Abstract
BackgroundPlant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments.ResultsWe designed novel hybrid signal sequences for multiple-compartment localization and characterize their function when fused to GFP in Nicotiana benthamiana leaf tissue. TriTag-1 and TriTag-2 use alternative splicing to generate differentially localized GFP isoforms, localizing it to the chloroplasts, peroxisomes and cytosol. TriTag-1 shows a bias for targeting the chloroplast envelope while TriTag-2 preferentially targets the peroxisomes. TriTag-3 embeds a conserved peroxisomal targeting signal within a chloroplast transit peptide, directing GFP to the chloroplasts and peroxisomes.ConclusionsOur novel signal sequences can reduce the number of cloning steps and the amount of genetic material required to target a heterologous protein to multiple locations in plant cells. This work harnesses alternative splicing and signal embedding for engineering plants to express multi-functional proteins from single genetic constructs.
Highlights
Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments
Various mRNAs produced from Arabidopsis protein-L-isoaspartate methyltransferase gene (PIMT2) are produced by alternative transcription initiation sites and alternative splicing events [16]
The spliceforms produced from the 3′ transcription initiation site target the protein to the chloroplast when the targeting sequence is retained, and to the cytosol when it is not
Summary
Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments. Re-engineering photorespiration [1,2] and isoprenoid synthesis [3,4] will involve both the chloroplasts and peroxisomes. A number of synthetic N-terminal and C-terminal extensions are readily available to target heterologous proteins to desired subcellular compartments, such as the chloroplast, peroxisome, mitochondrion, endoplasmic reticulum or the nucleus. We describe a simple technique for targeting of transgenic proteins to multiple organelles, the chloroplast, peroxisome, and cytosol. This combination of organelles is interesting due to their close functional association in photorespiration, isoprenoid biosynthesis, β-oxidation and other metabolic processes [3,13,14]
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