An efficient gene disruption method using a positive-negative split-selection marker and Agrobacterium tumefaciens-mediated transformation for Nomuraea rileyi
文献类型： 外文期刊
作者： Su, Yu 1 ; Wang, Zhongkang 1 ; Shao, Changwen 2 ; Luo, Yuanli 3 ; Wang, Li 1 ; Yin, Youping 1 ;
作者机构： 1.Chongqing Univ, Sch Life Sci, Chongqing 400030, Peoples R China
2.Linyi Univ, Coll Agr & Forestry, Linyi 276000, Peoples R China
3.Southeast Chongqing Acad Agr Sci, Chongqing 408000, Peoples R China
关键词： Nomuraea rileyi;Agrobacterium tumefaciens-mediated transformation (ATMT);Split-marker;beta-Glucuronidase;Negative selection marker
期刊名称：WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY （ 影响因子：4.0； 五年影响因子：4.3 ）
ISSN： 0959-3993
年卷期： 2018 年 34 卷 2 期
页码：
收录情况： SCI
摘要： 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 beta-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.
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