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1 Aarhus University 2 Department of Molecular Biology and Genetics - Afgrødegenetik og Bioteknologi, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University 3 Department of Molecular Biology and Genetics - Afgrødegenetik og Bioteknologi, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University
Background: When creating plant transformation vectors, full control of nucleotides flanking the insert in the final construct may be desirable. Modern ligase-independent methods for DNA-recombination are based on linearization by classical type II restriction endonucleases (REs) alone or in combination with nicking enzymes leaving residual nucleotides behind in the final construct. We here explore the use of type IIS and type IIB REs for vector linearization that combined with sequence and ligase-independent cloning (SLIC) overcomes this problem and promotes seamless gene-insertion in vectors. Providing the basis for a collection of biolistic plant transformation vectors ready to be cloned with different genes-of-interest, we present two vectors, where promoter and terminator are joined by a spacer. During spacer-removal linearization (SRL), type IIS and type IIB REs remove their own recognition sequences from the vector leaving no undesired, short sequences behind.Results: We designed two plant transformation vectors prepared for SRL in combination with SLIC, pAUrumII and pAUrumIII, harboring a spacer with recognition sites for a type IIS and IIB RE, respectively. The gene for a green fluorescent protein, gfp, was successfully cloned into both vectors; traces of pAUrumIII, however, contaminated the transformation due to incomplete linearization, an issue not encountered with the type IIS linearized pAUrumII. Both constructs, pAUrumII-gfp and pAUrumIII-gfp, were functional, when tested in vitro on wheat and barley endosperm cells for transient gfp expression.Conclusions: All nucleotides flanking an insert in a biolistic plant transformation vector can be customized by means of SRL in combination with SLIC. Especially type IIS REs promote an efficient cloning result. Based on our findings, we believe that the SRL system can be useful in a series of plant transformation vectors, favoring the presence of functional sequences for optimal expression over redundant cloning-site remnants. © 2014 Kronbak et al.; licensee BioMed Central Ltd.
Plant Methods, 2014, Vol 10, Issue 1, p. 1-10
Biolistics; Cereals; In-Fusion™; linearization; Plant transformation vector; Seamless cloning; Transient expression; Type IIB; Type IIS restriction endonucleases
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