State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; Xinjiang Agricultural University, Urumqi, Xinjiang 830000, China.
J Microbiol Methods. 2021 Oct;189:106313. doi: 10.1016/j.mimet.2021.106313. Epub 2021 Aug 25.
Dwarf bunt of wheat caused by Tilletia controversa Kühn has been identified an international quarantine disease, which replace the grain material into millions of teliospores. Agrobacterium tumefaciens-mediated transformation (ATMT) system is a powerful tool for fungi transformation with significant advantages of simple operation, high efficiency, and genetic stability of transformants. In this study, we constructed ATMT system for T. controversa. All the transformants were tested using Acetosyringone (AS) concentration at 150 μmol/l, hygromycin B at 25 μg/ml, 1 × 10 T. controversa hypha cells/ml, A. tumefaciens with OD of 0.5 co-cultivation at 16 °C for 48 h and culture was incubated at 16 °C for 20 days. Using the ATMT method, we cultivated 8 generations of transformants on complete medium (CM) containing hygromycin B antibiotic and validated by PCR, which indicate that T-DNA had been successfully inserted into each of T. controversa transformants. In addition, thermal asymmetric interlaced PCR (TAIL-PCR) evaluated the Ti element inserts were at random sites in the fungal genome. Thus, ATMT approach is an efficient tool for insertional mutagenesis of T. controversa.
由小麦黑粉菌（Tilletia controversa Kühn）引起的矮化小麦黑粉病已被确定为国际检疫性病害，它会将谷物材料转化为数百万个冬孢子。根癌农杆菌介导的转化（ATMT）系统是一种用于真菌转化的强大工具，具有操作简单、效率高和转化体遗传稳定性好的显著优势。在本研究中，我们构建了小麦黑粉菌的 ATMT 系统。所有转化体均采用 150 μmol/l 的乙酰丁香酮（AS）浓度、25 μg/ml 的潮霉素 B、1×10 个 T. controversa 菌丝细胞/ml、OD 值为 0.5 的根癌农杆菌在 16°C 下共培养 48 小时，在 16°C 下培养 20 天进行测试。使用 ATMT 方法，我们在含有潮霉素 B 抗生素的完全培养基（CM）上培养了 8 代转化体，并通过 PCR 进行验证，表明 T-DNA 已成功插入到每个小麦黑粉菌转化体中。此外，热不对称交错 PCR（TAIL-PCR）评估表明 Ti 元件插入是在真菌基因组的随机位点。因此，ATMT 方法是一种用于插入诱变小麦黑粉菌的有效工具。
