The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species.
The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species.
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DOI：
10.1186/1746-4811-5-6
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发表时间：
2009-05-20
期刊：
影响因子：
5.1
通讯作者：
Nebenführ A
中科院分区：
文献类型：
--
作者：
Li JF;Park E;von Arnim AG;Nebenführ A
植物基因组测序已经导致了大量未表征基因的鉴定。为了研究这些未知的基因功能，已经开发了几种瞬时转化系统作为冗长的转基因测定的快速和方便的替代方案。这些瞬时转化方法包括基因枪轰击法、原生质体转染法和农杆菌介导的瞬时转化法，根据不同的研究目的，各有优缺点。我们提出了一种新的瞬时测定的基础上共培养的年轻的拟南芥（拟南芥）幼苗与根癌农杆菌在表面活性剂的存在下，不需要任何专用设备，可以在一个星期内进行播种种子的蛋白质分析。这种快速农业介导的幼苗转化（FAST）已成功用于在不同遗传背景的拟南芥幼苗子叶（但不是根）中表达由不同启动子驱动的多种构建体。定位的三个以前未知的蛋白质进行了鉴定，通过与荧光细胞器标记的共转化。FAST程序需要最少的幼苗处理，也适用于96孔板。FAST方法的高转化效率使得能够通过免疫印迹从8个转化的幼苗中检测蛋白质。蛋白质-蛋白质相互作用，在这种情况下HY 5同源二聚化，很容易检测到Förster共振能量转移和双分子荧光互补技术在FAST处理的幼苗。初步试验表明，FAST程序也可应用于其他双子叶植物和单子叶植物物种，包括烟草、番茄、水稻和柳枝稷。FAST系统提供了一种快速、有效和经济的完整植物基因功能测定方法，只需最少的人工操作，无需专用设备。这种方法是未来自动化高通量分析的理想选择。
Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis.
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影响因子：
7.2
作者：
Campanoni, Prisca;Sutter, Jens Uwe;Blatt, Michael Robert
通讯作者：
Blatt, Michael Robert
影响因子：
6.2
作者：
Grant, JE;Cooper, PA;Dale, TM
通讯作者：
Dale, TM
影响因子：
14.9
作者：
Heazlewood, Joshua L.;Verboom, Robert E.;Millar, A. Harvey
通讯作者：
Millar, A. Harvey
DOI：
10.1007/bf00331014
发表时间：
1986-01-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
KONCZ, C;SCHELL, J
通讯作者：
SCHELL, J
影响因子：
6.9
作者：
Komatsu, Setsuko;Konishi, Hirosato;Hashimoto, Makoto
通讯作者：
Hashimoto, Makoto