙， </td></tr></tbody></table> 表一，主要模式植物的次生代谢产物。 <table border="1"><tbody><tr><td> 共表达数据库 </td><td> 地址 </td></tr><tr><td> 植物交叉规范IES </td><td></td></tr><tr><td>缔约方会议</td><td> <a href="http://webs2.kazusa.or.jp/kagiana/cop0911/" target="_blank">http://webs2.kazusa.or.jp/kagiana/cop0911/~~V</a> </td></tr><tr><td>沛</td><td> <a href="http://aranet.mpimp-golm.mpg.de/" target="_blank">http://aranet.mpimp-golm.mpg.de/</a> </td></tr><tr><td> 植物物种 </td><td></td></tr><tr><td> ATEED-II </td><td> <a href="http://atted.jp/" target="_blank">http://atted.jp/</a> </td></tr><tr><td>英美</td><td> <a href="http://142.150.214.117/welcome.htm" target="_blank">http://142.150.214.117/welcome.htm</a> </td></tr><tr><td>缔约方会议</td><td> <a href="http://webs2.kazusa.or.jp/kagiana/cop" target="_blank">http://webs2.kazusa.or.jp/kagiana/cop</a> </td></tr><tr><td> GeneCAT </td><td> <a href="http://genecat.mpg.de/" target="_blank">http://genecat.mpg.de/</a> </td></tr><tr><td> 拟南芥 </td><td></td></tr><tr><td>法</td><td> <a href="http://www.arabidopsis.leeds.ac.uk/act/coexpanalyser" target="_blank">http://www.arabidopsis.leeds.ac.uk/act/coexpanalyser</a> </td></tr><tr><td> AthCoR@CSB.DB </td><td> <a href="http://csbdb.mpimp-golm.mpg.de/csbdb/dbcor/ath.html" target="_blank">http://csbdb.mpimp-golm.mpg.de/csbdb/dbcor/ath.html</a> </td></tr><tr><td> CressExpress </td><td> <a href="http://cressexpress.org/" target="_blank">http://cressexpress.org/~~V</a> </td></tr><tr><td> PRIME </td><td> <a href="http://prime.psc.riken.jp/?action=coexpression_index" target="_blank">http://prime.psc.riken.jp/?action=coexpression_index</a> </td></tr><tr><td> 水稻 </td><td></td></tr><tr><td> RiceArrayNet </td><td> <a href="http://arraynet.mju.ac.kr/arraynet/" target="_blank">http://arraynet.mju.ac.kr/arraynet/~~V</a> </td></tr><tr><td>水稻阵列数据库</td><td> <a href="http://www.ricearray.org/coexpression/coexpression.shtml" target="_blank">http://www.ricearray.org/coexpression/coexpression.shtml</a> </td></tr></tbody></table> 表二。可用硅片共同表达分析基因表达的数据库。

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+required+for+cellulose+synthesis+by+regression+analysis+of+public+microarray+data+sets.">Identification of genes required for cellulose synthesis by regression analysis of public microarray data sets.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 8633-8638 (2005).</li><li>Tohge, T., Yonekura-Sakakibara, K., Niida, R., Watanabe-Takahashi, A., Saito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochemical+genomics+in+Arabidopsis+thaliana:+A+case+study+for+functional+identification+of+flavonoid+biosynthesis+genes.">Phytochemical genomics in Arabidopsis thaliana: A case study for functional identification of flavonoid biosynthesis genes.</a> Pure and Applied Chemistry. 79, 811-823 (2007).</li><li>Tohge, T., Fernie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Web-based+resources+for+mass-spectrometry-based+metabolomics:+A+user's+guide.">Web-based resources for mass-spectrometry-based metabolomics: A user's guide.</a> Phytochemistry. 70, 450-456 (2009).</li><li>Tohge, T., Fernie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+genetic+diversity,+informatics+and+metabolomics+to+facilitate+annotation+of+plant+gene+function.">Combining genetic diversity, informatics and metabolomics to facilitate annotation of plant gene function.</a> Nature Protocols. 5, 1210-1227 (2010).</li><li>Tohge, T., Kusano, M., Fukushima, A., Saito, K., Fernie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+and+metabolic+programs+following+exposure+of+plants+to+UV-B+irradiation.">Transcriptional and metabolic programs following exposure of plants to UV-B irradiation.</a> Plant Signal Behav. 6, (2011).</li><li>Tohge, T., Ramos, M. S., Nunes-Nesi, A., Mutwil, M., Giavalisco, P., Steinhauser, D., Schellenberg, M., Willmitzer, L., Persson, S., Martinoia, E., Fernie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+the+storage+metabolome:+profiling+the+barley+vacuole.">Towards the storage metabolome: profiling the barley vacuole.</a> Plant Physiol. , (2011).</li><li>Yonekura-Sakakibara, K., Tohge, T., Matsuda, F., Nakabayashi, R., Takayama, H., Niida, R., Watanabe-Takahashi, A., Inoue, E., Saito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+flavonol+profiling+and+transcriptome+coexpression+analysis+leading+to+decoding+gene-metabolite+correlations+in+Arabidopsis.">Comprehensive flavonol profiling and transcriptome coexpression analysis leading to decoding gene-metabolite correlations in Arabidopsis.</a> Plant Cell. 20, 2160-2176 (2008).</li></ol>

没有利益冲突的声明。

既然已经用了好几年，转录组和代谢组学技术，数据集成为辅助的基因注释代谢过程中通常开始一个新的峰代表一个未知的代谢产物的鉴定。这一事实导致了下一个阶段，这是评估的定量或认为是负责其合成的新的候选基因变异的代谢产物峰。然而，在本协议中所述的战略有三大问题）峰值标注的困难，二）通路预测的复杂性，三）基因信息和基因表达数据质量的决议。要对付的第一个问题，高峰注释应开展合作与标准化合物或从MS n分析，参考提取物，突变分析，代谢物数据库检索和文献调查（ 图12）2，组合的方式，利用信息洗脱。为Second问题，预测途径只能得到正确的峰值注释。然而，组织特异性代谢产物分析也可以支持峰值标注，应与相关基因的基因表达密切相关，因为代谢产物积累。因此结合不同的组织和生长条件的文件，可以帮助这第二个问题。第三个问题，关于基因信息的分辨率取决于序列数据的进展。在没有完成基因组序列的模型工厂的情况下，使用等模式植物中的同源基因的共同表达分析是有益的。详细的氨基酸序列对齐比较和进化树分析，可以支持连接到其他物种的模式生物。 该协议是适用于所有的新陈代谢。这是最有效的，在初中和高中的代谢分析，这是很好的特点是受到强烈的转录çontrol 1,5,11,16。在一些例子，硫同化执行成功的合作表达分析，基因β-氧化，支链氨基酸降解，叶绿素击穿，和赖氨酸代谢3，细胞壁代谢10,7和光信号级联14。通过联合基因组学，代谢和信息注释基因的功能不仅是生物合成基因和转录因子直接调节，同时也为了解生理过程及反应（例如图3 14）。 要发展这种方法从模式植物，作物品种，各地植物物种的代谢比较是在一些一般性的代谢功能强大的方法。例如，如果检测到相同的化合物在不同的植物物种，一些同源基因在这些植物物种中发现，跨物种共同使用同源基因的表达分析可以提供strong支持你的预测。这种方法可以在拟南芥，杨树，紫花苜蓿，除了重要的作物，如大麦，大米，小麦和大豆，植物物种的共同表达分析（6，行星： <a href="http://aranet.mpimp-golm.mpg.de/" target="_blank">http://aranet.mpimp-golm.mpg DE /</a> 9，缔约方会议： <a href="http://webs2.kazusa.or.jp/kagiana/cop0911/" target="_blank">http://webs2.kazusa.or.jp/kagiana/cop0911/</a> ;看一个例子，15岁 ）。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>蒸馏水的ULC / MS毕业生</td><td> BIOSOLVE </td><td> 23214102 </td></tr><tr><td>乙腈（ACN）的ULC / ms级</td><td> BIOSOLVE </td><td> 01204102 </td></tr><tr><td>甲醇（甲醇）的ULC / ms级</td><td> BIOSOLVE </td><td> 13684102 </td></tr><tr><td>蚁酸（甲酸）级的ULC / MS液相色谱</td><td> BIOSOLVE </td><td> 06914131 </td></tr><tr><td>标准化合物</td><td> EXTRASYNTHESE </td><td></td></tr><tr><td>线性离子阱（IT）的ESI-MS系统菲尼根 - 的LTQ </td><td>热电菲尼根</td><td></td></tr><tr><td>高效液相色谱系统测量师</td><td>热电菲尼根</td><td></td></tr><tr><td>分析列月神C18（2），2.0毫米直径，长度150毫米，100孔隙大小和3毫米的球形颗粒</td><td> phenomenex </td><td> 00F-4251-B0 </td></tr><tr><td> Xcalibur软件</td><td>热电菲尼根</td><td></td></tr></tbody></table>

annotation of plant gene function via combined genomics, metabolomics and informatics

由于模式植物物种完整的基因组序列和丰富的生物资源，如敲除突变体，野生种质和先进的繁殖种群数量不断扩大，有上升的负担，为基因功能注释。在这个协议中，使用联合共表达基因分析植物基因功能注释，代谢和信息提供（ 图1）。这种方法使用已知功能的靶基因，允许非注释基因的鉴定一定的代谢过程中可能会涉及与代谢目标化合物通过鉴定，理论基础。战略提出申请产生正向和反向遗传学的方法，尽管这些都不人口信息是毫不费力的。推论，这种方法也可以被用来作为一种方法来描述未知峰代表新的或特定的本身在有限的组织，植物或治疗压力，这是目前重要的试验，以了解植物的新陈代谢condary代谢产物。

Watch this Scientific Journal Video about Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics at JoVE.com

Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics

基因组学相结合，共同表达的基因分析和鉴定目标化合物通过新陈代谢，给基因功能注释。

联合基因组学，代谢组学和信息通过植物基因功能的注解

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as ...

annotation-plant-gene-function-via-combined-genomics-metabolomics

Research

JoVE Journal

Biology

19.4K 次观看.  Max-Planck-Institut. 基因组学相结合，共同表达的基因分析和鉴定目标化合物通过新陈代谢，给基因功能注释。

视频: Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

科研

生物学

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

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 &amp;#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 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

农杆菌介导的病毒诱导的基因沉默含量在棉花

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

高分辨率基因表达谱RNA的合成，加工和衰变的新转录RNA在细胞培养的代谢标记

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

分离绿色荧光蛋白系统可视化细菌在感染过程中传递的效应

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

mirMachine: A One-Stop Shop for Plant miRNA Annotation

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of gene expression, miRNAs play key roles in various cellular pathways, including both development and response to a/biotic stress, such as drought and diseases. Having high-quality reference genome sequences enabled identification and annotation of miRNAs in several plant species, where miRNA sequences are highly conserved. As computational miRNA identification and annotation processes are mostly error-prone processes, homology-based predictions increase prediction accuracy. We developed and have improved the miRNA annotation pipeline, SUmir, in the last decade, which has been used for several plant genomes since then. 
This study presents a fully automated, new miRNA pipeline, mirMachine (miRNA Machine), by (i) adding an additional filtering step on the secondary structure predictions, (ii) making it fully automated, and (iii) introducing new options to predict either known miRNA based on homology or novel miRNAs based on small RNA sequencing reads using the previous pipeline. The new miRNA pipeline, mirMachine, was tested using The Arabidopsis Information Resource, TAIR10, release of the Arabidopsis genome and the International Wheat Genome Sequencing Consortium (IWGSC) wheat reference genome v2.

Herein, we present a new and fully automated miRNA pipeline, mirMachine that 1) can identify known and novel miRNAs more accurately and 2) is fully automated and freely available. Users can now execute a short submission script to run the fully automated mirMachine pipeline.

mirMachine：植物miRNA注释的一站式商店

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of ...

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

通过BiFC-FRET-FLIM测定MADS盒转录因子和钙传感器蛋白两种单体之间的三方相互作用

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.