TALE N-Terminus for Faster Corn Transformation and Regeneration
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Solution Overview
Problem
Current plant transformation methods, such as Agrobacterium-mediated transformation, are time-consuming and have low transformation efficacy, necessitating improved techniques for faster and more efficient generation of transgenic plants.
Innovation Solution
The use of a truncated transcription activator-like effector (TALE) polypeptide, comprising the N-terminal region and optionally the Central Repeat Region, but lacking a transcriptional activation domain, is introduced into plant cells to enhance transformation efficacy, leading to faster growth of transformed calli and increased biomass of generated transgenic plants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Agrobacterium-mediated transformation is used, then stable integration of T-DNA into plant genome is achieved, but transformation efficacy is low and regeneration time is very time-consuming
Solution Approach 1:
The patent divides the TALE protein into functional segments: the N-terminal domain (amino acids 1-307) is separated from the C-terminal DNA-binding and activation domains. This segmented approach allows the N-terminal domain to be used independently to enhance transformation efficacy without requiring the complete TALE structure, thereby improving productivity while maintaining reliability through the natural Agrobacterium system.
Solution Approach 2:
The invention extracts and utilizes only the N-terminal domain of the TALE protein (amino acids 1-307), removing the C-terminal domains responsible for DNA binding and transcriptional activation. This extracted N-terminal domain alone is sufficient to enhance transformation efficacy, solving the contradiction by providing the beneficial effect without the complexity and time-consuming aspects of complete TALE-mediated transformation.
2Reliability
If complete TALE polypeptide is used, then transcriptional activation and DNA binding occur, but transformation procedure becomes more complex and time-consuming
Solution Approach 1:
The patent extracts only the N-terminal domain (amino acids 1-307) from the complete TALE polypeptide, removing the C-terminal domains that perform DNA binding and transcriptional activation. This extraction demonstrates that the N-terminal domain alone can enhance transformation efficacy, simplifying the transformation procedure while maintaining the essential function of improving transformation reliability.
Solution Approach 2:
By segmenting the TALE protein and using only the N-terminal portion, the invention reduces procedural complexity. The N-terminal domain functions independently to enhance transformation without requiring the additional steps or complexity of delivering and expressing complete TALE proteins with multiple functional domains.
3Measurement precision
If selection for transformed plant cells is carried out, then transformed cells can be identified among untransformed cells, but the process increases time consumption and reduces productivity
Solution Approach 1:
The invention extracts and uses only the N-terminal domain of TALE to enhance transformation efficacy, which indirectly improves the efficiency of selecting transformed cells. By increasing the proportion of successfully transformed cells through N-terminal domain-mediated enhancement, the selection process becomes more efficient, and regeneration can proceed faster without compromising selection accuracy.
Solution Approach 2:
The N-terminal domain of TALE performs a preliminary action by enhancing transformation efficacy before the selection and regeneration steps. This preliminary enhancement increases the number of successfully transformed cells, thereby reducing the time and effort required for subsequent selection and regeneration processes while maintaining measurement precision in identifying transformed cells.
Data Source
AI summary
The present invention is in the field of molecular biology and relates to improved methods for plant transformation and to polynucleotides and polypeptides for achieving the same.