Expression of biotin-binding proteins, avidin and streptavidin, in plant tissues using plant vacuolar targeting sequences

• Published in: Transgenic research 2002-04, Vol.11 (2), p.199-214
• Main Authors: MURRAY, Colleen, SUTHERLAND, Paul W, PHUNG, Margaret M, LESTER, Melissa T, MARSHALL, Richelle K, CHRISTELLER, John T
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Avidin - genetics; Avidin - metabolism; Biological and medical sciences; Biotechnology; Biotin - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic engineering; Genetic technics; Genetics and breeding of economic plants; Methods. Procedures. Technologies; Nicotiana - genetics; Nicotiana - metabolism; Pest animals; Pest resistance; Phenotype; Plant Leaves - metabolism; Plant Roots - metabolism; Plants, Genetically Modified; Protein Processing, Post-Translational; Protein Sorting Signals - genetics; Protein Transport; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Streptavidin - genetics; Streptavidin - metabolism; Transgenic animals and transgenic plants; Transgenic plants; Vacuoles - metabolism; Varietal selection. Specialized plant breeding, plant breeding aims
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1023/a:1015237610263

Tobacco plants have been developed which constitutively express high levels of the biotin-binding proteins, avidin and streptavidin. These plants were phenotypically normal and produced fertile pollen and seeds. The transgene was expressed and its product located in the vacuoles of most cell types in the plants. Targeting was achieved by use of N-terminal vacuolar targeting sequences derived from potato proteinase inhibitors which are known to target constitutively to vacuoles in potato tubers and, under wound-induction, in tomato leaves. Avidin was located in protein body-like structures within the vacuole and transgene protein levels remained relatively constant throughout the lifetime of the leaf. We describe two chimeric constructs with similar levels of expression. One comprised a potato proteinase inhibitor I signal peptide cDNA sequence attached to an avidin cDNA and the second a potato proteinase inhibitor II signal peptide genomic sequence (including an intron) attached to a core streptavidin synthetic sequence. We were unable to regenerate plants when transformation used constructs lacking the targeting sequences. The highest levels observed (up to 1.5% of total leaf protein) confirm the vacuole as the organelle of choice for stable storage of plant-toxic transgene products. The efficient targeting of these proteins did not result in any measured changes in plant biotin metabolism.