Name: co-expression of multiple chimeric fluorescent fusion proteins in an efficient way in plants
Uploaded: 2018-07-01T12:30:00
Description: Watch this Scientific Journal Video about Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants at JoVE.com

我们感谢王实验室的成员提供了有益的讨论和意见。这项工作得到了中国国家自然科学基金 (自然科学基金, 31570001 号赠款) 和广东省和广州市自然科学基金 (2016A030313401 和 201707010024) 的支持。

1. 底漆设计策略和 DNA 放大
<ol><li>设计 DNA 片段分子克隆的引物。该引物包括20个 bp 基因特异结合序列和20到 25 bp 5 '-端悬架序列, 这是互补重叠序列的相邻 DNA 分子 (见表 1例)。 注: 每个 DNA 片段的后续组装, 不同蛋白表达盒的连接, 以及与最终表达载体的整合都取决于相邻重叠序列的识别。</li>
	<li>扩增 DNA 片段, 包括启动子、荧光记者、靶基因和终结器, 这些都是用标准 PCR 反应构建...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-adapted+green+fluorescent+protein+is+a+versatile+vital+reporter+for+gene+expression,+protein+localization+and+mitosis+in+the+filamentous+fungus,+Aspergillus+nidulans.">Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans.</a> Mol Microbiol. 27 (1), 121-130 (1998).</li><li>Bruening, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+gene+silencing+regularized.">Plant gene silencing regularized.</a> Proc Natl Acad Sci U S A. 95 (23), 13349-13351 (1998).</li><li>Wassenegger, M., Pelissier, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+RNA-mediated+gene+silencing+in+higher+plants.">A model for RNA-mediated gene silencing in higher plants.</a> Plant Mol Biol. 37 (2), 349-362 (1998).</li><li>Dehio, C., Schell, J. <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+plant+genetic+loci+involved+in+a+posttranscriptional+mechanism+for+meiotically+reversible+transgene+silencing.">Identification of plant genetic loci involved in a posttranscriptional mechanism for meiotically reversible transgene silencing.</a> Proc Natl Acad Sci U S A. 91 (12), 5538-5542 (1994).</li><li>Zhong, G., Zhu, Q., Li, Y., Liu, Y., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Once+for+all:+A+novel+robust+system+for+co-expression+of+multiple+chimeric+fluorescent+fusion+proteins+in+plants.">Once for all: A novel robust system for co-expression of multiple chimeric fluorescent fusion proteins in plants.</a> Front Plant Sci. 8, 1071 (2017).</li><li>Li, X. 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在此, 我们展示了一种新的方法, 以有力地共同表达嵌合体荧光融合蛋白的植物。它既可用于瞬态表达和遗传转化, 又能与当前基于荧光蛋白的生物成像、分子、生物化学应用和技术相兼容9、10、13.此外, 它克服了传统方法的困难, 使用几个单独的表达质粒的蛋白质共同表达。相比之下, 它使用一个单一的表达载体, 其中包含多个?...

嵌合荧光融合蛋白被认为是研究细胞内动力学和亚型定位的有用工具, 进一步了解其生理功能和工作机制1,2,3,4. 与所讨论的蛋白质共同表达知名的细胞器报告蛋白, 以更好地说明其在单元格中膜系统中的时空原理、分布和功能, 尤其有益.4,5,6,7,8。
通过瞬时表达和稳定的遗传转化, 可以在植物中表达嵌合体荧光融合蛋白, 其各自的优点和局限性分别为9、10、11。蛋白质的瞬时表达是一种方便的方法, 包括枪法轰击-, 聚乙二醇 (PEG)-, 或电穿孔介导的 DNA 瞬时表达在原生质体和农杆菌介导的叶浸润完整的植物细胞, 如图 1A所示,B12,13,14,15,16。然而, 多嵌合体荧光融合蛋白在单个植物细胞中的联合表达需要多种独立表达质粒的混合物。因此, 在植物中使用多种质粒进行蛋白质共表达的缺点是, 由于几种粒度的几率大大降低, 同时进入同一细胞, 而与单粒,每种质粒的无控制随机量导致的蛋白质表达水平的变化转移到细胞17,18。此外, 在技术上有挑战性的是引入几个独立的表达质粒成一个单一的农杆菌蛋白共表达9,10,11。因此,农杆菌介导的烟叶浸润的蛋白质瞬时表达只能一次表达一种质粒, 如图 1B所示。相反, 产生荧光融合蛋白的转基因植物通常是由带有二进制转化载体的农杆菌实现的。然而, 将基因转移和插入到植物基因组中的二进制载体只能表达单一的荧光融合蛋白 (图 1B)9、10、12。生成一种能同时表达几种嵌合荧光蛋白的转基因植物需要多轮的基因交叉和筛查, 这可能需要数月到数年, 这取决于要共同表达的基因的数量。
就业一个单一表达载体的多种蛋白质的共同表达在植物已经报告了几个以前的研究19,20,21。然而, 在蛋白质共表达或过度表达的最终质粒的生成中, 通常需要对 dna 分子和主干载体进行多轮酶消化和 dna 结扎。在这里, 我们开发了一种新的和稳健的方法来共同表达多嵌合体荧光蛋白在植物中。它是一种高效、方便的方法, 在植物中实现了多种蛋白质的共同表达, 既具有瞬态表达, 又能稳定地转化为一种时间的方式。它使用一个单一的载体, 包含多个功能独立的蛋白表达盒, 为蛋白质的共同表达, 从而克服了传统方法的弊端。此外, 它是一个高度多才多艺的系统, 其中 dna 操作和组装是通过简单的一步优化反应, 没有额外的步骤 dna 消化和结扎。工作原理如图 2所示。此外, 它完全兼容目前的细胞, 分子和生物化学的方法, 基于嵌合体荧光融合蛋白。

<table>
	<tbody>
		<tr>
			<td>KOD-FX Polymerase</td>
			<td>TOYOBO</td>
			<td>KFX-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sma I</td>
			<td>NEB</td>
			<td>R0141L/S/V</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>BBI</td>
			<td>A600194-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>BBI</td>
			<td>A601336-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP</td>
			<td>NEB</td>
			<td>#N0447V</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>BBI</td>
			<td>C4H10O2S2</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>BBI</td>
			<td>A100159-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>NAD</td>
			<td>BBI</td>
			<td>A600641-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>T5 exonuclease</td>
			<td>Epicentre</td>
			<td>T5E4111K</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA polymerase</td>
			<td>NEB</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>NEB</td>
			<td>B9022S</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog Basal Salt Mixture(MS)</td>
			<td>Sigma</td>
			<td>M5524</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>BBI</td>
			<td>A500737-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>BBI</td>
			<td>A600560-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BBI</td>
			<td>A505255-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>BBI</td>
			<td>A614270-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold microcarrier particles</td>
			<td>Bio-Rad</td>
			<td>165-2263</td>
			<td>1.0 &#181;m</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>BBI</td>
			<td>CD0050-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrocarriers</td>
			<td>Bio-Rad</td>
			<td>165-2335</td>
			<td></td>
		</tr>
		<tr>
			<td>Rupture disk</td>
			<td>Bio-Rad</td>
			<td>165-2329</td>
			<td></td>
		</tr>
		<tr>
			<td>Stopping screen</td>
			<td>Bio-Rad</td>
			<td>165-2336</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>OXOID</td>
			<td>LP0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>OXOID</td>
			<td>LP0021</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BBI</td>
			<td>A610476-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>BBI</td>
			<td>A610440-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>BBI</td>
			<td>A600219-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Genview</td>
			<td>AH169-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Wortmannin</td>
			<td>Sigma</td>
			<td>F9128</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Sigma</td>
			<td>SML0975-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulphoxide (DMSO)</td>
			<td>BBI</td>
			<td>A600163-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Panasonic</td>
			<td>MLR-352H-PC</td>
			<td></td>
		</tr>
		<tr>
			<td>PSD-1000/He particle delivery system</td>
			<td>Bio-Rad</td>
			<td>165-2257</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Pulser</td>
			<td>Bio-Rad</td>
			<td>1652660</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvette</td>
			<td>Bio-Rad</td>
			<td>1652083</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Eppendorf</td>
			<td>5427000097</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>LSM 7 DUO (780&#38;7Live)</td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometers</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>EPS-300 Power Supply</td>
			<td>Tanon</td>
			<td>EPS 300</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Mshot</td>
			<td>MF30</td>
			<td></td>
		</tr>
		<tr>
			<td>Agrose</td>
			<td>BBI</td>
			<td>A600234</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>BBI</td>
			<td>A100339</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Diamine Tetraacetie Acid</td>
			<td>BBI</td>
			<td>B300599</td>
			<td></td>
		</tr>
	</tbody>
</table>

co-expression of multiple chimeric fluorescent fusion proteins in an efficient way in plants

关于蛋白质的时空亚细胞定位的信息对于了解细胞的生理功能至关重要。荧光蛋白和荧光融合蛋白的生成已被广泛地应用于直接可视化细胞内蛋白质定位和动力学的有效工具。这是特别有用的比较, 他们与知名的细胞器标记后, 共同表达与蛋白质的兴趣。然而, 植物中蛋白质共表达的经典方法通常涉及多个独立的表达质粒, 因此有缺点, 包括低的协同表达效率, 表达水平的变化, 和高的时间基因交叉和筛查的支出。在本研究中, 我们描述了一种健壮和新颖的方法, 共同表达的多嵌合体荧光蛋白在植物。它克服了传统方法的局限性, 采用了由多个半独立表达式盒组成的单表达载体。每个蛋白表达盒都含有其自身的功能蛋白表达元素, 因此可以灵活调整以满足不同的表达需求。同时, 通过优化一步反应, 无需额外的消化和结扎步骤, 在表达质粒中进行 DNA 片段的组装和操作是很容易和方便的。此外, 它完全兼容目前的荧光蛋白衍生生物成像技术和应用, 如烦恼和 BiFC。作为对该方法的验证, 我们采用了这种新的系统来共同表达荧光融合泡排序受体和分泌载体膜蛋白。结果表明, 它们的透视亚细胞定位与以往的研究一样, 都是由植物的瞬时表达和遗传转化引起的。

建立了一种健壮高效的多嵌合体荧光融合蛋白在植物中的联合表达方法。它突破了传统方法的障碍使用多种分离质粒进行蛋白质共表达, 如图 1AB所示,通过瞬时表达或稳定的遗传转化。在这一新的方法中, 我们生成一个单一的表达载体, 由多个蛋白表达盒组成, 以实现蛋白质共表达一次 (图 1C, D)。?...

Watch this Scientific Journal Video about Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants at JoVE.com

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

我们开发了一种新的方法来共同表达多嵌合体荧光融合蛋白在植物中克服传统方法的困难。它利用单一表达质粒, 包含多功能独立蛋白表达盒, 以实现蛋白质的共同表达。

一种高效的植物多嵌合体荧光融合蛋白的协同表达

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Research

JoVE Journal

Chemistry

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

聚苹果酸为基础的生物聚合物纳米靶向的多种肿瘤标志物：用于个性化医疗的机会？

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

科研

化学

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

通过RAFT控制聚合轻便和高效制备三组分荧光Glycopolymers

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

在水中水合蛋白原位表征SALVI和TOF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

点击化学可视化植物木动力学: 双标记是幸福!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

基质辅助激光解吸/电离质谱中增强碳水化合物离子信号的高效样品制备方法

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of FÃƒÆ’Ã‚Â¢Ãƒâ€¹Ã¢â‚¬Â '

由 F− 310 Nm 支队所示的阴离子光电子成像

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

纳米金刚石的生物激发多巴胺表面改性及其对银纳米粒子的还原

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...