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) 液体培养基与适当的抗生素在50毫升瓶。</li>
	<li>在37° c 的瓶子上一夜之间孵育瓶。在室温下离心 1.2万 x g 的悬浮液收集细菌5分钟, 丢弃上清。按照制造商的质粒准备试剂盒的程序提取和纯化质粒。</li>
</ol>3. 原生质体分离
<ol><li>从10天的老大豆幼苗 (图 1) 中切下新扩展的单叶叶。 注: 在适宜的发育阶段选择叶片是大豆原生质体制备成功的关键。只有在早期发育阶段才使用扩大叶。随着树叶的成熟, 细胞壁变得更加难以消化。</li>
	<li>用新鲜的剃刀刀片, 从单叶叶中取出中脉, 然后将残余切成 0.5-1 毫米的条纹。 注意: 两个单叶的叶子在10毫升的酶溶液中消化, 将为超过10的转化提供足够的原生质。</li>
	<li>使用一对镊子, 立即和轻轻地转移到10毫升的新准备的酶溶液 (表 1) 在15毫升管的叶子条。真空在室温下渗透15分钟的叶子条。</li>
	<li>在室温下, 用柔和的搅拌 (40 rpm) 在低光照下孵育酶溶液中的叶子条, 4-6 小时。确保当原生质体释放时, 酶溶液变成黄绿色。检查显微镜下的酶/原生质体溶液 (X10)。 注意: 释放的原生质体是球形的, 而未消化的细胞有不规则或椭圆形的形状。</li>
	<li>将10毫升的酶/原生质体溶液在50毫升的试管中轻轻地浇注并加入10毫升的 W5 溶液 (表 1) 在室温下, 以停止消化。轻轻地倒置管几次。将酶/原生质体溶液轻轻倒入一个干净的75µm 尼龙网格上, 放在50毫升管上, 除去未消化的叶子组织。</li>
	<li>将流经的酶/原生质体溶液离心于50毫升的 100 x g, 室温下为1-2 分钟。用10毫升的血清吸管轻轻移除上清液, 不干扰原生质体颗粒。</li>
	<li>通过在显微镜下 hemacytometer 下的原生质体数 (x10), 在 W5 溶液中重原生质体并稀释到 2 x 105 mL-1的浓度。将原生质体保持在冰上30分钟。</li>
	<li>离心悬浮在 100 x g 在室温下1-2 分钟, 用1毫升吸管轻轻地移除 W5 溶液, 而不干扰原生质体颗粒。重 MMG 溶液 (表 1) 中的原生质, 在室温下浓度为 2 x 105 mL-1 。</li>
</ol>4. 原生质体转化
<ol><li>100µL 等分原生质体 (2 x 104原生质体在 2 x 105 ml-1) 在1.5 毫升低粘离心管使用未切割的200µL 吸管提示。把一个分放在一旁作为负控制。在原生质体的其余分中加入10µL 质粒 (10-20 µg)。</li>
	<li>在 1.5 mL 离心管的内壁上缓慢地加入110µL 的新制备的 PEG 溶液 (表 1), 然后轻轻地倒置和旋转管, 直到溶液变得均匀。</li>
	<li>室温下孵育转化混合物15分钟。</li>
	<li>为了停止转换, 慢慢地在室温下将400µL 的 W5 溶液加到1.5 毫升的管中, 然后轻轻地倒置管子, 直到溶液变得均匀。离心管在100克在室温下1-2 分钟, 用吸管丢弃上清液。</li>
	<li>将1毫升的 WI 解决方案 (表 1) 添加到试管和重中, 轻轻移1-2 次。添加1毫升的 5% (卷/卷) 无菌小牛血清在每个井的 6-良好的组织培养板, 以大衣的表面, 并防止原生质体黏附在板块。</li>
	<li>几秒钟后, 用吸管将小牛血清丢弃。将悬浮原生质体转移到培养板的井中。用盖子盖住盘子。</li>
</ol>5. 原生质体培养和收获
<ol><li>在室温下在黑暗中孵育原生质1-2 天。 注意: 我们发现, 两天的孵化产生更强的荧光蛋白信号一般与一天的孵化相比, 和荧光蛋白信号可能持续长达3-4 天。</li>
	<li>将原生质体溶液转移到1.5 毫升低粘离心管。离心管在 100 x g 为1-2 分钟在室温下收获原生质体。用吸管移除上清液, 并将10µL 原生质体转移到玻片上。</li>
	<li>在荧光或共聚焦显微镜下观察荧光信号。使用非转化的原生质体作为阴性控制 (没有信号应该被观察)。</li>
</ol>

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A., 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=The+NAC+domain-containing+protein,+GmNAC6,+is+a+downstream+component+of+the+ER+stress-and+osmotic+stress-induced+NRP-mediated+cell-death+signaling+pathway.">The NAC domain-containing protein, GmNAC6, is a downstream component of the ER stress-and osmotic stress-induced NRP-mediated cell-death signaling pathway.</a> BMC Plant Biol. 11 (1), 129 (2011).</li><li>Kidokoro, S., 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=Soybean+DREB1/CBF-type+transcription+factors+function+in+heat+and+drought+as+well+as+cold+stress-responsive+gene+expression.">Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression.</a> Plant J. 81 (3), 505-518 (2015).</li><li>Sun, X., 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=Targeted+mutagenesis+in+soybean+using+the+CRISPR-Cas9+system.">Targeted mutagenesis in soybean using the CRISPR-Cas9 system.</a> Sci Rep-UK. 5, 10342 (2015).</li><li>Karimi, M., Inz&#233;, D., Depicker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATEWAY&#8482;+vectors+for+Agrobacterium-mediated+plant+transformation.">GATEWAY&#8482; vectors for Agrobacterium-mediated plant transformation.</a> Trends Plant Sci. 7 (5), 193-195 (2002).</li><li>Xia, Z., 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=Positional+cloning+and+characterization+reveal+the+molecular+basis+for+soybean+maturity+locus+E1+that+regulates+photoperiodic+flowering.">Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering.</a> P. Natl. A. Sci. USA. 109 (32), E2155-E2164 (2012).</li></ol>

作者声明他们没有竞争的金融利益。

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><tbody><tr><td>MES</td><td>Sigma Aldrich</td><td>M8250-100G</td><td></td></tr><tr><td>Cellulase CELF</td><td>Worthington Biological Corporation</td><td>LS002611</td><td></td></tr><tr><td>Pectolyase Y-23</td><td>BioWorld</td><td>9033-35-6</td><td></td></tr><tr><td>CELLULASE &quot;ONOZUKA&quot; R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>MACEROZYME R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>Mannitol</td><td>ICN Biomedicals</td><td>152540</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C79-500g</td><td></td></tr><tr><td>BSA</td><td>NEB</td><td>R3535S</td><td></td></tr><tr><td>DTT</td><td>Sigma Aldrich</td><td>D5545-5G</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653-1kg</td><td></td></tr><tr><td>KCl</td><td>Fisher</td><td>P217-500g</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M8266-100g</td><td></td></tr><tr><td>PEG4000</td><td>Fluka</td><td>81240</td><td></td></tr><tr><td>nylon mesh</td><td>carolina</td><td>652222N</td><td></td></tr><tr><td>Tissue Culture Plates</td><td>USA Scientific</td><td>CC7682-7506</td><td></td></tr><tr><td>Razor Blades</td><td>Fisher</td><td>12-640</td><td></td></tr><tr><td>hemacytometer</td><td>hausserscientific</td><td>1483</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>EZNA plasmid miniprep kit</td><td>Omega</td><td>D6942-01</td><td></td></tr><tr><td>GeneJET Plasmid Miniprep Kit</td><td>Thermo Scientific</td><td>K0502</td><td></td></tr><tr><td>Centrifuge 5810</td><td>eppendorf</td><td>5811000827</td><td></td></tr><tr><td>Centrifuge 5424</td><td>eppendorf</td><td>22620401</td><td></td></tr><tr><td>Jencons Powerpette Plus Pipet Controller</td><td>Jencons</td><td>14526-202</td><td></td></tr><tr><td>Zeiss 710 Confocal Microscope</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td><td>Ambion</td><td>AM12450</td><td></td></tr><tr><td>15 mL Centrifuge Tubes</td><td>Denville</td><td>C1018-P</td><td></td></tr><tr><td>50 mL Centrifuge Tubes</td><td>Denville</td><td>C1060-P</td><td></td></tr><tr><td>Newborn Calf Serum</td><td>Thermo Scientific</td><td>16010159</td><td></td></tr><tr><td>Soil</td><td>Ingram&#039;s Nursery</td><td></td><td></td></tr><tr><td>perlite</td><td>Vigoro</td><td>100521091</td><td></td></tr><tr><td>Torpedo Sand</td><td>JKS Ventures</td><td></td><td></td></tr><tr><td>LB Broth, Lennox (Powder)</td><td>Fisher</td><td>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. ...

a-simple-method-for-isolation-soybean-protoplasts-application-to

Research

JoVE Journal

Biology

24.8K 次观看.  California State University, Northridge. 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。

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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

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 ...

环境科学

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

烟草 Benthamiana叶在制备鳞片上的瞬态表达

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

生物化学

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

生物学

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

转基因大豆毛状根的高效生产

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

作者聚焦:用于基因功能分析的大豆毛状根转化  

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

大豆毛根转化基因功能分析

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...

生物工程

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

细菌，昆虫细胞和植物系统:重组蛋白表达在不同的生物工厂的比较分析

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

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