Golden Gate Cloning-Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants

Area of Science:

• Plant genetic engineering
• Synthetic biology
• Molecular biology

Background:

• Multigene expression in plants is crucial for genetic engineering and synthetic biology.
• Self-cleaving 2A peptides facilitate polycistronic gene expression, simplifying transgene regulation.
• Existing vector assembly systems lack optimization for 2A peptide-mediated polycistronic expression.

Purpose of the Study:

• To develop efficient Golden Gate cloning-compatible modular systems for constructing plant polycistronic expression vectors.
• To assess the impact of transgene position on protein expression levels in 2A peptide-mediated cassettes.
• To compare the efficacy of different acceptor backbones for enhancing gene expression.

Main Methods:

• Designed modular genetic components including 2A peptides (T2A, P2A) and acceptor backbones (pGO-DV1, pGO-DV2).
• Utilized Golden Gate cloning to assemble six tricistronic vectors with combinatorially placed fluorescent protein genes (mCherry, eYFP, eGFP).
• Performed transient expression assays in tobacco leaves to evaluate protein expression.

Main Results:

• Protein expression levels were comparable among fluorescent proteins regardless of their position in the tricistronic cassette.
• The pGO-DV2 acceptor backbone, designed as a DNA replicon, enhanced protein expression levels by up to 71% compared to pGO-DV1.
• The developed system allows rapid and flexible construction of polycistronic expression vectors.

Conclusions:

• The novel Golden Gate cloning system effectively supports 2A peptide-mediated polycistronic expression in plants.
• Transgene position does not significantly affect expression levels in these tricistronic cassettes.
• The pGO-DV2 backbone offers a significant advantage for increasing protein yield in plant expression systems.