Name: a simple method for isolation of soybean protoplasts and application to transient gene expression analyses
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这项工作得到了来自国家科学基金会 (NSF-PGRP-IOS-1339388) 的植物基因组研究项目的支持。

1. 植物的生长
<ol><li>母猪 5-10 大豆种子 (威廉斯 82) 在一个 13 cm 罐在温室在长的天情况下 (16 h 光在1500µmol m-2 s-1) 在25° c 在风俗土壤混合为大豆 (1:1: 1 土、珍珠岩和鱼雷砂的比值。</li>
</ol>2. 质粒 DNA 的制备
<ol><li>使用无菌吸管尖端或牙签, 选择一个单一的殖民地或冷冻甘油库存的大肠杆菌携带的质粒含有的利益基因和接种它在20毫升 Luria Bertani (LB) 液体?...

<ol>
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本方案对大豆原生质体的分离及在瞬态表达研究中的应用进行了全面的测试, 并在实验室中得到了很好的效果。程序简单易得, 需要普通设备和最低成本。我们的协议产生了大量的均匀, 高质量的原生质相比, 以前报告的方法34,35,36,37,38。然而, 由于有许多因素影响原生质体的?...

原生质体是细胞壁被移除的植物细胞。由于它们保持植物细胞的大部分功能和活动, 原生质体是一个良好的模型系统, 观察和评价各种细胞事件, 是有价值的工具, 研究体细胞杂交1和植物再生2。原生质体也被广泛用于植物转化3,4,5, 因为细胞壁会阻止 DNA 进入细胞。原生质体具有完整植物的一些生理反应和细胞过程, 因此为研究亚细胞蛋白定位的基础研究提供了基本的价值6,7,8,蛋白质-蛋白质相互作用9,10和启动子活动11,12,13在活细胞中。
首次报道了植物原生质体的分离, 在 1960年14 , 对原生质体分离和转化的协议进行了开发和优化。原生质体分离的标准程序包括叶片的切割和细胞壁的酶消化, 然后分离出未被消化的组织碎片释放出的原生质体。转换策略包括电穿孔15,16, 微注射17,18, 和聚乙二醇基 (PEG)4,5,19方法。广泛的物种已报告原生质体分离成功, 包括柑桔20, 芸薹21, 茄22和其他观赏植物系列23,24。虽然不同的组织类型用于各种物种, 一个系统的瞬态表达在拟南芥叶肉原生质体 (TEAMP) 分离从模型植物的叶子拟南芥已经建立良好的25并广泛应用于不同的应用程序。
大豆 (甘氨酸最大值(L.)含笑.) 是最重要的蛋白质和油料作物之一26。与拟南芥和大米不同, 获得转基因大豆植株是相当困难和低效率的。农杆菌介导的浸润已普遍用于烟草表皮细胞的瞬态基因表达研究27和幼苗在拟南芥28,29, 而农杆菌杆菌已用于大豆毛状根的转化,30。病毒诱导的基因沉默方法已被用于下调的目标基因31,32和瞬态表达式33系统的方式。原生质体为这些方法提供了一个有价值的和多才多艺的替代品。原生质体可以从大豆的地上材料中获得, 并允许快速、同步的转基因表达。然而, 自从大豆原生质体在 198334初期成功分离后, 大豆原生质体的应用有了有限的报告35、36、37、38, 主要是由于大豆原生质体的产量相对较低。
本文介绍了一种简单有效的大豆原生质体分离方法及其在瞬态基因表达研究中的应用。利用大豆幼苗的幼单叶叶, 我们能够在几个小时内获得大量的重要原生质体。此外, 我们还优化了 PEG-钙介导的转化法, 使 DNA 转化为高效的大豆原生质体是简单、低成本的。

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">MES</td>
			<td style="width:181px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">M8250-100G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulase CELF</td>
			<td>Worthington Biological Corporation</td>
			<td>LS002611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pectolyase Y-23</td>
			<td>BioWorld</td>
			<td>9033-35-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CELLULASE &#34;ONOZUKA&#34; R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACEROZYME R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td>ICN Biomedicals&#160;</td>
			<td>152540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher&#160;</td>
			<td>C79-500g&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>Sigma Aldrich&#160;</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma Aldrich&#160;</td>
			<td>S7653-1kg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher&#160;</td>
			<td>P217-500g&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma Aldrich&#160;</td>
			<td>M8266-100g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PEG4000</td>
			<td>Fluka</td>
			<td>81240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nylon mesh</td>
			<td>carolina</td>
			<td>652222N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Tissue Culture Plates&#160;</td>
			<td>USA Scientific</td>
			<td>CC7682-7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Razor Blades</td>
			<td style="width:181px;">Fisher</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hemacytometer</td>
			<td>hausserscientific</td>
			<td>1483</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:181px;">Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">EZNA plasmid miniprep kit</td>
			<td style="width:181px;">Omega</td>
			<td>D6942-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">GeneJET Plasmid Miniprep Kit</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>K0502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Centrifuge 5810</td>
			<td style="width:181px;">eppendorf</td>
			<td style="width:119px;">5811000827</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Centrifuge 5424</td>
			<td style="width:181px;">eppendorf</td>
			<td>22620401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Jencons Powerpette Plus Pipet Controller</td>
			<td style="width:181px;">Jencons</td>
			<td>14526-202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:256px;">Zeiss 710 Confocal Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:256px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td>
			<td style="width:181px;">Ambion</td>
			<td style="width:119px;">AM12450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">15 mL Centrifuge Tubes</td>
			<td style="width:181px;">Denville</td>
			<td style="width:119px;">C1018-P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">50 mL Centrifuge Tubes</td>
			<td style="width:181px;">Denville</td>
			<td style="width:119px;">C1060-P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Newborn Calf Serum</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:119px;">16010159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Soil</td>
			<td style="width:181px;">Ingram&#39;s Nursery</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">perlite</td>
			<td style="width:181px;">Vigoro</td>
			<td style="width:119px;">100521091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Torpedo Sand</td>
			<td style="width:181px;">JKS Ventures</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LB Broth, Lennox (Powder)</td>
			<td style="width:181px;">Fisher</td>
			<td style="width:119px;">BP1427-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple method for isolation of soybean protoplasts and application to transient gene expression analyses

大豆 (甘氨酸最大值(L.)含笑) 是一种重要的农作物品种, 已成为研究遗传和生物化学途径的豆科动物模型。因此, 建立高效的大豆瞬时基因表达系统是十分重要的。在这里, 我们报告了一个简单的协议, 大豆原生质体的制备及其应用的瞬态功能分析。我们发现, 大豆幼苗的幼单叶叶片产生了大量高质量的原生质体。通过优化 PEG-钙介导的转化方法, 利用大豆单叶原生质体实现了高转化率。该系统为检测活大豆细胞中复杂的调控和信号机制提供了一个高效、通用的模型, 有助于更好地了解豆类的不同细胞、发育和生理过程。

对10天的老大豆不同器官进行了原生质体准备 (图 1), 并在显微镜下观察了产量 (图 2)。从下胚轴和上的细胞壁几乎没有被消化, 有些细胞相互连接 (图 2B, 2C)。在子叶 (图 2D) 和根 (图 2A) 中, 单元格壁仅在单元格的一小部分中被移除。相比之下, 在使用...

Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

我们开发了一个简单和有效的协议, 以制备大量的大豆原生质体研究复杂的调控和信号机制的活细胞。

大豆原生质体分离的简易方法及其在瞬态基因表达分析中的应用

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Research

JoVE Journal

Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

蛋白通过流体动力基因传递在小鼠体内瞬时表达

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...