A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies
A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies
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DOI：
10.1111/j.1365-313x.2010.04322.x
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发表时间：
2010-10-01
期刊：
影响因子：
7.2
通讯作者：
Blatt, Michael R.
中科院分区：
文献类型：
--
作者：
Grefen, Christopher;Donald, Naomi;Blatt, Michael R.
蛋白质的荧光标记和共聚焦成像技术已成为分析亚细胞水平蛋白质分布和动态特性的首选方法。通常使用的是许多瞬时表达策略，这些策略大大减少了成像前的准备时间，但它们的应用仅限于少数可处理的物种和组织。我们之前开发了一种在拟南芥根表皮和根毛中瞬时表达荧光标记蛋白的简单方法。我们在这里描述了一组具有荧光标签的网关兼容载体，其中包含拟南芥的泛素-10基因启动子（P-UBQ10），该载体在短暂转化后可以在烟草和拟南芥组织中延长荧光标记蛋白的表达，并且对于产生稳定转化的品系同样有用。作为原理证明，我们利用荧光标记对整体质膜蛋白SYP121（一种囊泡运输蛋白SNARE家族成员）和细胞质蛋白DHAR1（一种促进活性氧清除的蛋白）进行了转化。我们还对SYP121及其相互作用伙伴KC1 K+通道进行了转化，以证明该方法在双分子荧光互补（BiFC）中的实用性。利用农杆菌共培养方法对拟南芥进行瞬时转化，获得了包括根毛和保护细胞在内的所有表皮细胞的表达。比较研究表明，P-UBQ10启动子的表达水平与天然SYP121启动子的表达水平相似，忠实地再现了亚细胞水平上蛋白质分布的特征。与35s驱动构建体不同，P-UBQ10启动子下的表达在短暂转化后超过2周的时间内保持升高。这个载体和荧光标签的工具箱为研究膜动力学和细胞发育，以及与保护细胞中的环境刺激和根中的营养获取相关的事件提供了显著的优势。
P>Fluorescent tagging of proteins and confocal imaging techniques have become methods of choice in analysing the distributions and dynamic characteristics of proteins at the subcellular level. In common use are a number of strategies for transient expression that greatly reduce the preparation time in advance of imaging, but their applications are limited in success outside a few tractable species and tissues. We previously developed a simple method to transiently express fluorescently-tagged proteins in Arabidopsis root epidermis and root hairs. We describe here a set of Gateway-compatable vectors with fluorescent tags incorporating the ubiqutin-10 gene promoter (P-UBQ10) of Arabidopsis that gives prolonged expression of the fluorescently-tagged proteins, both in tobacco and Arabidopsis tissues, after transient transformation, and is equally useful in generating stably transformed lines. As a proof of principle, we carried out transformations with fluorescent markers for the integral plasma membrane protein SYP121, a member of the SNARE family of vesicle-trafficking proteins, and for DHAR1, a cytosolic protein that facilitates the scavenging of reactive oxygen species. We also carried out transformations with SYP121 and its interacting partner, the KC1 K+ channel, to demonstrate the utility of the methods in bimolecular fluorescence complementation (BiFC). Transient transformations of Arabidopsis using Agrobacterium co-cultivation methods yielded expression in all epidermal cells, including root hairs and guard cells. Comparative studies showed that the P-UBQ10 promoter gives similar levels of expression to that driven by the native SYP121 promoter, faithfully reproducing the characteristics of protein distributions at the subcellular level. Unlike the 35S-driven construct, expression under the P-UBQ10 promoter remained elevated for periods in excess of 2 weeks after transient transformation. This toolbox of vectors and fluorescent tags promises significant advantages for the study of membrane dynamics and cellular development, as well as events associated with environmental stimuli in guard cells and nutrient acquisition in roots.