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目的:旨在敲除禾谷镰刀菌Fusarium graminearum Fg PDE1基因,确定其缺失突变体表型,从而分析该基因的生物学功能。方法:应用Split-marker技术构建含有潮霉素基因敲除盒,通过PEG介导原生质体转化,PCR筛查抗潮霉素转化子以获得缺失突变体ΔFg PDE1,根据突变体表型变化及致病性的检测对Fg PDE1基因的功能进行分析。结果:采用Split-marker技术,成功构建了Fg PDE1基因敲除盒;PEG介导转化禾谷镰刀菌原生质体后成功获得转化子。经PCR筛查,得到3个PCR确认的敲除突变体;表型观察发现,ΔFg PDE1菌落的外型及菌落生长速度与野生型没有明显差异。孢子侵染西红柿果实实验证明:以西红柿为侵染宿主,相对于野生型,突变体致病性没有明显减弱;但突变体分生孢子产量显著下降。结论:Fg PDE1基因可能与禾谷镰刀菌分生孢子的形成有关。
OBJECTIVE: To knock out the Fusarium graminearum Fg PDE1 gene from Fusarium graminearum and determine the phenotype of its deletion mutants to analyze the biological function of this gene. Methods: The knock-out cassette containing hygromycin gene was constructed by split-marker technique. PEG-mediated protoplast transformation was used to screen the hygromycin-resistant transformants by PCR to obtain the mutant ΔFg PDE1. According to the phenotypic changes and The pathogenicity test analyzed the function of the Fg PDE1 gene. Results: Fg PDE1 knockout cassette was successfully constructed by Split-marker technique. Transformants were successfully obtained after PEG-mediated transformation of Fusarium graminearum protoplasts. Three PCR-confirmed knockout mutants were obtained by PCR screening. Phenotypic observation showed no significant difference in the appearance and colony growth rate of ΔFg PDE1 colonies compared with wild type. Spore infestation of tomato fruit experiments show that: Tomato as the host infection, compared with the wild-type, mutant pathogenicity was not significantly reduced; but conidia mutant significantly decreased. Conclusion: Fg PDE1 gene may be related to the formation of Fusarium graminearum conidia.