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Updated: Jun 10, 2026
08:31
Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
Published on: February 5, 2021
Engineering the K+ uptake regulatory pathway by MultiRound Gateway.
Fei Ren1, Qi-Jun Chen, Min Xie
1State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Journal of Plant Physiology
|August 17, 2010
Summary
Improved MultiRound Gateway vectors efficiently assemble multiple plant genes for enhanced potassium nutrition. Transgenic Arabidopsis lines showed tolerance to low potassium and calcium conditions, demonstrating vector effectiveness.
Area of Science:
- Plant Molecular Biology
- Plant Physiology
Background:
- Previous work introduced improved MultiRound Gateway vectors.
- Calcineurin B-like (CBL) pathways are crucial for potassium (K+) nutrition in plants.
Purpose of the Study:
- To demonstrate the effectiveness of improved MultiRound Gateway vectors.
- To engineer a CBL pathway for K+ nutrition using these vectors.
Main Methods:
- Utilized two improved entry vectors (pL12R34H-Ap, pL34R12-Cm) for Gateway recombination.
- Generated five pMDC99-derived binary vectors with multiple genes (CIPK23, CBL1, CBL9, AKT1).
- Transformed vectors pK31, pK39, and pK4 into Arabidopsis and analyzed transgene presence and expression.
Main Results:
- PCR and qRT-PCR confirmed stable co-existence and high expression of multiple transgenes.
- Transgenic Arabidopsis lines (K31, K39, K4) exhibited significant tolerance to low K+ and low K+/Ca2+ conditions.
- No significant phenotypic differences were observed among the K31, K39, and K4 lines.
Conclusions:
- Improved MultiRound Gateway vectors efficiently assemble multiple transgenes.
- These vectors facilitate stable inheritance and expression of multiple genes, even under the same promoter and terminator.
- The engineered CBL pathway demonstrates the utility of these vectors for plant nutrition studies.
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