Name: polysome purification from soybean symbiotic nodules
Uploaded: 2022-07-01T15:00:00
Description: Watch this Scientific Journal Video about Polysome Purification from Soybean Symbiotic Nodules at JoVE.com

这项研究由CSIC I + D 2020年第282号资助，FVF 2017年第210号资助和PEDECIBA（玛丽亚·玛莎·塞恩斯）资助。

1. 植物生长和根瘤菌接种
<ol><li>为了产生所需的结节，请在受控条件下将所选的大豆种子播种在生长室的选定基质中。 注意：在该协议中，种子播种在装有沙子：蛭石（1：1）混合物的0.5升塑料瓶中。生长室的昼/夜循环温度分别为28 °C / 20 °C，光/暗光周期分别为16 h/8 h。光合有效辐射强度为620 μmol·m−2·s−1.</li>
	<li>提前准备液体酵母提取物甘露醇（YEM）培养基?...

<ol>
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在翻译水平上研究基因表达调控对于更好地理解不同的生物学过程至关重要，因为细胞基因表达的终点是蛋白质丰度13，14。这可以通过分析感兴趣的组织或生物体的翻译组来评估，其中应纯化多体部分并分析其相关的mRNA。
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豆科植物，如大豆（Glycine max），可以与称为根瘤菌的特定土壤细菌建立共生关系。这种互惠关系导致植物根部形成新的器官，即共生结节。结节是寄存细菌的植物器官，由宿主细胞组成，其细胞质定植于一种称为拟杆菌的特殊形式的根瘤菌。这些拟杆菌催化大气氮（N 2）还原成氨，氨被转移到植物中以换取碳水化合物1，2。
尽管这种固氮共生是研究最充分的植物-微生物共生之一，但许多方面仍有待更好地理解，例如受到不同非生物胁迫条件的植物如何调节它们与共生伙伴的相互作用以及这如何影响结节代谢。通过分析结节翻译组（即主动翻译的信使RNA[mRNA]的子集），可以更好地理解这些过程。多核糖体或多核糖体是与mRNA相关的多个核糖体的复合物，通常用于研究翻译3。多聚体分析方法包括分析与多聚体相关的mRNA，并已成功用于研究控制不同生物过程中发生的基因表达的转录后机制4，5。
从历史上看，基因组表达分析主要集中在确定mRNA丰度6，7，8，9。然而，由于基因表达转录后调控的不同阶段，特别是翻译10，11，12，转录本和蛋白质水平之间缺乏相关性。此外，在转录组水平上的变化与在翻译组水平上发生的变化之间没有观察到依赖性13。对正在翻译的mRNA集的直接分析可以比仅分析mRNA水平时获得的细胞基因表达（其终点是蛋白质丰度）更准确和完整的测量14，15，16。
该协议描述了如何通过两层蔗糖垫的差异离心从完整的大豆结节中纯化植物来源的多聚体（图1）。然而，由于拟杆菌衍生的核糖体也存在于结节中，因此核糖体和RNA物种的混合物被纯化，即使真核糖体占主要部分（90%-95%）。还描述了随后的RNA分离、定量和质量控制（图1）。该协议与RNA-seq相结合，应提供有关复杂组织（例如共生结节）中真核mRNA的翻译调节的实验结果。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64269/64269fig01.jpg"> 图1：从共生结节纯化真核多体的拟议方法的示意图概述。 该方案概述了方案中遵循的步骤，从（1）植物生长和（2）结节收获到（3）制备胞质提取物，（3）获得总样品和（4）PAR样品，以及（5）RNA提取和质量控制。缩写：PEB = 多聚体提取缓冲液;RB = 重悬缓冲液;总 = 总核糖核酸;PAR = 多聚体相关的 mRNA。 <a href="https://www.jove.com/files/ftp_upload/64269/64269fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plant growth and rhizobia inoculation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Daihan Scientific</td>
			<td></td>
			<td>Model SHO-1D</td>
		</tr>
		<tr>
			<td>YEM-medium</td>
			<td>Amresco</td>
			<td>J850 (yeast extract) 0122 (mannitol)</td>
			<td></td>
		</tr>
		<tr>
			<td>Water deficit treatment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>Merck</td>
			<td>221295</td>
			<td></td>
		</tr>
		<tr>
			<td>Porometer</td>
			<td>Decagon Device</td>
			<td></td>
			<td>Model SC-1</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of cytosolic extracts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brij L23</td>
			<td>Sigma-Aldrich</td>
			<td>P1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sigma</td>
			<td></td>
			<td>Model 2K15</td>
		</tr>
		<tr>
			<td>Chloranphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>DOC</td>
			<td>Sigma-Aldrich</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA 360</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1.04936</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tissue grinder</td>
			<td>Fisher Scientific</td>
			<td>12649595</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>PTE</td>
			<td>Sigma-Aldrich</td>
			<td>P2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Invitrogen</td>
			<td>15504-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing dish&#160;</td>
			<td>Deltalab</td>
			<td>1911103</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of sucrose cushions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Invitrogen</td>
			<td>15503022</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 40 Ti rotor</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td>Optima L-100K</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman-Coulter</td>
			<td>344059</td>
			<td>13.2 mL tubes</td>
		</tr>
		<tr>
			<td>RNA extraction and quality control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Thermo scientific</td>
			<td>R0492</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td></td>
			<td>Model 2100. Eukaryote total RNA nano assay</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>DI</td>
			<td>41191</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Dorwil</td>
			<td>UN1170</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Mallinckrodt</td>
			<td>3032-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Sigma</td>
			<td>10814-010</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol LS</td>
			<td>Ambion</td>
			<td>102960028</td>
			<td></td>
		</tr>
		<tr>
			<td>Miscellaneous</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 15 mL</td>
			<td>Biologix</td>
			<td>10-0152</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 10 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>321-4050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 1000 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>361-1050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 20 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>341-4050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 200 &#181;L</td>
			<td>Tarsons</td>
			<td>528104</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5 mL</td>
			<td>Tarsons</td>
			<td>500010-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 2.0 mL</td>
			<td>Tarsons</td>
			<td>500020-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing company</td>
			<td>Macrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 250 mL flask</td>
			<td>Marienfeld</td>
			<td>4110207</td>
			<td></td>
		</tr>
	</tbody>
</table>

polysome purification from soybean symbiotic nodules

该协议的目的是为研究大豆（甘氨酸max）共生结节的真核翻译组提供策略。本文描述了优化的方法，以分离植物来源的多核糖体及其相关的mRNA，以使用RNA测序进行分析。首先，通过在保存多小体和RNA的条件下从整个冷冻大豆结节中匀浆获得细胞质裂解物。然后，通过低速离心清除裂解物，并使用15%的上清液进行总RNA（TOTAL）分离。剩余的澄清裂解物用于通过两层蔗糖垫（12%和33.5%）超速离心分离多聚体。重悬后从多粒体沉淀中纯化多聚体相关mRNA（PAR）。TOTAL和PAR均通过高灵敏度毛细管电泳进行评估，以满足RNA-seq测序文库的质量标准。作为下游应用的一个例子，测序后，基因表达分析的标准管道可用于获得转录组和翻译组水平的差异表达基因。总之，该方法与RNA-seq相结合，可以研究复杂组织（例如共生结节）中真核mRNA的翻译调节。

使用上述程序纯化的TOTAL和PAR级分的数量和质量评估是决定其成功与否的关键，因为对于大多数下游应用，例如RNA测序，高质量样品是文库制备和测序的基础。此外，RNA分子的完整性允许在样品收集时捕获基因表达谱的快照18。在这种情况下，使用生物分析仪进行这些测量时会获得RNA完整性数（RIN）。RIN 用于以稳健、可靠且独立于用户的方式分配完整性值，范围从 10（完整）到 1...

Watch this Scientific Journal Video about Polysome Purification from Soybean Symbiotic Nodules at JoVE.com

Polysome Purification from Soybean Symbiotic Nodules

该协议描述了从完整的大豆结节中纯化真核多体的方法。测序后，基因表达分析的标准管道可用于鉴定转录组和翻译组水平的差异表达基因。

大豆共生结节多聚体纯化

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

该协议意义重大，因为据我们所知，它是第一个描述一种新的离心机金属，用于从大豆共生结核中动态多体纯化。该技术的最大优点是与RNA-Seq结合，允许研究在诸如共生结节的复杂组织中组织的关系。首先，在称量盘中称量约0.2克完整的结节，并将它们转移到预冷的两毫升管中。然后加入1.2毫升多聚体提取缓冲液，让样品解冻两分钟，并用组织研磨机匀浆，直到结节完全破碎和均质化。接下来，将样品在冰上轻轻搅拌孵育10分钟或直到所有样品都经过处理，并在4摄氏度下以16， 000倍G离心15分钟以沉淀碎片。然后，回收上清液，并重复离心步骤。仔细回收澄清的胞质提取物后，将200微升等分试样转移到干净的1.5毫升微量离心管中进行完全分离。然后，将4.5毫升33.5%蔗糖层倒入离心管中，并用P-1000微量移液器小心缓慢地加入4.5毫升12%层。接下来，将试管放在冰上，直到加入澄清的胞质提取物。小心地移液到管的侧壁上，将一毫升澄清的胞质提取物装载到蔗糖垫的顶部。然后，将超离心管转移到预冷的桶中，并在 217， 874 G 的温度下在 4 摄氏度下离心两个小时。丢弃剩余的胞质提取物和蔗糖垫后，用200微升预冷的重悬缓冲液重新悬浮多体沉淀。接下来，在冰上孵育30分钟，然后将多体重悬转移到1.5毫升预冷管中进行RNA提取。用750微升RNA分离试剂匀浆样品后，在室温下孵育5分钟。然后，加入200微升冷氯仿，用力摇动试管15秒。接下来，在室温下孵育 10 分钟。在4摄氏度下以12，000倍G离心15分钟进行相分离，并将500微升上层水相转移到干净的管中，而不会干扰粉红色有机相。然后，加入375微升冷异丙醇和0.5微升RNA游离糖原。之后，通过上下移液彻底混合。将混合物在 4 摄氏度下孵育 10 分钟，并以 12， 000 倍 G 离心 15 分钟。然后，弃去上清液，并用一毫升冷的75%乙醇洗涤RNA沉淀。通过短暂的涡旋混合。接下来，在 7， 500 G 下在 4 摄氏度下离心五分钟。弃去上清液，风干RNA沉淀。然后，将沉淀溶解在50微升RNA游离水中，并在65摄氏度下孵育五分钟。使用高灵敏度毛细管电泳和/或在 2%RNA 游离琼脂糖凝胶上进行电泳评估 RNA 浓度和完整性。估计样品体积后，加入0.1体积的三摩尔乙酸钠，三体积的冷乙醇和0.5微升RNA，游离糖原。然后，将它们彻底混合。对几种总和多体相关RNA样品级分进行了毛细管电泳，显示出清晰的核糖体RNA条带，没有任何拖尾的外观。但是，这并未反映在RIN值中，因为它们的范围在5.9和7.5之间，对应于非完整样品。生物分析仪未能识别电泳图中显示的18S和25S峰，包括总和多体相关RNA样品。没有样品降解的视觉迹象。但是，对样品进行了分析，以可视化几乎完全降解的样品的结果，以便进行比较。最重要的是在整个协议期间在多聚体和RNA保存条件下工作。测序后，所分析基因表达的总和帕尔RNA级分标准模式可用于鉴定转录和翻译水平的差异表达基因。

Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

从胚胎干细胞衍生心脏祖细胞

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

从非转化人成纤维细胞增生四倍体细胞的建立

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

新生小鼠胰腺胰岛功能的有效隔离

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Imaging Dpp Release from a Drosophila Wing Disc

Imaging Dpp Release from a Drosophila Wing Disc

成像 Dpp 释放从果蝇翼光盘

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

从斑马鱼胚胎中产生幼稚的胚泡外植体

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Isolation of Preadipocytes from Broiler Chick Embryos

从肉鸡雏鸡胚胎中分离出前脂肪细胞

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. ...