Split Intein Selectable Marker for Plant Co-Transformation
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Solution Overview
Problem
Current plant co-transformation methods rely on multiple selectable gene markers, requiring careful adjustment of antibiotic concentrations and suffering from differences in selection efficacy, which complicates the stable transformation of large DNA fragments and optimal gene expression in plants.
Innovation Solution
A split selectable marker system using split inteins is introduced, allowing for single-selectable-marker-gene dependent co-transformation in plants. This system comprises two vectors, each containing a promoter, a nucleotide sequence encoding a fragment of a selectable marker protein, and a terminator, with the intein fragments joining to form a peptide bond upon expression in a plant cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple selectable gene markers are used for plant co-transformation, then transformation efficiency is improved, but device complexity and operational difficulty increase due to requiring careful adjustment of multiple antibiotic concentrations
Solution Approach 1:
The patent merges multiple selectable marker functions into a single split selectable marker system. Two separate vectors each carry a fragment of the same selectable marker gene (e.g., nptII for kanamycin resistance), and only when both vectors co-transform into the same plant cell do the fragments complement to form a functional marker that confers antibiotic resistance. This eliminates the need to manage multiple different antibiotics and their concentration adjustments.
Solution Approach 2:
The selectable marker gene is segmented into two separate functional fragments (N-terminal and C-terminal portions) that are distributed across two different transformation vectors. Each fragment alone is non-functional, but when both vectors are present in the same cell, the fragments complement each other to restore full marker function. This segmentation enables single-marker-based selection of co-transformation events.
2Quantity of substance
If large DNA fragments are used for multigene engineering, then complete synthetic pathways can be introduced, but T-DNA stability decreases leading to truncation at left and/or right ends
Solution Approach 1:
Large DNA fragments containing complete synthetic pathways are segmented into smaller manageable units distributed across multiple separate binary vectors. Each vector contains a portion of the total genetic material along with a fragment of the split selectable marker. This segmentation makes the DNA more stable during Agrobacterium-mediated transformation while still enabling delivery of the complete pathway through co-transformation.
Solution Approach 2:
The split selectable marker system serves multiple functions simultaneously: it enables selection of co-transformed cells, facilitates delivery of large DNA pathways through smaller vector units, and provides a standardized method for multi-gene stacking. The same split marker system can be applied to different synthetic pathways and gene combinations.
3Productivity
If conventional co-transformation methods are used, then multiple genes can be transformed, but selection efficacy varies between different selectable markers requiring careful optimization
Solution Approach 1:
The split selectable marker system provides a universal selection approach that works across different plant species and transformation contexts. By using the same split marker system (e.g., split nptII for kanamycin resistance or split hpt for hygromycin resistance) regardless of the specific genes being co-transformed, researchers eliminate the need to optimize different marker combinations and can directly compare transformation efficiencies.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The split selectable marker system enables efficient co-transformation of multiple genes into plant cells, reducing the complexity of constructing long T-DNA molecules and improving the capability for pathway engineering and genetic improvement of polygenic traits.
Implementation Method 1
the N-terminal fragment and the C-terminal fragment of the intein join the N-terminal fragment and the C-terminal fragment of the selectable marker protein to form a peptide bond
Data Source
AI summary
The current disclosure relates to a split-intein-based gene-stacking system through split-selectable-marker-enabled co-transformation in Arabidopsis thaliana and poplar. The disclosure is also directed to methods of co-transforming plant cells, comprising delivering DNA vectors into a plant cell.