Generation and Screening of T-DNA Insertion Mutants Mediated by Agrobacterium tumefaciens in the Garden Asparagus Stem Blight Pathogen Phomopsis asparagi

文献类型： 外文期刊

第一作者： Zhang, Yueping

作者： Zhang, Yueping;Qu, Huaxiang;Zhao, Ping;Tang, Yongping;Zhou, Jingsong;Luo, Shaochun;Yin, Yuling;Chen, Guangyu

作者机构：

期刊名称：CURRENT MICROBIOLOGY （ 影响因子：2.188； 五年影响因子：2.197 ）

ISSN： 0343-8651

年卷期： 2017 年 74 卷 11 期

页码：

收录情况： SCI

摘要： The garden asparagus stem blight caused by filamentous fungus Phomopsis asparagi exposes a serious threat on asparagus production globally. However, to present, we understand poorly about the molecular mechanisms of fungal pathogenicity. To facilitate functional genomics research of P. asparagi, here we developed a highly efficient and stable Agrobacterium tumefaciens-mediated transformation approach which yielded 150-200 transformants per 1 x 10(6) conidia. Our results indicated that 25 A degrees C, acetosyringone concentration of 150 mu mol/L, and 72 h were recommended as optimal co-cultivation conditions for the transformation. Using this transformation approach, we constructed a T-DNA insertion mutant library containing 1253 strains. Twenty randomly selected T-DNA insertion mutants were able to grow on 0.2 x PDA selective media after five successive subcultures without selective pressure, indicating that the exogenous T-DNA was stably integrated into the P. asparagi genome. We confirmed several randomly selected mutants using PCR with primers specific to the hph gene. Southern blots suggested that three out of the five selected mutants have a single T-DNA insertion. Interestingly, multiple mutant candidates with growth defects were obtained from the growth assay. Moreover, several mutants were selected for further analysis on the T-DNA flanking sequences through TAIL-PCR analysis. A sequence comparison of total junction fragments implied that the insertion of T-DNA within P. asparagi genome appeared to be a random event. The transformation technology and genetic resources developed here will facilitate studies of pathogenic mechanisms in this devastating filamentous fungal pathogen of garden asparagus.

分类号：

  • 相关文献
作者其他论文 更多>>

微信小程序