禾谷镰刀菌中FgPDE1基因的敲除及其功能的研究

Research on Gene Knockout and Function of FgPDE1 in Fusarium graminearum

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： The purpose is to knock out Fg PDE1 gene in Fusarium graminearum,so as to identify the function of the gene through analysis of the phenotype of the deletion mutants. Methods： The Splitmarker strategy is applied to build knockout cassette containing hygromycin phosphotransferase gene and antihygromycin transformants are obtained by using PEG-mediated protoplast transformation. The Fg PDE1 gene deletion mutants are screened by the absent of the PCR products of the Fg PDE1 gene. The function of the gene is analyzed according to the mutant phenotype and pathogenicity detection. Results： The Split-marker knockout cassette is successfully constructed and the transformants are obtained after PEG-mediated transformation of the protoplasts of PH-1 and then,three Fg PDE1 gene deletion mutants are obtained through PCR screening. The phenotypic analysis revealed that there is no significant difference between ΔFg PDE1 and wild type in terms of colony phenotype and growth rate. The virulence assay by fruit of tomato infected by conidiospores show that the mutants do not decrease greatly in pathogenicity. However,microscopic observation show that the conidiospore amount of ΔFg PDE1 reduce significantly. Conclusions： Fg PDE1 gene might be related to conidia formation of the Fusarium graminearum.