A gateway cloning vector set for high-throughput functional analysis of genes in planta
A gateway cloning vector set for high-throughput functional analysis of genes in planta
复制标题
DOI：
10.1104/pp.103.027979
复制
发表时间：
2003-10-01
期刊：
影响因子：
7.4
通讯作者：
Grossniklaus, U
中科院分区：
文献类型：
--
作者：
Curtis, MD;Grossniklaus, U
目前的挑战是，既然两个植物基因组已经被测序，那么为越来越多的预测基因分配一个功能。在拟南芥中，大约55%的基因可以被赋予一个假定的功能，然而，只有不到8%的基因被直接的实验证据赋予了一个功能。为了鉴定这些功能，必须对许多基因进行全面的分析，这将包括用于组成型或诱导型异位表达的嵌合转基因的产生、用于反义或显性负表达的嵌合转基因的产生、用于亚细胞定位研究的嵌合转基因的产生、用于启动子分析的嵌合转基因的产生以及用于基因互补研究的嵌合转基因的产生。这种转基因的生产通常受到依赖于限制性消化和连接的费力的常规克隆技术的阻碍。为了提供高通量基因分析的工具，我们已经产生了一个网关兼容的农杆菌双元载体系统，有助于快速和可靠的DNA克隆。该载体集合可免费获得，用于非商业目的，并且可用于组成型或诱导型基因的异位表达。所述载体可用于表达与维多利亚水母绿色荧光蛋白和β-葡糖醛酸糖苷酶蛋白的蛋白质融合体，从而可鉴定蛋白质的亚细胞定位。它们还可用于产生启动子-报告基因构建体并促进基因组DNA片段的有效克隆以进行互补实验。所有载体均衍生自pCambia T-DNA克隆载体，除了化学诱导型载体之外，用于农杆菌属（Agrobacterium sp.）介导的广泛植物物种的转化。
The current challenge, now that two plant genomes have been sequenced, is to assign a function to the increasing number of predicted genes. In Arabidopsis, approximately 55% of genes can be assigned a putative function, however, less than 8% of these have been assigned a function by direct experimental evidence. To identify these functions, many genes will have to undergo comprehensive analyses, which will include the production of chimeric transgenes for constitutive or inducible ectopic expression, for antisense or dominant negative expression, for subcellular localization studies, for promoter analysis, and for gene complementation studies. The production of such transgenes is often hampered by laborious conventional cloning technology that relies on restriction digestion and ligation. With the aim of providing tools for high throughput gene analysis, we have produced a Gateway-compatible Agrobacterium sp. binary vector system that facilitates fast and reliable DNA cloning. This collection of vectors is freely available, for noncommercial purposes, and can be used for the ectopic expression of genes either constitutively or inducibly. The vectors can be used for the expression of protein fusions to the Aequorea victoria green fluorescent protein and to the beta-glucuronidase protein so that the subcellular localization of a protein can be identified. They can also be used to generate promoter-reporter constructs and to facilitate efficient cloning of genomic DNA fragments for complementation experiments. All vectors were derived from pCambia T-DNA cloning vectors, with the exception of a chemically inducible vector, for Agrobacterium sp.-mediated transformation of a wide range of plant species.