An efficient gene disruption method using a positive-negative split-selection marker and Agrobacterium
Yu Su1,2, Zhongkang Wang1, Changwen Shao3
1School of Life Science, Chongqing University, Chongqing, 400030, China.
Abstract:
Targeted gene disruption via Agrobacterium tumefaciens-mediated transformation (ATMT) and homologous recombination is the most common method used to identify and investigate the functions of genes in fungi. However, the gene disruption efficiency of this method is low due to ectopic integration. In this study, a high-efficiency gene disruption strategy based on ATMT and the split-marker method was developed for use in Nomuraea rileyi. The β-glucuronidase (gus) gene was used as a negative selection marker to facilitate the screening of putative transformants. We assessed the efficacy of this gene disruption method using the NrCat1, NrCat4, and NrPex16 genes and found that the targeting efficiency was between 36.2 and 60.7%, whereas the targeting efficiency using linear cassettes was only 1.0-4.2%. The efficiency of negative selection assays was between 64.1 and 82.3%. Randomly selected deletion mutants exhibited a single copy of the hph cassette. Therefore, high-throughput gene disruption could be possible using the split-marker method and the majority of ectopic integration transformants can be eliminated using negative selection markers. This study provides a platform to study the function of genes in N. rileyi.
Related Concept Videos
Positive, Negative, and Zero Work
Positive and Negative Feedback Loops
Chromatin Position Affects Gene Expression
Topologically Associated Domains (TADs)
The 3-dimensional positioning of chromatin in the nucleus influences the...
Position-effect Variegation
Bacterial Transformation
Griffith made an unexpected discovery when he killed the pathogenic strain and mixed its remains with the live, non-pathogenic strain. Not only did the mixture kill host mice, but it also contained living pathogenic bacteria that...
Velocity and Position by Integral Method
Consider an example to calculate the velocity and position from the acceleration function. A motorboat is traveling at a constant velocity of 5.0 m/s when it starts to decelerate to arrive at the dock. Its acceleration is...


