Abstract
BackgroundWhen 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.ResultsWe 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.ConclusionsAll 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.
Highlights
When creating plant transformation vectors, full control of nucleotides flanking the insert in the final construct may be desirable
Creating plant transformation vectors As the first two of a collection of plant transformation vectors prepared for spacer-removal linearization (SRL) used with In-FusionTM cloning, pAUrumII and pAUrumIII were designed (Figure 1)
We investigated here the possibility of starting a collection of biolistic plant transformation vectors based on sequence and ligase-independent cloning (SLIC) ( In-FusionTM) ready to be cloned with different genes-of-interest, while having the full control of any single nucleotide present in the final construct
Summary
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. In recent years methods have been developed, which overcome most of the obstacles mentioned [1] These methods are in general based on longer matching overhangs than most classical type II REs offer, and include ligase-independent cloning (LIC) [2], sequence and ligase-independent cloning (SLIC) [3] (equivalent to the commercially available In-FusionTM cloning system, Clontech Laboratories, [4,5]), uracil specific excision reagent (USERTM, New England Biolabs) cloning [6,7], circular polymerase extension cloning (CPEC) [8] and one-step isothermal in vitro recombination (Gibson AssemblyTM, Synthetic Genomics) [9]. The assembled hybrid DNA only contains nicks or short gaps around the assembled sequences, which will be repaired and ligated by E. coli after transformation [4,5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
