Chlorophyll Binding Ab Regulatory Elements for Multi-Transgene Expression
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Solution Overview
Problem
The challenge in plant biotechnology is the homology-based gene silencing (HBGS) that occurs when multiple transgenes with high sequence identity promoters are introduced into a genome, leading to reduced efficacy of transgenic products due to gene silencing and polynucleotide rearrangements, which complicates the expression of multiple transgenes in a single locus.
Innovation Solution
The use of regulatory elements from the Glycine max chlorophyll binding Ab gene, including promoters, 5' UTRs, and terminators, to create expression cassettes that can be linked with transgenes, allowing for diverse expression patterns and minimizing the use of identical promoters, thereby reducing the likelihood of HBGS.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple transgenes with high sequence identity promoters are introduced into a genome, then the quantity of transgenes expressed is increased, but homology-based gene silencing occurs leading to reduced expression efficacy
Solution Approach 1:
The patent changes the promoter sequence parameter by using regulatory elements from chlorophyll binding Ab genes with low sequence identity (less than 80% identity to each other) while maintaining functional equivalence. This parameter change allows multiple transgenes to be expressed simultaneously without triggering homology-based gene silencing, thus resolving the contradiction between increasing transgene quantity and maintaining expression efficacy
Solution Approach 2:
The patent segments the promoter region into distinct regulatory elements (5' UTR, promoter, 3' UTR, terminator) from different chlorophyll binding Ab genes. By using segmented regulatory elements with low sequence identity among them, the system can accommodate multiple transgenes without homology-based silencing, enabling both high transgene quantity and reliable expression
2Ease of manufacture
If the same promoter is repeatedly used in multi-transgene stacks, then the ease of construction is improved, but homology-based gene silencing occurs reducing transgene expression
Solution Approach 1:
The patent identifies chlorophyll binding Ab genes as a universal resource that provides multiple functional regulatory elements (promoters, 5' UTRs, 3' UTRs, terminators) that can be used across different transgene constructs. By using this universal family of regulatory elements with low mutual sequence identity, the system maintains construction simplicity while avoiding homology-based silencing
Solution Approach 2:
The patent changes the sequence identity parameter of promoters from high (repeated use of same promoter) to low (use of different chlorophyll binding Ab regulatory elements) while maintaining the functional parameter of promoter activity. This allows diverse transgene stacks to be constructed with reliable expression without sacrificing ease of manufacture
3Stability of the object's composition
If regulatory elements with high sequence identity are used, then the consistency of expression pattern is improved, but polynucleotide rearrangements occur complicating transgene expression
Solution Approach 1:
The patent changes the sequence identity parameter from high to low (less than 80% identity) among regulatory elements from different chlorophyll binding Ab genes. This parameter change prevents polynucleotide rearrangements while the functional equivalence of these regulatory elements maintains consistent expression patterns across transgenes
Solution Approach 2:
The patent creates a composite regulatory system by combining elements (promoters, UTRs, terminators) from multiple different chlorophyll binding Ab genes. This composite approach using low sequence identity elements prevents genomic rearrangements while maintaining stable and consistent transgene expression patterns
Data Source
Figure 1~1B
Figure 2
Figure 2
AI summary
Provided are compositions and methods for expressing a transgene in plant cells and/or plant tissues using regulatory elements, including the promoters, 5'UTR, 3' UTRs, and/or terminators isolated from Glycine max chlorophyll binding Ab genes.