尖孢镰刀菌亚麻专化型Folprp4基因参与调控菌丝生长和分生孢子发生

Folprp4 Gene Involved in the Conidiogenesis and Mycelial Growth in Fusarium oxysporum f.sp.lini

目的:通过对尖孢镰刀菌中Folprp4基因的鉴定,揭示其在尖孢镰刀菌中的功能及致病相关性。方法:基于同源重组原理,根据测定出的Folprp4基因序列,应用Split-Marker重组技术构建含有潮霉素抗性基因(hph)的基因缺失盒。将基因缺失盒经PEG介导转化到野生型原生质体中,在含有潮霉素B的TCC培养基上筛选转化子,通过PCR正负筛查获得Folprp4基因缺失突变株(ΔFolprp4)。构建含有Folprp4基因的载体pZDH1,并将其转化到敲除突变体中进行互补测验。结果:与野生型(hm)和异位插入突变体(ecFolprp4)相比,敲除突变体菌丝生长受到严重阻碍,当野生型和异位插入突变体长满整个平板时,敲除突变体菌落呈小点状。敲除突变体的另一个显著变化是ΔFolprp4的分生孢子产量显著下降。侵染实验表明,ΔFolprp4对亚麻幼苗的毒力显著降低。互补实验表明,该互补载体的回复子(Folprp4-C)在菌落形态、生长速率、分生孢子产量和毒力方面均恢复到了野生型菌株。结论:Folprp4基因与尖孢镰刀菌的菌丝生长、分生孢子发生和致病性有关。

Objective:To identify the Folprp4 gene in Fusarium oxysporum f.sp.lini and reveal its function and pathogenic correlation in Fusarium oxysporum f.sp.lini.Methods:Based on the principle of homologous recombination,the Split-Marker strategy was applied to construct the gene deletion cassette containing hygromycin resistance gene(hph)according to the genomic sequence of Folprp4 gene determined.The constructs were transformed into wild-type protoplasts mediated by PEG and the transformants were screened on TCC medium containing hygromycin B.The Folprp4 gene deletion mutants were confirmed by PCR with positive and negative primers.To do a complement test,the vector pZDH1 containing Folprp4 gene was constructed,and transformed into the deletion mutants.Results:Compared with wild type(hm)and ectopic insertion mutant(ecFolprp4),the growth of the mycelia of the deletion mutants was seriously obstructed and its colony looked like a tiny dot when the wild type and ectopic insertion mutant extended full of the plate.Another striking change of the deletion mutant was that the conidial production ofΔFolprp4 were significantly decreased.The infection assay showed that the virulence ofΔFolprp4 on flax seedlings was significantly reduced.The complement test indicated that the transformants of the complementary vector(Folprp4-C)recovered in colony morphology,growth rate,conidial yield and virulence of wild-type strain.Conclusion:The results revealed that Folprp4 gene was tightly associated with mycelial growth,conidial formation and pathogenicity in Fusarium oxysporum f.sp.lini.