A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta

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 ␤-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.

Global translational reprogramming is a fundamental layer of immune regulation in plants

“…The 35S:ccdB cassette-LUC-NOS construct was generated by fusing PCR fragments of the 35S promoter from pMDC140 23 , the ccdB cassette and the NOS terminator from pRNAi-LIC 24 and LUC from pGWB235 22 . The 35S:ccdB cassette-LUC-NOS was then inserted into pCAMBIA1300 via Pst I and EcoR I and designated as pGX301 for cloning 5′ leader sequences through replacement of the Apa I-flanked ccdB cassette 24 .…”

“…The 35S:ccdB cassette-LUC-NOS was then inserted into pCAMBIA1300 via Pst I and EcoR I and designated as pGX301 for cloning 5′ leader sequences through replacement of the Apa I-flanked ccdB cassette 24 . Similarly, the 35S:RLUC-HA-rbs terminator construct was made through fusion of PCR fragments of 35S from pMDC140 23 , RLUC from pmirGLO (Promega, E1330) and rbs terminator from pCRG3301 25 . The 35S:RLUC-HA-rbs fragment flanked with EcoR I was inserted into pTZ-57rt (Thermo fisher, K1213) via TA cloning to generate pGX125.…”

Large-scale analysis of the GRAS gene family in Arabidopsis thaliana

“…Each PCR product was subcloned into either pENTR Directional TOPO or pDONR221 vector using the Gateway recombination cloning technology according to the manufacturer's instructions (INVITROGEN, http://www.invitrogen.com), except for the promoter region of SCL23. Subsequently, each promoter fragment was transferred to the pMDC162 Gateway-compatible binary vector (Curtis and Grossniklaus, 2003) by recombination reactions, according to the manufacturer's instruction (INVITROGEN, http://www. invitrogen.com).…”

“…Each fragment was cloned into the pENTR Directional TOPO vector as described above. Subsequently, each error-free full-length ORF in pENTR was transferred to the pMDC43 Gateway-compatible binary vector by recombination reactions to generate N-terminal GFP translational fusions as previously described (Curtis and Grossniklaus 2003). Arabidopsis thaliana Col-0 plants were transformed using standard procedures for floral dipping (Clough and Bent 1998).…”

A stable tRNA-like molecule is generated from the long noncoding RNA GUT15 in Arabidopsis

“…The mutagenesis of the GUT15 final intron splice sites was performed using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies). To detect the expression in plants, the At2g18440 and At3g12587 gene versions were cloned into the pMDC32 Gateway binary vector [41] using the Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific).…”