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

Title
Golden Gate Cloning-Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants
Authors
정제형이제훈원효준오은석오만호
Keywords
Golden Gate cloning; Replicon; 2A peptides; polycistronic expression; plant metabolic engineering
Issue Date
2020-10
Publisher
Frontiers in plant science
Citation
VOL 11, 559365
Abstract
The expression of multiple proteins and high-throughput vector assembly system are highly relevant in the field of plant genetic engineering and synthetic biology. Deployment of the self-cleaving 2A peptide that mediates polycistronic gene expression has been an effective strategy for multigene expression, as it minimizes issues in coordinated transgene regulation and trait staking in plants. However, efficient vector assembly systems optimized for 2A peptide-mediated polycistronic expression are currently unavailable. Furthermore, it is unclear whether protein expression levels are influenced by the transgene position in the polycistronic expression cassette. In this article, we present Golden Gate cloning-compatible modular systems allowing rapid and flexible construction of polycistronic expression vectors applicable for plants. The genetic modules comprised 2A peptides (T2A and P2A)-linked tricistron expression cassette and its acceptor backbones, named pGO-DV1 and pGO-DV2. While both acceptor backbones were binary T-DNA vectors, pGO-DV2 was specially designed to function as a DNA replicon enhancing gene expression levels. Using the Golden Gate cloning, a set of six tricistronic vectors was constructed, whereby three transgenes encoding fluorescent proteins (mCherry, eYFP, and eGFP) were combinatorially placed along the expression cassette in each of the binary vectors. Transient expression of the construct in tobacco leaves revealed that the expression levels of three fluorescent proteins were comparable each other regardless of the gene positions in the tricistronic expression cassette. pGO-DV2-based constructs were able to increase protein expression level by up to 71%, as compared to pGO-DV1-based constructs.
URI
http://pubs.kist.re.kr/handle/201004/71953
ISSN
1664-462X
Appears in Collections:
KIST Publication > Article
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