Nat1 Gene Selection Marker for Oomycete Genetic Transformation
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Solution Overview
Problem
Current screening markers for oomycete genetic transformation are limited, particularly for gene complementation, and the extensive use of existing pesticides leads to drug resistance, necessitating the development of novel markers for efficient genetic manipulation.
Innovation Solution
The application of the Nat1 gene as a selection marker for oomycete transformation, which confers nourseothricin resistance, allowing for high screening efficiency and supporting gene complementation and multi-knockout experiments by using a nourseothricin-containing culture medium.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the NPT II gene is used as a screening marker for oomycete transformation, then the transformant can be screened with high reliability using geneticin resistance, but the transformant cannot be reused for gene complementation due to G418 resistance
Solution Approach 1:
The patent changes the selection parameter from geneticin (G418) to nourseothricin by using the Nat1 gene instead of NPT II gene. This parameter change allows the transformant to be screened with high reliability while remaining reusable for gene complementation experiments, as nourseothricin resistance does not interfere with subsequent G418-based selections.
2Productivity
If pesticide resistance genes are used as screening markers, then the screening efficiency is improved, but the extensive use of these pesticides induces strains to develop drug resistance
Solution Approach 1:
The patent uses nourseothricin, an antibiotic, as a selectable marker instead of pesticide resistance genes. This disposable selection method achieves high screening efficiency without the harmful side effect of inducing pesticide resistance in oomycete strains, as the antibiotic selection does not cross-resist with agricultural pesticide applications.
3Ease of manufacture
If limited screening markers are used for oomycete transformation, then the transformation process can be completed, but the lack of available markers for gene complementation restricts further genetic manipulation
Solution Approach 1:
The Nat1 gene serves as a universal selectable marker that can be used in multiple genetic manipulation scenarios including both initial transformation and subsequent gene complementation experiments. This multi-functional marker system enables flexible and diverse genetic manipulation of oomycetes without being restricted by marker exhaustion or resistance conflicts.
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 Nat1 gene achieves a screening efficiency of 90% or more, reducing labor consumption and providing a diversified selection marker for oomycete transformation, effectively enabling gene complementation and multi-knockout processes.
Implementation Method 1
N-cetyltransferase 1 (Nat1) gene... encoding gene of a mutated protein that can induce the oomycete to produce more than 500 times resistance to the oxathiapiprolin
Data Source
AI summary
Disclosed is an application of Nat1 gene as a screening marker in the genetic transformation of an oomycete, where the Nat1 gene is employed as a screening marker to screen an oomycete transformant. A nucleotide sequence of the Nat1 gene is shown in SEQ ID NO: 1. This disclosure further provides a Nat1 gene-related biological material, which is a Nat1 gene-containing expression cassette, a recombinant vector containing the Nat1 gene or the expression cassette, a recombinant microorganism containing the Nat1 gene, the expression cassette or the recombinant vector, or a genetically-modified oomycete cell line containing the Nat1 gene, the expression cassette or the recombinant vector.