A novel chloroplast transformation vector compatible with the Gateway® recombination cloning technology

Abstract

To analyze the suitability of Gateway(®) vectors for transformation of chloroplasts, we converted a standard plastid transformation vector into a Gateway(®) destination vector containing the necessary recombination sites attR1 and attR2. Insertion of the green fluorescent protein (GFP) coding sequence with associated T7g10 ribosome binding site into this destination vector created the expression vector for transformation of tobacco chloroplasts with the biolistic method. Correct integration of the transgene into the plastid genome was verified by PCR and the homoplasmic nature of the transformed plants was confirmed by Southern Blot analysis. Expression of the GFP reporter protein was monitored by confocal laser scanning microscopy (CLSM) and quantification by western blot analysis showed a GFP accumulation level of 3% total soluble protein (TSP). The presented results clearly demonstrate that the Gateway(®) recombination sites are compatible with all steps of plastid transformation, from generation of transplastomic plants to expression of GFP. This is the first report of a plastid transformation vector made by the Gateway(®) recombinant cloning technology, which proves the suitability of this system for use in chloroplasts.

The Engineered Chloroplast Genome Just Got Smarter

“…One method used the Gateway system for construction of chloroplast vectors to simplify vector construction and improve vector design [28]. Another group used modular design of genetic elements to construct chloroplast vectors to build transcriptional units as well as target any homologous recombination site of choice [29].…”

Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development

“…For the intermediary vector pMA‐lettuce, the sequences corresponding to the trnI and trnA region of the Lactuca sativum plastid genome (Ruhlman et al ., ) flanked by Kpn I and Sac II restriction sites were custom synthesized and introduced into the company's standard backbone vector (GeneArt, Germany). In order to create pDEST‐PN‐L, the complete Gateway ® cloning cassette together with the aadA expression cassette was excised from pDEST‐PN‐T (Gottschamel et al ., ) and inserted into pMA‐lettuce using the restriction enzymes Kpn I and Sac II. The sequence of the synthetic fusion gene ( ediii ‐1‐4) consists of all four DENV‐EDIII sequences (order: ediii ‐1, ediii ‐3, ediii ‐4, ediii ‐2) linked by penta‐glycine linkers (Etemad et al ., ; Gottschamel et al ., ).…”

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems

“…In these vectors, the transgene is controlled by the strong tobacco plastid rRNA operon promoter (Prrn; Svab and Maliga 1993) fused with the 5 0 UTR of gene 10 from bacteriophage T7 and the 3 0 UTR of the plastid rbcL gene. The Gateway Ò -compatible plastid transformation vector pDEST-T7 was obtained by excision of the Prrn promoter sequence (Ye et al 2001) from plasmid pDEST-PN-T (Gottschamel et al 2013). The exclusive presence of the phage T7-derived promoter (Tabor and Richardson 1985) in the new vector was confirmed by sequencing.…”

“…Several vectors for transient or stable plant transformation (Buntru et al 2013;Dubin et al 2008Dubin et al , 2010Earley et al 2006;Karimi et al 2007Karimi et al , 2013Lyska et al 2013) have been reported including a plastid transformation vector for constitutive transgene expression (Gottschamel et al 2013).…”