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CHINESE JOURNAL OF OIL CROP SCIENCES ›› 2021, Vol. 43 ›› Issue (6): 1115-1125.doi: 10.19802/j.issn.1007-9084.2020256

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Optimization of T-DNA insertion-mediated mutagenesis of Leptosphaeria biglobosa and mutant screening

Yue-han YANG(), Jing ZHANG, Long YANG, Ming-de WU, Guo-qing LI()   

  1. Hubei Key Laboratory of Crop Disease Monitoring and Safety Control, Plant Science and Technology College, Huazhong Agricultural University, Wuhan 430070, China
  • Received:2020-08-26 Online:2021-12-22 Published:2021-12-23
  • Contact: Guo-qing LI E-mail:329017660@qq.com;guoqingli@mail.hzau.edu.cn

Abstract:

Blackleg of oilseed rape (Brassica spp.) is caused by Leptosphaeria biglobosa, which has been found to occur widely in oilseed rape-producing areas in our country, resulting in a substantial seed yield loss in the industry of oilseed rape. However, the mechanisms for ecological adaptation and pathogenesis of L. biglobosa have not been elucidated. Therefore, it is necessary to conduct in-depth studies on molecular mechanisms for ecological adaptation and pathogenesis of L. biglobosa. In order to fulfill this objective, this study was done to mutate L. biglobosa by Agrobacterium tumeficens-mediated transformation (ATMT). The contents included optimization of the factors affecting the ATMT, evaluation of the quality of the transformants, and screening of mutants. The results showed that the optimum ATMT factors for L. biglobosa Lb731 were: hygomycin B concentration at 50 μg/mL, transformation recipients (conidia) from the 15-day-old cultures (20℃), conidial concentration at 2 × 107-8 conidia/mL, co-incubation of A. tumeficens-recipients at 25℃ for 72 h. The transformation efficiency reached 80 transformants per million conidia on average. The T-DNA insertion frequency reached up to 100% based on PCR assays with 72.7% single-copy insertion among the transformants based on Southern blottings. Results also showed that the hygromycin-resistance trait in the transformants inherited stably through nuclear mitosis. A total of 32 mutants were screened from 2136 transformants, including 11 mutants with suppressed mycelial growth, 7 mutants with deficiency in pigment biosynthesis, and 14 mutants with deficiency in conidial production. Meanwhile, 7 out of the 32 mutants were identified to completely lose pathogenicity (virulence) on oilseed rape. Using the hiTAIL-PCR technique, three DNA sequences flanking the T-DNA-insertion sites were obtained from three mutants. Overall, these results laid a solid foundation for further elucidation of the molecular mechanisms for ecological adaptation and pathogenesis in L. biglobosa.

Key words: Leptosphaeria biglobosa, Agrobacterium tumeficiens-mediated transformation (ATMT), T-DNA insertional mutagenesis, mutant screening

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