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Química

一种高效的植物多嵌合体荧光融合蛋白的协同表达

Published: July 01, 2018
doi:
10.3791/57354
, ,
1College of Life Sciences,South China Agricultural University

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1. 底漆设计策略和 DNA 放大 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。 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…

Representative Results

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)。?…

Discussion

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?…

Declarações

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

Co-expressionChimeric Fluorescent Fusion ProteinsPlant Molecular BiologyCell BiologyProtein ExpressionProtein LocalizationPCRIsothermal AssemblyVector ConstructionTransient ExpressionGenetic Transformation
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Peng, X., Zhong, G., Wang, H. Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants. J. Vis. Exp. (137), e57354, doi:10.3791/57354 (2018).
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