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

• Published in: Transgenic research Vol. 22; no. 6; pp. 1273 - 1278
• Main Authors: Gottschamel, Johanna, Waheed, Mohammad Tahir, Clarke, Jihong L, Lössl, Andreas G
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer-Verlag 01.12.2013; Springer Netherlands; Springer
• Subjects: Animal Genetics and Genomics; Binding Sites; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biotechnology; Capsid Proteins - genetics; chloroplasts; Chloroplasts - genetics; confocal laser scanning microscopy; Fundamental and applied biological sciences. Psychology; Genetic Engineering; Genetic technics; Genetic Vectors; Genome, Plant; green fluorescent protein; Green Fluorescent Proteins - genetics; Life Sciences; Methods. Procedures. Technologies; Molecular Medicine; Nicotiana - genetics; Plant Genetics and Genomics; Plants, Genetically Modified - genetics; plastid DNA; polymerase chain reaction; Recombination, Genetic; Ribosomes - genetics; Southern blotting; tobacco; Transformation, Genetic; transgenes; Transgenic animals and transgenic plants; Transgenics; Western blotting; GFP; recombinant cloning technology; Plastid transformation; Gateway; Recombination; Technology; Green fluorescent protein; Plastid; Vector; Chloroplast
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1007/s11248-013-9726-3

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.