Maize Regulatory Elements for Transgene Co-expression Stability
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Solution Overview
Problem
The repeated use of similar promoters in transgenic plants for co-expression of multiple transgenes can lead to homology-based gene silencing and instability, particularly in Agrobacterium systems, due to recombination issues with repetitive DNA sequences.
Innovation Solution
The use of purified maize gene regulatory elements, such as the chlorophyll a/b promoter and 3′ untranslated regions, which are operably linked to create expression vectors that can drive the expression of transgenes independently, reducing the likelihood of gene silencing and enhancing the stability of transgene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the same promoter is repeated multiple times in a transgene stack to drive co-expression of multiple transgenes, then the expression of multiple transgenes is achieved, but homology-based gene silencing occurs and plasmid stability decreases due to recombination
Solution Approach 1:
The patent divides the promoter sequence into multiple segments by using different promoters for each transgene in the stack. Instead of repeating the same promoter sequence, each transgene is driven by a distinct promoter segment (e.g., promoter A for transgene 1, promoter B for transgene 2, etc.), thereby eliminating homology-based recombination while maintaining co-expression capability
Solution Approach 2:
The patent changes the sequence parameter of the promoter by selecting promoters with different sequence identities. By using promoters that have low sequence similarity to each other (different sequence parameters), the patent prevents homology-based gene silencing and recombination events that would otherwise occur with repeated identical promoter sequences
2Productivity
If repetitive DNA sequences are used within a transgene construct to achieve multiple transgene expression, then co-expression is enabled, but homology-based gene silencing frequently occurs in transgenic plants
Solution Approach 1:
The patent segments the regulatory sequences by assigning unique promoters to each transgene. This segmentation eliminates the repetitive DNA sequences that cause homology-based gene silencing, while still enabling co-expression of multiple transgenes through the use of multiple distinct regulatory elements
Solution Approach 2:
The patent applies local quality by making each promoter region unique in sequence composition. Each promoter has distinct local sequence characteristics that prevent homology-based interactions, while collectively they provide the necessary regulatory function for multi-gene expression
3Device complexity
If similar DNA sequences are used in transgene constructs to drive multiple transgenes, then the transgene stack is created, but recombination and plasmid instability occur in Agrobacterium systems
Solution Approach 1:
The patent segments the transgene construct by inserting unique promoter sequences between each transgene. This segmentation breaks up repetitive DNA patterns that trigger recombination in Agrobacterium, thereby maintaining plasmid stability while preserving the multi-gene stack architecture
Solution Approach 2:
The patent uses unique promoter sequences as intermediary elements between adjacent transgenes. These intermediary promoter sequences act as buffers that prevent recombination between flanking transgenes, maintaining plasmid stability in Agrobacterium transformation systems
Data Source
AI summary
Provided are vector constructs and methods for expressing a transgene in plant cells and/or plant tissues using gene regulatory elements, including the promoters, 5′-UTRs, and/or 3′-UTRs, isolated from Zea mays.