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. 2003 Oct;133(2):462-9.
doi: 10.1104/pp.103.027979.

A gateway cloning vector set for high-throughput functional analysis of genes in planta

Mark D Curtis  1 , Ueli Grossniklaus
Affiliations

A gateway cloning vector set for high-throughput functional analysis of genes in planta

Mark D Curtis et al. Plant Physiol. 2003 Oct.

Abstract

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.

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Figures

Figure 1.
Figure 1.
A schematic illustrating the structure of the Gateway-compatible cloning vectors, showing the recombination sites flanked by AscI and PacI eight-nucleotide recognition sites. A, pMDC32, constitutive expression vector, harboring a dual 35S promoter, and pMDC30, a heat shock-inducible vector; B, pMDC7, derived from PER8, an estrogen-inducible vector. C, pMDC45, pMDC44, and pMDC43, for the construction of C-terminal GFP6 fusions. D, pMDC83, pMDC84, and pMDC85, for the construction of N-terminal GFP6his-tagged fusions. E, pMDC139, pMDC140, and pMDC141, for the construction of N-terminal GUS fusions. F, pMDC107, pMDC111, and pMDC110, for the construction of promoter-reporter (native promoter-gene fusion) GFP6 vectors. G, pMDC162, pMDC163, and pMDC164, for the construction of promoter-reporter (native promoter-gene fusion) GUS vectors. H, pMDC99, pMDC100, and pMDC123, for complementation of mutants with genomic fragments.
Figure 2.
Figure 2.
Nucleotide sequence adjacent to each Gateway cassette showing the reading frame for fusions with GFP (a and b) and GUS (c). These sequences also show the stop codons in the vectors where the attR2 site is followed by the PacI site but not in vectors where the attR2 site is followed by the AscI site.
Figure 3.
Figure 3.
17-β-Estradiol induced WUSCHEL expression (a-c) in 31-d-old seedlings of three independent transformants showing germinating somatic embryos growing at the primary and lateral root tips. Noninduced seedlings show no somatic embryo development (data not shown). The same WUSCHEL gene from the same entry clone was used to make C- and N-terminal fusions with GFP and N-terminal fusions with GUS. d, f, and h show light microscope images, and e and g show fluorescent microscope images. e, Fluorescent microscope image of pMDC114 expression in bombarded onion epidermal cells (WUSCHEL cDNA, fused to C-terminus of GFP, in pMDC43). g, Fluorescent microscope image of pMDC116 expression in bombarded onion epidermal cells (WUSCHEL cDNA, fused to N-terminus of GFP, in pMDC84). h, Light microscope image of pMDC153 expression in bombarded epidermal onion cells (WUSCHEL cDNA, fused to N-terminus of GUS, in pMDC141). All show that the marker protein was localized to the nucleus.
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