我们感谢雅各布维尔为他提供了有益的意见和建议。我们还感谢安妮在生物信息学分析方面的帮助。Yanglei 和智博李得到中国奖学金委员会 (CSC) 的支持。我们感谢 NWO-TTW Perspectief Programma Back2Roots (TKI-AF-15510) 对 OPK 的财政支持。

1. 杀菌条件下的马铃薯发芽
<ol><li>用无菌水冲洗马铃薯表面。将马铃薯浸泡在70% 乙醇中, 然后在3% 次氯酸钠中沐浴, 每人5分钟。再用无菌水冲洗, 除去剩下的次氯酸钠。</li>
	<li>准备发芽和生长马铃薯块茎所需的材料;将塑料罐、植入篮子、蛭石和水热处理121摄氏度以20分钟消毒。 注: 确保所有使用的材料都耐高压。否则, 使用其他杀菌方法。</li>
	<li>将表面杀菌的马铃薯放入植入篮中, 放入含湿蛭石的蒸压锅中。</li>
	<li>把锅放在24摄氏度的气候室里三周。设置它的周期16小时的光 (120 µmol m-2s-1) 和 8 h 的黑暗。为了避免微生物污染, 用一个大的玻璃纤维盒盖住锅。</li>
</ol>2. 马铃薯根系分泌物的收集
<ol><li>当芽苗发芽时, 将篮子与整个马铃薯转移到一个被消毒的烧杯中, 装满150毫升的蒸压去离子水, 土豆块茎放在水面上, 根埋在水中 (见图 1)。保持幼苗在气候室, 并使用相同的设置之前 [24 °c; 周期的16小时的光 (120 µmol m-2s-1), 8 h 的黑暗]。</li>
	<li>从第二天起, 收集含有分泌物的水, 用无菌的去离子水将烧杯填满。每天进行取样, 直到转移苗后的第七天。</li>
	<li>将每个样品分别存放在4摄氏度。对于每个样品, 在 Luria Bertani (磅; 1% 胰蛋白胨, 0.5% 酵母萃取物, 0.5% NaCl) 琼脂板上传播100µL, 以检查微生物污染物。丢弃被污染的样品。</li>
	<li>将所采集的样品组合成一个幼苗, 并将其浓缩为-40 °c, 最后体积为150毫升。</li>
</ol>3. 细菌生长
<ol><li>将 a 的丝状菌株从-80 摄氏度甘油储存到 LB 琼脂板上, 并在30摄氏度的一夜内孵化出盘子。</li>
	<li>接种一个磅液体培养基与一个单一的殖民地从板块, 并种植的文化在一个颤抖的孵化器在 200 rpm 隔夜在30摄氏度。</li>
</ol>4. 细菌的治疗和取样
<ol><li>将根分泌物处理的细菌生长曲线与阴性对照进行比较, 制备出含有50毫升 LB 液培养基的一系列烧瓶。在所有烧瓶中加入0.5 毫升的隔夜细菌培养, 并分别将0%、5%、10% 或 15% (v/v) 根渗出物或无菌去离子水添加到培养基中。</li>
	<li>使用具有去离子水的文化代替根分泌物作为控制。测量光学密度在600毫微米 (OD600) 以后每 1 h。通过绘制 OD600值与时间来生成增长曲线。 注: 我们看到10% 的根分泌物不影响B. 丝状的生长 (见图 2);该比值用于 RNA 序列实验。</li>
	<li>用90毫升预热的 LB 培养基, 将一夜之间的丝状文化稀释到600毫升0.05 的最初 OD。添加10毫升的根分泌物的培养和孵化它在30°c 1 小时。</li>
	<li>使用10毫升无菌去离子水处理作为控制。以 9000 x g 为2分钟, 在4摄氏度时, 从养殖中收集细胞。丢弃上清液, 立即将颗粒冻结在液氮中, 然后将其贮存在-80 摄氏度, 直至使用。</li>
</ol>5. RNA 分离
注意: 在开始隔离之前, 准备工作台、机架和吸管, 方法是用 RNase 净化液清洗它们 (见材料表)。随时戴上手套, 确保所有的管子、小贴士和解决方案都是 RNase 的。尽可能把样品放在冰上。
<ol><li>添加 0.1% (v/v) 的焦碳酸二乙基 (DEPC) 到超纯水 (100 µL DEPC 每100毫升溶液), 搅拌好, 并保持在室温下过夜。在隔夜治疗后, 将混合物 DEPC 灭菌。使用此 DEPC 处理的超纯水制备 TE 缓冲器 [10 毫米三盐酸 (pH = 8); 1 毫米 EDTA] 和 10% SDS (w/v) 溶液。</li>
	<li>在冰上解冻细胞颗粒, 在400µL (DEPC) 缓冲中悬浮。将悬浮液转移到2毫升螺钉盖管中。</li>
	<li>预混料300µL 氯仿-异戊醇 (24:1 伏/五) 和300µL 苯酚 (酸苯酚, RNA 级), 并允许混合物站立5分钟。用500µL 的有机上相添加到悬浮细胞。然后加入50µL 10% SDS 和0.5 克玻璃珠 (0.5 µm) 入管。</li>
	<li>牢固地关闭盖子并且安置管子在一个珠磨房均质机 (参见材料表)。执行3×四十五年代脉冲均匀化与1分钟间隔在冰上。</li>
	<li>离心样品为10分钟在 1.1万 x g (4 °c), 并且转移上部阶段到新的管子。</li>
	<li>加入500µL 氯仿-异戊醇 (24:1) 到上阶段从步骤5.5 和离心机混合物5分钟在 1.1万 x g (4 °c)。 注: 以下步骤是从制造商的说明中修改的, 它是基于商用玻璃纤维过滤器的 RNA 隔离套件 (见材料表)。</li>
	<li>将上部相的500µL 转移到一个新鲜的管子上, 加入2卷 (1 毫升) 的裂解/结合缓冲液, 并将其与吹打上下混合。</li>
	<li>结合过滤器和收集管 (从 RNA 隔离套件) 和吸管的混合物从步骤5.7 到过滤管。离心管为十五年代在 8000 x g 和放弃流动通过。</li>
	<li>准备1.5 毫升离心管, 100 µL DNase 缓冲器, 10 µL DNase I, µL 抑制剂 5 RNase (见材料表)。在过滤管的过滤器上添加准备好的溶液组合, 在15-25 摄氏度时孵化 20-30 分钟。</li>
	<li>按照制造商的指示执行洗涤步骤。使用50µL 的洗脱缓冲洗脱 RNA。</li>
	<li>保存洗脱 RNA 在-80 摄氏度, 并在它周围放置一个塑料石蜡膜。同时, 将一些微升的样品转移到一个1.5 毫升的离心管上进行质量检查。</li>
</ol>6. RNA 质量检查和排序
<ol><li>用 microvolume 分光光度计检查 RNA。 注: 吸光度率在260/280 以上应高于 1.8, 比例为260/230 以上至少 1.8, 最好在2.0 以上。</li>
	<li>使用推荐的 rna 分析试剂盒在自动电泳仪器中运行纯化 rna (见材料表);RNA 完整性号 (玲) 必须至少在7以上才能继续。</li>
	<li>根据制造商的建议, 每样使用3µg 的 rna, 生成具有 rna 序列库准备套件 (见材料表) 的库。 注: 在高通量测序平台上对库的准备工作进行了排序, 生成了配对端读取。</li>
</ol>7. 使用基于 Web 的管道 T 雷克斯的数据分析
<ol><li>使用 FASTX 工具包版本 0.0.13 (Phred 质量分数 > 20) 执行质量筛选。用 Trimmomatic 工具修剪来自适配器序列的原始 RNA 顺序读取。 注: 所有下游分析都是以高质量的干净数据为基础的。</li>
	<li>下载B. 丝状ATCC 6462 NCBI 基因组数据库 (NCBI 加入号: CP009692.1), 并将其作为参考基因组, 用于映射使用 Bowtie2/2.2. 3 的清洁读数。</li>
	<li>使用 HTSeq v0.6.1 计数映射到每个基因的读取数字和每 kilobase 每百万的转录读数 (RPKM) 的每个基因根据该基因的长度和读取计数映射到这个基因。</li>
	<li>为了分析 T 雷克斯管道的数据, 请使用 RPKM 表作为输入, 以及三描述性文件: (i) 一个因素文件来描述实验的因素, (ii) 对比文件, 以确定哪些因素将比较的差异基因表达和 (iii) 一个类文件来描述感兴趣的基因组。 注意: 描述性文件是 in.txt 格式。例子可以在 T-雷克斯 (http://genome2d.molgenrug.nl/) 的网页上找到。一个详细的数据分析指令, 使用 T 雷克斯可以在以前的出版物中找到. 14。</li>
</ol>

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作者声明他们没有竞争的财政利益。

植物-微生物相互作用被假设是由一个微妙地调整的平衡在细菌和植物之间决定。这种相互作用是高度复杂和难以研究的自然系统, 其中包括多种微生物物种, 可能作为联营集团。本文介绍了一种简化的协议, 以研究在良好的控制条件下, 根分泌物的细菌反应。在暴露于根分泌物时, 细菌的转录剖面提供了细菌对根际生态位的适应的详细信息。这种根分泌物收集协议不需要复杂的程序和专用设备。然?...

植物可能渗出多达20% 的碳固定在光合作用中通过根进入根际1,即, 土壤狭窄的区域附近的根。由于养分的可用性较高, 根际是一种适合多种微生物的栖息地, 包括植物生长促进细菌。根分泌物含有一系列无机化合物, 如离子、无机酸、氧和水。然而, 大多数根系分泌物是由有机物质形成的, 可分为低分子量化合物和高分子量化合物。低分子量化合物包括氨基酸, 有机酸, 糖, 酚类化合物, 脂肪酸, 和一系列次生代谢物。高分子量化合物包括粘液和蛋白质2,3。根际微生物可以将其中一些化合物用作生长和发育的能量来源。根系分泌物在 rhizobacterial 群落的形成过程中起着重要的作用, 因为植物产生的化合物能通过影响特异基因的表达影响根际相关细菌的行为。
了解细菌对根分泌物的反应是破译植物-微生物相互作用机制的关键步骤。由于细菌对植物-微生物相互作用的反应是差异基因表达的产物, 可以通过转录分析来研究。使用这种方法, 以前的研究发现了几个重要的基因参与植物-微生物的相互作用。在铜绿假单胞菌中, 参与代谢、趋化和 II. 型分泌的基因对甜菜根分泌物4有反应。风扇等。5研究了amyloliquefaciens FZB42 对玉米根系分泌物的 transcriptomic 分析。结果表明, 在根分泌物强烈诱发的基因中, 有几个组参与了与养分利用、趋化、运动和非核糖体合成抗菌肽和 polyketides 有关的代谢途径。
这些研究的准确性依赖于根系分泌物的收集。虽然有几种方法描述了用于不同目的的根分泌物的收集, 但它们要么要求尖端仪器, 要么在受控条件6、7、8中不执行。此外, 根际抑制微生物可以影响植物细胞膜通透性和损害根组织, 特别是在微生物联合体9的情况下, 对根分泌物的组成有很显著的作用。在调查微生物对根分泌物的反应时, 重要的是使用明确的条件, 以避免其他微生物对化合物的改变10。此外, 基于 rna 序列的转录研究需要高质量的 rna。然而, 当处理非模型细菌菌株时, 标准协议或商业套件通常由于未知因素或特殊生长特性而效率低下。
这里描述的协议是使用丝状, 这是一个革兰阳性, 孢子形成细菌的 Firmicute。它在各种植物的根际中普遍存在。本文报道了几种植物生长促进特性, 包括甜菜11的系统抗性 (ISR) 的诱导, 对黄瓜12的禾草病原菌的抑制作用, 以及氮固定在向日葵根际13。然而, 其与寄主植物相互作用的分子机制还没有得到很好的研究。
本文的实验目的是研究 endosphere 分离的丝状对马铃薯根系分泌物的 transcriptomic 反应。简而言之, 该议定书由以下步骤组成: 一是在无菌条件下收集马铃薯根系分泌物。然后, 从细菌细胞中提取高质量的 RNA, 用根分泌物处理。最后一步是使用基于 web 的 T 雷克斯管道14的数据分析。该协议用于识别丝状基因, 表明在接触到根系分泌物时表达水平的变化, 从而可能在植物微生物相互作用中发挥重要作用。

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			<td height="21" style="height:21px;width:260px;">sodium hypochlorite</td>
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			<td style="width:152px;">&#160;CAS: 7681-52-9&#160;</td>
			<td style="width:339px;">10-15%&#160; active chlorine</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Luria-Bertani (LB) broth</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">incubater</td>
			<td style="width:236px;">New Brunswick Scientific</td>
			<td style="width:152px;">Innova 4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">spectrophotometer</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">Genesys 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">liquid nitrogen</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">glass beads</td>
			<td style="width:236px;">Sigma</td>
			<td style="width:152px;">G8893</td>
			<td>0.5 &#181;m</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">2.0 ml tube with screw cap</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>RNase free</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1.5 ml and 2.0 ml eppendorf tube</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>RNase free</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Bead mill homogenizer</td>
			<td>BioSpec</td>
			<td style="width:152px;">607</td>
			<td>Mini_beadbeater</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">centrifuge</td>
			<td style="width:236px;">Eppendorf</td>
			<td style="width:152px;">5430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Diethyl pyrocarbonate (DEPC)</td>
			<td style="width:236px;">sigma</td>
			<td style="width:152px;">CAS: 1609-47-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:236px;">sigma</td>
			<td style="width:152px;">CAS: 151-21-3&#160;</td>
			<td>10% solution prepared with DEPC treated MQ water</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">TE buffer</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>10 mM Tris-HCl; 1 mM EDTA, pH=8</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">phenol</td>
			<td>Sigma</td>
			<td style="width:152px;"></td>
			<td>RNA grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">chloroform-isoamyl alcohol&#160;</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>prepare 24:1 of chloroform:isoamyl alcohol, store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">High pure RNA isolation kit</td>
			<td style="width:236px;">Roche</td>
			<td style="width:152px;">11828665001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNase Decontamination Solution</td>
			<td style="width:236px;">Invitrogen</td>
			<td style="width:152px;">AM9780</td>
			<td>RNase-Zap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Automated electrophoresis instrument</td>
			<td style="width:236px;">Agilent</td>
			<td style="width:152px;">2100</td>
			<td>Bioanalyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microvolume spectrophotometer</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">Nanodrop ND-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNA quality analysis kit</td>
			<td style="width:236px;">Agilent</td>
			<td style="width:152px;">RNA 6000 Nano kit&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNase inhibitor</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">RiboLock</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Directional RNA library Prep kit</td>
			<td style="width:236px;">NEB</td>
			<td style="width:152px;">Ultra</td>
			<td style="width:339px;">For Illumina</td>
		</tr>
	</tbody>
</table>

plant-microbe interaction: transcriptional response of bacillus mycoides to potato root exudates

有益植物相关的细菌在促进植物生长和预防疾病中起着重要作用。应用植物生长促进细菌 (PGPR) 作为生物肥料或生防剂已成为使用常规肥料的有效替代品, 并能以较低的成本提高作物生产率。植物-微生物的相互作用取决于寄主植物分泌的信号以及它们伴生细菌的反应。然而, 分子机制的有益细菌如何响应其相关的植物衍生信号是没有充分了解。评估细菌对根分泌物的 transcriptomic 反应是确定际条件下细菌基因表达和调控的有力途径。这种知识对于了解植物-微生物相互作用所涉及的基本机制是必要的。本文描述了一个详细的协议, 以研究丝状EC18 的 transcriptomic 反应, 从马铃薯 endosphere 的菌株, 马铃薯根系分泌物。在最近的高通量测序技术的帮助下, 该协议可以在几个星期内执行, 并产生大量的数据集。首先, 我们收集在不育条件下的根分泌物, 然后将它们添加到B. 丝状文化中。这些文化中的 RNA 是用苯酚/氯仿方法与商业试剂盒相结合, 并通过自动电泳仪器进行质量控制而分离出来的。在测序后, 采用基于 web 的 T 型雷克斯管道进行数据分析, 确定了一组差异表达基因。这种方法是一个有用的工具, 以促进新发现的细菌基因所涉及的植物-微生物相互作用。

植物相关的微生物可以积极地影响植物的生长和健康。然而, 植物与微生物共生的复杂相互作用机制还没有得到充分的理解。根系分泌物在调节 rhizobacterial 活动和行为方面起着重要作用, 一般认为根系的微生物定植与微生物对根系分泌物的吸引有关。本研究的目的是探讨 rhizobacterial B 丝状对马铃薯根系分泌物的 transcriptomic 反应。为实现这一目的, 马铃薯块茎在蒸压?...

Watch this Scientific Journal Video about Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates at JoVE.com

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

本议定书的目的是研究 endosphere 分离芽孢杆菌丝状对马铃薯根系分泌物的 transcriptomic 反应。这种方法有助于鉴定与植物微生物相互作用有关的重要细菌基因, 并原则上适用于其他菌和植物, 并作小的调整。

植物-微生物相互作用:丝状芽孢杆菌对马铃薯根系分泌物的转录反应

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant ...

plant-microbe-interaction-transcriptional-response-bacillus-mycoides

Research

JoVE Journal

Biology

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

12.4K 次观看.  University of Groningen. 本议定书的目的是研究 endosphere 分离芽孢杆菌丝状对马铃薯根系分泌物的 transcriptomic 反应。这种方法有助于鉴定与植物微生物相互作用有关的重要细菌基因, 并原则上适用于其他菌和植物, 并作小的调整。

视频: Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

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

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

马铃薯和尼科蒂亚娜本塔米亚纳的农业渗透和PVX农业感染

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

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

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

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

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

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

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

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

方法发现小鼠BCRP1的另类用法子和转录调控

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...